Novel Vaccination Strategies

Novel Vaccination Strategies Edited by Stefan H. E. Kaufmann

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Novel Vaccination Strategies Edited by Stefan H. E. Kaufmann

Editor : Prof. Dr. Stefan H. E. Kaufmann Max-Planck-Institute for Infection Biology Department of Immunology Schumannstraûe 21/22 10117 Berlin Germany

& This book was carefully produced. Never-

theless, authors, editor and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data : A catalogue record for this book is available from the British Library. Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at .  Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2004 All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Printed in the Federal Republic of Germany Printed on acid-free paper Composition ProSatz Unger, Weinheim Printing betz-druck gmbh, Darmstadt Bookbinding Litges & Dopf Buchbinderei GmbH, Heppenheim ISBN

3-527-30523-8

V

Contents Colour Plates

XXXIII

Part I 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8

Part II 2

2.1 2.2 2.2.1 2.2.2 2.2.2.1 2.2.2.2 2.2.3 2.3 2.3.1

Challenges for the Vaccine Developer, including Correlates of Protection Gustav J. V. Nossal Introduction 3 Mechanisms of Protection within the Immune System 4 Protection against Viruses 5 HIV/AIDS as an Example of a Persisting Virus 8 Protection against Extracellular Bacteria 9 Protection against Intracellular Bacteria 11 Protection against Parasites 12 Conclusions 14 References 15

3

Vaccination and Immune Response Shaping Adaptive Immunity against Pathogens: The Contribution of Innate Immune Responses 19 Stefan Ehlers and Silvia Bulfone-Paus Introduction 19 Activation of Innate Immunity: Sensing the Enemy 20 Pathogen-associated Molecular Patterns 21 Host Cellular Sensors 24 Dendritic Cells 24 Mast Cells 25 Nonpeptide MHC Ligands Triggering Invariant T-cell Receptors 26 Translating Innate Immune Activation into Regulatory Circuits: Molecular Pathways Shaping Adaptive Immunity 27 TLR-initiated Signaling Cascades 27

VI

Contents

2.3.2 2.3.2.1 2.3.2.2 2.3.3 2.3.3.1 2.3.3.2 2.4

Molecules Involved in Recruiting Effector Cells 28 Defensins 28 Chemokines 30 Molecules Involved in T and B Cell Differentiation 31 Th1-inducing Cytokines 32 Th2-inducing Cytokines 35 Implications for Vaccine Development 36 References 38

3

Adjuvant-induced Th2- and Th1-dominated Immune Responses in Vaccination 51 James M. Brewer and Kevin G. J. Pollock Introduction 51 The Two-Signal Model of Adjuvant-induced Immune Activation 53 Th1 and Th2 Induction by Vaccine Adjuvants 56 Antigen Dose Effects 57 The Three-signal Model of Adjuvant-induced Immune Activation 58 Th2 Induction by Adjuvants 61 Differential Activation of DCs 63 Inappropriate Th1/Th2 Responses to Vaccines 64 Human Th2 vaccines 65 Human Th1 Vaccines 65 Conclusion 66 References 67

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11

4 4.1 4.2 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.4 4.4.1 4.4.2 4.4.3 4.4.4 4.5 4.5.1 4.5.2 4.6

Memory 73 Alexander Ploss and Eric G. Pamer Introduction 73 Characteristics of Memory Cells 74 CD8+ T Cell Memory 75 Phenotyping Memory CD8+ T Cells 75 Enhanced Responsiveness of Memory CD8+ T cells: Potential Mechanisms 76 Generation of Memory CD8+ T Cells 76 Maintaining CD8+ T Cell Memory 78 Models of CD8+ T cell Memory Generation 79 CD4+ T Cell Memory 82 Differentiation of Effector and Memory CD4+ T Cells 82 Phenotype of Memory CD4+ T Cells 83 Memory Generation and Maintenance 83 Trafficking of Memory CD4+ T Cells 84 B cell Memory 84 Generation of B Cell Memory 84 Maintenance of B Cell Memory 85 Conclusions 86

Contents

Acknowledgements 86 References 86 5

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8

T Cell-based Vaccines 89 Katharina M. Huster, Kristen M. Kerksiek, and Dirk H. Busch Summary 89 Introduction 89 Ex-vivo Detection of Antigen-specific T Cells 91 In-vivo Kinetics of Antigen-specific T Cell Responses 95 Effector Function and Subtypes of Effector T Cells 97 T Cell Receptor Repertoire, Avidity Maturation, and Epitope Competition 99 Functional Heterogeneity of T Cell Memory 101 Vaccination Strategies and Their Efficacy for T Cell-based Vaccination 103 Concluding Remarks 106 References 107

Part III

Adjuvants

6

Microbial Adjuvants 115 Klaus Heeg, Stefan Zimmermann, and Alexander Dalpke Introduction 115 Microbial Danger Signals 117 Toxins (CT and LT) 117 Toll-like Receptor-dependent Microbial Adjuvants 118 Lipopolysaccharide and Lipid A Derivatives 118 Peptidoglycan and Lipoteichoic Acid 119 Other Microbial Components (Lipopeptides, Flagellin) 119 Bacterial DNA 119 Toll-like Receptor-dependent Synthetic Compounds 121 Synthetic CpG DNA 121 Other Synthetic TLR ligands 123 Low Molecular Weight TLR Agonists 124 Conclusion 125 References 126

6.1 6.2 6.2.1 6.2.2 6.2.2.1 6.2.2.2 6.2.2.3 6.2.2.4 6.2.3 6.2.3.1 6.2.3.2 6.2.3.3 6.3

7 7.1 7.2 7.2.1 7.2.2 7.2.3

Host-derived Adjuvants 129 Norbert Hilf, Markus Radsak, and Hansjörg Schild Introduction 129 Heat Shock Proteins in Immunology 130 General Remarks 130 Heat Shock Proteins Are Immunogenic 131 Heat Shock Proteins Bind Peptides 131

VII

VIII

Contents

7.2.4 7.2.5 7.2.6 7.2.7 7.2.8 7.2.9 7.3 7.4

Receptor-mediated Uptake of HSPs 133 Cross-presentation Pathways for HSP–Peptide Complexes 135 Danger Signals – The Importance of the Second Signal 137 Heat Shock Proteins as Danger Signals 137 Heat Shock Proteins as Endogenous Adjuvants 139 Clinical Use of Heat Shock Proteins 140 Cytokines as Adjuvants 141 Concluding Remarks 142 References 142

8

Microparticles as vaccine adjuvants and delivery systems 147 Derek T. O’Hagan and Manmohan Singh Introduction 147 The Role of Adjuvants in Vaccine Development 148 Immunostimulatory Adjuvants 150 MPL 150 CpG 150 QS21 150 Cytokines 151 Particulate Vaccine Delivery Systems 151 Lipid-based Particles as Adjuvants 152 The Adjuvant Effect of Synthetic Particles 153 Uptake of Microparticles into APC 153 Microparticles as Adjuvants for Antibody Induction 153 The Induction of Cell-mediated Immunity with Microparticles 155 Microparticles as Delivery Systems for DNA Vaccines 155 Microparticles as Delivery Systems for Adjuvants 157 Microparticles as Single-dose Vaccines 157 Alternative Particulate Delivery Systems 159 Alternative Routes of Immunization 159 Mucosal Immunization with Microparticles 160 Microparticles as Delivery Systems for Mucosal Adjuvants 160 Adjuvant for Therapeutic Vaccines 162 Future Developments in Vaccine Adjuvants 162 Acknowledgments 163 References 163

8.1 8.2 8.3 8.3.1 8.3.2 8.3.3 8.3.4 8.4 8.4.1 8.4.2 8.4.3 8.4.4 8.4.5 8.4.6 8.4.7 8.4.8 8.4.9 8.5 8.5.1 8.5.2 8.6 8.7

9

9.1 9.2 9.2.1 9.2.1.1

Liposomes and ISCOMs 173 Gideon Kersten, Debbie Drane, Martin Pearse, Wim Jiskoot, and Alan Coulter Introduction 173 Liposomes and Related Structures 176 Composition, Characteristics, and Preparation Methods of Liposomes 176 Composition and Characteristics of Liposomes 176

Contents

9.2.1.2 9.2.2 9.2.2.1 9.2.2.2 9.2.2.3 9.2.3 9.3 9.3.1 9.3.1.1 9.3.1.2 9.3.1.3 9.3.2 9.3.2.1

9.3.2.2 9.3.3 9.3.3.1 9.3.3.2 9.4

10

Liposomes 176 Transfersomes 176 Niosomes 177 Virosomes 177 Proteosomes and Outer Membrane Vesicles 178 Archaeosomes 179 Cochleates 179 Preparation Methods of Liposomes 179 Mechanisms of Action of Liposomes 180 Protection, Stabilization, and Mimicry 181 Targeting 182 Enhanced or Controlled Processing 182 Liposome Performance and Products 183 ISCOMs 184 Composition, Characteristics, and Preparation Methods of ISCOMs 184 Composition 184 Characteristics of ISCOMs 185 Preparation of ISCOMs 186 Immunology and Mode of Action of ISCOM Vaccines 187 Immune Responses to ISCOM Vaccines 187 Parenteral Immunization of Mice 187 Parenteral Immunization of Nonhuman Primates 188 Mucosal Immunization 189 Effective Immunization with ISCOM Vaccines in the Presence of Preexisting Antibody 189 Mode of Action of ISCOM Vaccines 189 Performance and Products 191 Protection Afforded by ISCOM Vaccines in Animal Models 191 Human Clinical Trials with ISCOMs 191 Perspectives 193 References 194

Virosomal Technology and Mucosal Adjuvants 197 Jean-François Viret, Christian Moser, Faiza Rharbaoui, Ian C. Metcalfe, and Carlos A. Guzmán 10.1 Overview 197 10.2 Mucosal Adjuvants 200 10.2.1 Introduction 200 10.2.2 Families of Mucosal Adjuvants 200 10.2.3 Administration Strategies 204 10.2.3.1 Direct Admixing of Antigen and Adjuvants 204 10.2.3.2 Covalent Linkage of the Adjuvant and Antigen or Adjuvant Incorporation into other Mucosal Delivery Systems 205 10.2.3.3 Adjuvant in Prime–Boost Vaccination Strategies 205 10.2.4 Interaction of Mucosal Adjuvants with the Innate Immune System 206

IX

X

Contents

10.2.5 10.3 10.3.1 10.3.2 10.3.2.1 10.3.2.2 10.3.2.3 10.3.2.4 10.3.3 10.3.4

Conclusion 207 Virosomal Technology 208 Introduction 208 Adjuvant Properties of Virosomes 209 Virosome Structure and Immunopotentiation 209 Depot Effect 210 The Pivotal Role of Fusion-active Virosomal Hemagglutinin 210 Effect of Pre-existing Immunity to Influenza Virus 211 Validation of the Virosomal Vaccine Concept 212 Conclusion 213 References 214

Part IV

Classical and Novel Vaccination Strategies : A Comparison

11

Classical Bacterial Vaccines 221 Thomas Ebensen, Claudia Link, and Carlos A. Guzmán Bacterial Vaccines: Introductory Remarks 221 Inactivated Vaccines 223 Methods of Inactivation 223 Advantages and Limitations of Inactivated Vaccines 223 Live Vaccines 224 Attenuation 225 Advantages and Limitations of Live Bacterial Vaccines 225 Vaccines for Human Bacterial Diseases 226 Anthrax (Bacillus anthracis) 226 Cholera (Vibrio cholerae) 227 Enterotoxigenic Escherichia coli 228 Plague (Yersinia pestis) 229 Shigellosis (Shigella species) 229 Tuberculosis (Mycobacterium tuberculosis) 230 Typhoid Fever (Salmonella enterica serovar Typhi) 231 Tularemia (Francisella tularensis) 232 Whooping cough (Bordetella pertussis) 232 Veterinary Bacterial Vaccines 233 Infections Caused by Bordetella and Pasteurella Species 234 Brucellosis (Brucella spp.) 235 Porcine Pleuropneumonia (Actinobacillus pleuropneumoniae) 237 Diseases Caused by Mycoplasma spp. 237 Salmonellosis in Animals 238 Leptospirosis (Leptospira spp.) 239 Other Commercially Relevant Animal Diseases 239 Conclusions 240 Acknowledgements 240 References 240

11.1 11.2 11.2.1 11.2.2 11.3 11.3.1 11.3.2 11.4 11.4.1 11.4.2 11.4.3 11.4.4 11.4.5 11.4.6 11.4.7 11.4.8 11.4.9 11.5 11.5.1 11.5.2 11.5.3 11.5.4 11.5.5 11.5.6 11.5.7 11.6

Contents

12 12.1 12.2 12.3 12.3.1 12.3.2 12.3.2.1 12.3.2.2 12.3.2.3 12.4 12.4.1 12.4.2 12.4.2.1 12.4.2.2 12.4.2.3 12.4.2.4 12.4.2.5 12.4.3 12.4.4

13 13.1 13.2 13.2.1 13.2.2 13.3 13.4 13.4.1 13.4.2 13.5 13.5.1 13.5.2 13.5.3 13.6 13.7 13.7.1 13.7.2 13.7.3 13.7.4 13.8

Subunit Vaccines and Toxoids 243 Maria Lattanzi, Giuseppe Del Giudice, and Rino Rappuoli Introduction 243 Toxoids 243 Subunit Vaccines: Conventional Vaccinology Approach 244 Polysaccharide Vaccines 244 Recombinant DNA Technology for Subunit Vaccines 245 HBV Vaccine 246 Acellular Pertussis Vaccine 246 Lyme Disease Vaccine 247 The Future of Subunit Vaccine Development: The Genomic Approach 248 When Theory Becomes Reality: The MenB Example 248 Further Applications of the Genomic Approach to Vaccine Development 253 Streptococcus pneumoniae 253 Staphylococcus aureus 254 Porphyromonas gingivalis 254 Streptococcus agalactiae 254 Chlamydia pneumoniae 255 The Genomic Approach to Parasite Vaccines 255 The Genomic Approach to Viral Vaccines 256 References 258 Engineering Virus Vectors for Subunit Vaccines Joseph Patrick Nkolola and Tomas Hanke Introduction 265 Adenoviruses 266 Replication Incompetent Adenoviruses 266 Replication-selective Adenoviruses 267 Adeno-associated Viruses 269 Poxviruses 269 Mammalian Poxviruses 271 Avipoxvirus Vectors 272 Herpes Simplex Viruses 272 Recombinant HSV Vectors 273 Amplicon Vectors 273 Disabled Infectious Single-cycle HSV 275 Retroviruses 275 Alphaviruses 276 Full-length Infectious Clones 277 RNA Replicons 277 DNA Plasmid Replicons 277 Particle-based Replicons 279 Polioviruses 279

265

XI

XII

Contents

Rhabdovirus Vectors 281 Heterologous Prime–Boost Vaccination Strategies 282 Cell Lines Acceptable for Growing Human Recombinant Subunit Vaccines 282 13.11.1 History and General Characteristics of the Cell Line 283 13.11.2 The Cell Bank System 283 13.11.3 Quality Control Testing 283 13.12 Conclusion 284 References 284 13.9 13.10 13.11

14

14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8

15

15.1 15.2 15.3 15.3.1 15.3.2 15.3.3 15.4 15.5

16

16.1 16.2

Update on antiviral DNA vaccine research (2000– 2003) 289 Daniel Franke, Jovan Pavlovic, Tillmann S. Utesch, Max von Kleist, Jan Schultz, Guenter Dollenmaier, and Karin Moelling Summary 289 Effect of Antiviral DNA Vaccines in Mice 289 Effect of Antiviral DNA Vaccines in Larger Species 301 Genetic Adjuvants 302 CTL-Epitope Immunization 303 Targeting DNA Vaccines to Cellular Compartments or the Cell Surface 304 DNA for Chimeric Antigens 304 DNA-prime–Protein/Viral-boost Immunization 305 Age-dependent Effectiveness of DNA Vaccines 306 References 307 Live Recombinant Bacterial Vaccines 319 Simon Clare and Gordon Dougan Summary 319 Introduction 319 Early Efforts to Generate Recombinant Live Bacterial Vaccines 322 Clinical Studies Involving the Development of Live Recombinant Vaccines 325 Live Recombinant Salmonella Vaccines 325 Live Cholera Vaccines 328 Live Shigella Vaccines 329 Expression of Heterologous Antigens in Live Bacterial Vectors 330 The Future 333 Acknowledgements 334 References 334 Mucosal Vaccination 343 Wieslawa Olszewska and Peter J. M. Openshaw Summary 343 Introduction 343 Goals of Mucosal Vaccination 344

Contents

16.3 16.3.1 16.3.2 16.3.3 16.3.4 16.3.5 16.4 16.4.1 16.4.1.1

16.4.1.2 16.4.1.3 16.4.2 16.4.2.1 16.4.2.2 16.4.2.3 16.4.2.4 16.4.2.5 16.4.2.6 16.4.2.7 16.5 16.5.1 16.6 16.7

17

17.1 17.2 17.3 17.4 17.5 17.6 17.7 17.7.1 17.7.2 17.7.3 17.8

Benefits of Mucosal Vaccination 345 Main Features of the Common Mucosal Immune System 345 Distinctive Characteristics of Mucosal Immunity 346 Multivalent Mucosal Vaccines 347 Edible Vaccines 347 Overcoming Preexisting Immunity or Tolerance 348 Challenges for Mucosal Immunization 349 Mucosal Delivery Systems 349 Live Bacterial Vectors 349 Commensal Flora as Expression Vectors 349 Pathogens as Expression Vectors 349 Virosomes 350 Mucosal DNA Vaccines 350 Mucosal Adjuvants 351 Biodegradable Polymeric Particles 351 Bacterial Toxins 351 CpG Oligodinucleotides 352 Cytokines and Chemokines 353 Saponins 353 Immune Stimulating Complexes (ISCOMS) 353 MF59 355 Vaccination via the Respiratory Tract 355 Applications of Nasal Vaccination 355 Oral Vaccines 357 Conclusions 357 Acknowledgements 357 Reference List 359 Passive Vaccination and Antidotes : A Novel Strategy for Generation of Wide-spectrum Protective Antibodies 365 Antonio Cassone and Luciano Polonelli Introduction and Definitions 365 Emergence of New Agents of Disease 367 Passive Vaccination and Antidotes: Advantages and Disadvantages 368 Passive Vaccination: Implementation and Obstacles 370 A Novel Strategy for Passive Vaccination: Concept and Relevance of Killer Antibodies 371 Fungi and Fungal Infections 372 A Novel Approach to Passive Vaccination through the Merging of Killer Phenomenon and Idiotypic Network 373 The Killer Phenomenon 373 Antibodies and the Idiotypic Network 374 Yeast Killer Toxin Anti-idiotypes 375 Microbicidal IdAb: Consequences and Extensions 377

XIII

XIV

Contents

17.9 17.10 17.11

18 18.1 18.2 18.2.1 18.2.2 18.2.3 18.3 18.3.1 18.3.2 18.3.3 18.4 18.4.1 18.4.1.1 18.4.1.2 18.4.2 18.4.2.1 18.4.2.2 18.4.2.3 18.4.2.4 18.4.3 18.4.3.1 18.4.3.2 18.5 18.5.1 18.5.2 18.5.3 18.6

19

19.1 19.2 19.2.1

Passive Vaccination with Single-chain Variable-fragment Antibodies Carried or Secreted by a Mucosal Live Bacterial Vector 378 Antibody Peptide Fragments as Wide-spectrum Anti-infectives 380 Conclusions and Perspectives 382 Acknowledgements 383 References 383 Plant-based Oral Vaccines 387 Kan Wang, Rachel Chikwamba, and Joan Cunnick Introduction 387 Mucosal Immunization 387 Vaccination Strategies for Infectious Diseases 388 Mucosal Immunization vs. Parenteral Immunization 389 Mucosal Immunization and Adjuvants 390 Plant-derived Edible Vaccines 391 Advantages of the Plant-based System 391 Transient and Stable Systems for Production of Plant-derived Proteins 392 Choice of Plants and Plant Tissues 393 Plant-expression Systems for Antigen Production 394 Transcriptional Level 396 Choice of Promoters 396 Transcriptional Gene Silencing 396 Post-transcriptional Level 397 Introns 397 mRNA Stability and 3´ Terminator 397 An Optimal Start Context and 5´-end Enhancer for Translation 398 Codon Usage 398 Post-translational Level and Beyond 399 Targeting and Retention Signals 399 Stability of Gene Expression and Transmission of the Transgene 400 Maize as Production and Delivery System 400 Antigen Production in Endosperm Tissue of Maize Seed 402 Antigen Production in Embryo (Germ) Tissue of Maize Seed 403 Pharmaceutical Crop Production and Containment 405 Concluding Remarks 406 References 407 Virus-like Particles : Combining Innate and Adaptive Immunity for Effective Vaccination 415 Martin F. Bachmann and Gary T. Jennings Summary 415 Immunology of Vaccines 415 Immunology of VLPs 416 B Cell Responses 416

Contents

19.2.2 19.3 19.4 19.4.1 19.4.2 19.4.3 19.5 19.5.1 19.5.2 19.5.3 19.5.4

T Cell Responses 417 VLPs as Viral Vaccines 419 VLPs as Carriers of B and/or T Cell Epitopes 420 Fused Epitopes 420 Coupled Epitopes 422 Targeting Self Molecules by using VLPs 424 Clinical Development 425 Hepatitis B Virus VLP Vaccine 425 Human Papilloma VLP Vaccines 426 Norwalk Virus VLP Vaccines 428 VLPs Presenting Foreign Epitopes 429 References 430

Part V

Vaccines for Specific Targets

20

Helicobacter pylori 435 Paolo Ruggiero, Rino Rappuoli, and Giuseppe Del Giudice Introduction 435 Epidemiology of H. pylori Infection 436 H. pylori-related Diseases 436 H. pylori Antigens Relevant in Virulence and Pathogenesis 437 Eradication of H. pylori: the Pros and Cons 439 Current Therapies against H. pylori: Efficacy and Limits 439 Why Develop a Vaccine against H. pylori 440 Animal Models of H. pylori Infection 441 Mice and Other Rodents 441 Ferrets 442 Gnotobiotic Piglets 442 Monkeys 443 Dogs 443 The feasibility of Vaccination in Animal Models 443 The Mechanisms of Protective Immunity against H. pylori 444 Vaccination against H. pylori in Humans 446 Vaccination with Purified Recombinant Urease 446 Salmonella-vectored Urease 447 Inactivated Whole-cell Vaccines 448 Parenteral Multi-component Vaccines 449 Conclusions 450 References 451

20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.8.1 20.8.2 20.8.3 20.8.4 20.8.5 20.9 20.10 20.11 20.11.1 20.11.2 20.11.3 20.11.4 20.12

21 21.1 21.2

Novel Vaccination Strategies against Tuberculosis 463 Stefan H. E. Kaufmann Introduction 463 Mechanisms underlying Infection and Immunity 464

XV

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Contents

21.3 21.4 21.5 21.5.1 21.5.2 21.5.3 21.6

Rational Vaccine Design: Basic Considerations 469 Protective Antigens and Knockout Targets 469 The Major Strategies: Subunit, Attenuated, and Combination Vaccines 471 Subunit Vaccines 471 Attenuated Vaccines 472 Combination Vaccines 474 Concluding Remarks 474 Acknowledgements 474 References 475

22

Rationale for Malaria Vaccine Development 479 Allan Saul,Victor Nussenzweig, and Ruth S. Nussenzweig 22.1 Introduction 479 22.2 Preerythrocytic Vaccines 481 22.2.1 Rationale for Vaccines that Elicit Antibody-mediated Protection 481 22.2.2 Rationale for Vaccines that Elicit Cell-mediated Immunity 482 22.2.3 Human Vaccine Trials 485 22.3 Asexual Stage Vaccines 488 22.3.1 Red Cell Surface Antigens 490 22.3.2 Antigens Eliciting Antibody-dependent Cellular Inhibition 491 22.3.3 Antitoxin Vaccines 492 22.3.4 Antibody-independent Mechanisms 493 22.4 Mosquito-stage Vaccines 494 22.4.1 Targets of Transmission-blocking Vaccines 495 22.4.1.1 The 6-Cys Malaria Gamete Surface Antigens 495 22.4.1.2 The P25 and P28 EGF Domain Zygote, Ookinete, and Oocyst Antigens 496 22.4.1.3 Chitinase 497 22.5 Conclusion 497 References 498 23

23.1 23.2 23.3 23.3.1 23.3.2 23.3.3 23.3.4 23.4 23.5 23.6 23.6.1

Vaccine for Specific Targets : HIV 505 R. Kay, Edmund G.-T. Wee, and Andrew J. McMichael Introduction 505 Antibody Vaccines 506 The T Cell Response 509 CTL-inducing Vaccines 510 Studies in Humans 511 CD4+ T Cell Help 512 The Dynamics of the CD8+ T Cell Response 513 Innate Immunity 513 Mucosal Immunity 515 Vaccine Design 516 Attenuated and Killed Vaccines 516

Contents

23.6.2 23.6.2.1 23.6.2.2 23.6.2.3 23.6.2.4 23.6.2.5 23.6.3 23.6.4 23.6.5 23.7

Subunit Vaccines 517 DNA 517 Viral and Bacterial Vectors 517 Delivery: Prime–Boost Regimen 517 Whole Protein-based or Epitope-based Vaccines 518 Clades 518 Measurement of CTL Responses 520 Phase 1 and 2 Trials 521 Phase 3 Trials 521 Conclusion 522 Acknowledgements 522 References 522

24

Vaccines against Bioterror Agents 529 Karen L. Elkins, Drusilla L. Burns, Michael P. Schmitt, and Jerry P. Weir Introduction and Overview 529 Vaccination against Smallpox 531 Vaccination against Viral Hemorrhagic Fevers 532 Vaccination against Anthrax 533 Vaccination against Plague 536 Vaccination against Tularemia 538 Vaccination against Botulinum Toxin 540 Vaccination against Category B and C Pathogens 542 Vaccine Development and Regulation for ‘Low-incidence’ Pathogens, including Bioterror Pathogens and Emerging Diseases 542 Perspectives 543 Acknowledgements 544 References 544

24.1 24.2 24.3 24.4 24.5 24.6 24.7 24.8 24.9 24.10

Part VI

Vaccines in the Real World: Safety, Cost Efficiency and Impact of Vaccination

25

Imperfect Vaccines and the Evolution of Pathogen Virulence Paul W. Ewald Introduction 549 Virulence-antigen Vaccines against Bacteria 551 Corynebacterium diphtheriae 551 Bordetella pertussis 553 Hemophilus influenzae 555 Virulence-antigen Vaccines against Viruses 556 Circumventing Social Barriers to Vaccination 558 A Call for Field Experiments 559 References 560

25.1 25.2 25.2.1 25.2.2 25.2.3 25.3 25.4 25.5

549

XVII

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Contents

26 26.1 26.2 26.3 26.3.1 26.3.2 26.3.3 26.3.4 26.3.5 26.3.6 26.3.7 26.4 26.4.1 26.4.2 26.4.3 26.4.4 26.4.5 26.4.6 26.4.7 26.4.8 26.4.9 26.4.10 26.4.11 26.4.12 26.5 26.5.1 26.5.2 26.5.3 26.6 26.6.1 26.6.2 26.7

26.7.1 26.7.2 26.7.2.1 26.7.2.2 26.7.2.3 26.7.3 26.7.3.1 26.7.3.2 26.7.3.3 26.7.3.4

Cost-Effectiveness of Vaccinations 567 Thomas D. Szucs Introduction 567 Differences between Vaccines and Medicines 570 Analytic Methods 570 Elements of an Economic Evaluation 570 The Input 570 Direct Medical Costs 571 Direct Nonmedical Costs 571 Indirect Costs 572 The Output: Consequences and Outcomes 573 Economic Evaluation Methodology 573 Cost-of-Illness Studies 574 Cost-minimization Analyses 574 Cost–Benefit Analyses 575 Cost-effectiveness Analyses 575 Cost–Utility Analyses 576 The Importance of the Perspective 576 The Use of Models 577 Why Discounting? 578 Dealing with Uncertainty 578 Target Populations 578 The Timing of Economic Studies 579 Collecting Economic Data during a Clinical Trial 580 Post-marketing Studies and Pharmacoeconomics 580 Areas of Controversy 581 Measuring Indirect Costs 581 Externalities 582 Methodologic Quality 582 Challenges of the Future 583 Limitations and Ethical Issues 584 Strategic Outlook for the Vaccine Industry 584 Case Study for Illustration and Education: Economic Evaluation of Vaccination of Children Against Hepatitis A and Hepatitis B in Germany 585 Objective 585 Methodology 585 Determination of Costs 585 Determination of Effectiveness 586 Determination of Cost-effectiveness 586 Results 586 Costs 586 Effectiveness 586 Cost-effectiveness 587 Which Strategy Saves the Most Money? 589

Contents

26.7.3.5 Which Strategy is the Most Effective in Terms of Disease Prevention? 589 26.7.4 Discussion 590 26.7.4.1 Limitations of the Study 591 References 592 27 27.1 27.2 27.3 27.3.1 27.3.1.1 27.3.1.2 27.3.1.3 27.3.1.4 27.3.1.5 27.3.1.6 27.3.2 27.3.3 27.3.3.1

27.3.3.2

27.3.3.3 27.4 27.4.1 27.4.2 27.4.3 27.4.4 27.5

Index

Immunological Safety of Vaccines: Facts, Hypotheses and Allegations 595 Michel Goldman and Paul-Henri Lambert Introduction 595 Recognized Adverse Effects of Vaccines: a Brief Overview 596 Autoimmunity Triggered by Infection or Immunization: an Increasing Concern 598 Mechanisms of Autoimmunity Induction 598 Molecular Mimicry 598 Enhanced Presentation of Self-antigens 599 Bystander Activation 599 Polyclonal B Cell Activation 599 Antibodies 599 Regulatory T Cells 600 Autoimmune Pathology in the Course of Infectious Diseases 600 The Risk of Vaccine-associated Autoimmunity 601 Vaccine-attributable Autoimmune Diseases 601 Guillain–Barré Syndrome and Influenza Vaccine 601 Measles–Mumps–Rubella Vaccine and Thrombocytopenia 602 Vaccine-related Allegations of Autoimmune Adverse Effects 602 Hepatitis B and Multiple Sclerosis 602 Vaccination and Diabetes 603 New-generation Vaccines and Autoimmunity: Approaches to Early Risk Assessment 604 Other Unsubstantiated Allegations 605 Measles–Mumps–Rubella Vaccine and Autism 606 Thiomersal and Neurological Disorders 606 Aluminum and Macrophagic Myofasciitis 606 Multiple Vaccinations and Allergies 607 Concluding Remarks 607 References 607 613

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Novel Vaccination Strategies “We are not only responsible for what we do – but also for what we do not do.” Voltaire

Preface

Of last year’s 56 million instances of premature death, almost 17 million were due to infectious diseases. This translates into one dead individual every two seconds. Annual instances of death caused by each of the two leading infections, AIDS and tuberculosis, exceed the mortalities caused by injuries, diabetes, Alzheimer’s disease, Parkinson's disease, multiple sclerosis, breast cancer, and rheumatic diseases together. Similarly, almost 40 % of all life-years lost by disability are due to infectious diseases; this is more than all losses due to injuries, neuropsychiatric disorders, cardiovascular diseases, and cancer that follow in these dreadful statistics. Yet, there is another side to the picture, and that is that we have an extraordinarily effective measure for prevention of infectious diseases at hand. These are vaccines, which annually save more than 8 million lives, which translates into one person saved every 5 seconds. Vaccination is not only effective but it is also the most cost-efficient measure in medicine. Unfortunately, 2 to 3 million additional lives are currently being lost due to the fact that already-existing vaccines are not being made available to everybody. Wherever broadscale vaccination programs have been implemented, their success rates are remarkable. Incidences of measles, polio, rubella, mumps, pertussis, and diphtheria have all been dramatically reduced in countries where broadscale vaccination programs exist. The vaccines against these diseases were developed mostly by trial and error, and therefore could only be successful for pathogens that cause disease in a direct way. Pathogens that use more tricky strategies and subvert, impair, or misdirect the host immune response cannot be prevented by such a strategy. The next generation of vaccines has to be designed in a rational way, on the basis of our increasing knowledge of immunology and molecular genetics at the interface between pathogen and host. Fortunately, basic sciences have advanced dramatically during the past decade, and we now have available the genomic blueprints of all major pathogens as well as of the human host and the most-favored experimental animal model, the mouse.

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Preface

It is the goal of this book, “Novel Vaccination Strategies”, to benefit from recent achievements in basic research for the rational design of novel vaccination strategies against diseases that have thus far evaded successful control. In addition, it includes a kind of retrospective review of vaccine examples that have already demonstrated their great efficacy, so that we can learn from these experiences. The immune system is the target of all vaccination strategies and, hence, a great part of this book tries to decipher the immune mechanisms underlying control of the plethora of infectious agents. It is generally accepted that the acquired immune response, which accounts for specificity and memory, is the ultimate target of vaccination. Yet, the acquired immune response often fails to directly attack pathogens, and rather does so by activating innate immune mechanisms. Hence, the innate immune response is under the stringent control of the acquired immune response. More recently, we have also learnt that the acquired immune response is not activated by pathogens directly, but rather through mediation of the innate immune system. The innate immune response senses invading pathogens or vaccines and then instructs the acquired immune system to develop the most appropriate response against the homologous pathogen. Unfortunately, many pathogens have developed tricks to deviate host responses from the default direction. Here lies a major Achilles’ heel of the immune system and also a chance for rational vaccine design. For many infections that cannot be controlled by vaccines thus far, it will be important to induce an immune response that is better than the one stimulated by natural infection. Much can be expected from novel adjuvants capable of stimulating the most adequate immune response for a given pathogen. Adjuvants will be required particularly for subunit vaccines, whether they be based on naked DNA, on protein, or on carbohydrate antigens. The right formulation comprising both the protective antigen and the appropriate adjuvant will define the success of a future subunit vaccine. Yet, it is likely that for certain diseases, subunit vaccines comprising only a few specific antigens will be insufficient. In these cases viable vaccines will be required, and the choice of the most appropriate recombinant vaccine carrier will be equally difficult. The list of nature's scourges is headed by the ‘big three‘, that is, AIDS, tuberculosis, and malaria, which represent major challenges for rational vaccine design. Therefore, specific chapters are devoted to vaccine development against these diseases, as well as to vaccination strategies against Helicobacter pylori, which is responsible not only for gastric ulcers but also for certain forms of stomach cancer. Moreover, a chapter on vaccines against bioterror agents has been included, since we have become aware of the dreadful possibilities infectious agents offer to those who want to pervert our increasing knowledge about infectious diseases. We have to be aware that we live in a world that has been populated by microorganisms for more than 3 billion years, while the beginning of mankind dates back only 5 million years. Hence, it would be unreasonable to think that we can conquer all microbes successfully. Rather, we have to accept that the survival strategies of most microbes, which are based on a combination of rapid replication and rapid change, are highly successful and that our current knowledge may not be sufficient for designing vaccines against the most devious pathogens. Moreover, the undesired

Preface

possibility needs to be considered that novel, more hazardous strains may evolve under the pressure of imperfect vaccines. By design, this book focuses on the scientific basis of rational vaccine design. This is not, however, meant to underestimate the importance of subsequent development, safety assessment, and clinical trials. Two chapters have been included that deal with two important aspects downstream of vaccine research: safety assessment and costefficiency aspects of vaccination. It has become clear that the complete process of vaccine development is most successful as a close interaction between basic research, mostly done at public academic institutions, and development, best done by private industry. Unfortunately, vaccine development is not always high on the list of interests of industry. After all, the major goal of a successful vaccine is eradication of the targeted disease. Consequently, the most successful vaccine will concomitantly eradicate its own market. Moreover, many vaccines are needed for diseases that are most prevalent in countries that have the least financial resources. As a consequence, the return on investment for some vaccines may be too low to attract industry partners. On the other hand, public health systems would receive a profound return on investment. This has been proven impressively by the vaccines currently available. For every dollar that is spent on vaccination against measles, mumps, rubella, diphtheria, pertussis, or tetanus the public hand saves $10 to $20. Vaccination against childhood tuberculosis, tetanus, polio, measles, and hepatitis B prolongs healthy life by one year for a cost of $10 to $40. It is obvious that, not only the protected individual, but also the general public benefits enormously from vaccination. Currently available vaccines have proven their great cost-efficiency and success in an impressive way. Their availability has been made possible by efforts dating back several decades. Recent achievements in basic research have now laid a new foundation for rational design of novel vaccines, and the general public will benefit from such vaccines in future decades, provided appropriate efforts are undertaken now. Stefan H. E. Kaufmann

Berlin, August 2003

Acknowledgements

I thank the contributors of this book for sharing their insights into vaccine research, Yvonne Bennett for her excellent secretarial help, and Andreas Sendtko and PriscaMaryla Henheik, the publishing editors of Wiley-VCH, for their continuous efforts.

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List of Contributors Martin F. Bachmann Cytos Biotechnology AG Wagistr. 25 8952 Zürich-Schlieren, Switzerland James M. Brewer Division of Immunology, Infection and Inflammation University of Glasgow, Western Infirmary Glasgow, G11 6NT, Scotland Silvia Bulfone-Paus Research Center Borstel, Center for Medicine and Biosciences Dept. of Immunology and Cell Biology Parkallee 22 23845 Borstel Germany Drusilla L. Burns Division of Bacterial, Parasitic, and Allergenic Products Center for Biologics Evaluation and Research 1401 Rockville Pike, HFM 431 Rockville, MD 20852, USA

Dirk Hans Busch Institute for Medical Microbiology, Immunology, and Hygiene Technical University Munich Trogerstr. 9 81675 Munich, Germany Antonio Cassone Department of Infectious, Parasitic and Immune-Mediated Diseases Istituto Superiore di Sanit™ Viale Regina Elena, 299 00161 Rome, Italy Rachel Chikwamba Plant Biology Department Arizona State University Tempe, AZ 85287, USA Simon Clare Centre for Molecular Microbiology and Infection Department of Biological Sciences Imperial College London Exhibition Road London SW7 2AZ, UK

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Alan Coulter Pharmaceutical R&D CSL Limited 45 Poplar Road Parkville,VIC., 3052 Australia

Debbie Drane Pharmaceutical R&D CSL Limited 45 Poplar Road Parkville,VIC., 3052 Australia

Joan Cunnick Department of Microbiology Iowa State University Ames, IA 50011 USA

Thomas Ebensen Vaccine Research Group GBF-German Research Centre for Biotechnology Division of Microbiology Mascheroder Weg 1 38124 Braunschweig Germany

Alexander Dalpke Institute of Medical Microbiology and Hygiene Philipps University Marburg Pilgrimstein 2 35037 Marburg Germany Giuseppe Del Giudice IRIS Research Center, Chiron Srl Via Fiorentina 1 53100 Siena Italy Guenter Dollenmaier Institute of Medical Virology University of Zurich 8028 Zurich Switzerland Gordon Dougan Centre for Molecular Microbiology and Infection Department of Biological Sciences Imperial College London Exhibition Road London SW7 2AZ UK

Stefan Ehlers Research Center Borstel, Center for Medicine and Biosciences Division of Molecular Infection Biology Parkallee 22 23845 Borstel Germany Karen L. Elkins Division of Bacterial, Parasitic, and Allergenic Products Center for Biologics Evaluation and Research 1401 Rockville Pike, HFM 431 Rockville, MD 20852 USA Paul W. Ewald Department of Biology University of Louisville Louisville, KY 40292 USA Daniel Franke Institute of Medical Virology University of Zurich 8028 Zurich Switzerland

List of Contributors

Michel Goldman Department of Pathology and Pediatrics Geneva University 1 Rue Michel Servet Genè ve 27 Switzerland Carlos A. Guzmán Vaccine Research Group GBF-German Research Centre for Biotechnology Division of Microbiology Mascheroder Weg 1 38124 Braunschweig Germany Tomas Hanke Medical Research Council Human Immunology Unit Weatherall Institute of Molecular Medicine University of Oxford, The John Radcliffe Hospital Headley Way Oxford OX3 9DS UK Klaus Heeg Institute of Medical Microbiology and Hygiene Philipps University Marburg Pilgrimstein 2 35037 Marburg Germany Norbert Hilf Institute for Cell Biology Department of Immunology University of Tübingen Auf der Morgenstelle 15 72076 Tübingen Germany

Katharina M. Huster Institute for Medical Microbiology, Immunology, and Hygiene Technical University Munich Trogerstr. 9 81675 Munich Germany Gary T. Jennings Cytos Biotechnology AG Wagistr. 25 8952 Zürich-Schlieren Switzerland Wim Jiskoot Utrecht Institute for Pharmaceutical Sciences Department of Pharmaceutics P.O. Box 80.082 3508 TB Utrecht The Netherlands Stefan H. E. Kaufmann Max Planck Institute for Infection Biology Department of Immunology Schumannstraûe 21–22 10117 Berlin Germany R. Kay Medical Research Council Human Immunology Unit Weatherall Institute of Molecular Medicine University of Oxford The John Radcliffe Hospital Headley Way Oxford OX3 9DS UK

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Kristen M. Kerksiek Institute for Immunology Ludwig Maximilian University Trogerstr. 9 81675 Munich, Germany Gideon Kersten Product and Process Development Netherlands Vaccine Institute Antonie van Leeuwenhoeklaan 11 3720 BA Bilthoven The Netherlands Paul-Henri Lambert Department of Pathology and Pediatrics Geneva University 1 Rue Michel Servet Genève 27 Switzerland Maria Lattanzi IRIS Research Center Chiron Srl Via Fiorentina 1 53100 Siena Italy Claudia Link Vaccine Research Group GBF-German Research Centre for Biotechnology Division of Microbiology Mascheroder Weg 1 38124 Braunschweig Germany

Andrew J. McMichael Medical Research Council Human Immunology Unit Weatherall Institute of Molecular Medicine University of Oxford, The John Radcliffe Hospital Headley Way Oxford OX3 9DS UK Ian C. Metcalfe Berna Biotech Ltd Rehhagstrasse 79 3018, Bern Switzerland Karin Mölling Institute of Medical Virology University of Zurich 8028 Zurich Switzerland Christian Moser Berna Biotech Ltd Rehhagstrasse 79 3018, Bern Switzerland Joseph Patrick Nkolola Medical Research Council Human Immunology Unit Weatherall Institute of Molecular Medicine University of Oxford, The John Radcliffe Hospital Headley Way Oxford OX3 9DS UK

List of Contributors

Gustav J. V. Nossal Department of Pathology The University of Melbourne Victoria 3010 Australia Ruth S. Nussenzweig Department of Medical and Molecular Parasitology New York University School of Medicine New York, NY 10016 USA Victor Nussenzweig Department of Pathology New York University School of Medicine New York, NY 10016 USA Derek T. O'Hagan Chiron Vaccines Chiron Corp. 4560 Horton St. Emeryville, CA 94608 USA Wieslawa Olszewska Department of Respiratory Medicine National Heart and Lung Institute Imperial College London UK Peter J. M. Openshaw Department of Respiratory Medicine National Heart and Lung Institute Imperial College London UK

Eric G. Pamer Infectious Diseases Service Department of Medicine and Laboratory of Antimicrobial Immunity Immunology Program, Sloan-Kettering Institute Memorial Sloan-Kettering Cancer Center 1275 York Avenue New York, NY 10021 USA Jovan Pavlovic Institute of Medical Virology University of Zurich 8028 Zurich Switzerland Martin Pearse Pharmaceutical R & D CSL Limited 45 Poplar Road Parkville,VIC., 3052 Australia Alexander Ploss Infectious Diseases Service Department of Medicine and Laboratory of Antimicrobial Immunity Immunology Program, Sloan-Kettering Institute Memorial Sloan-Kettering Cancer Center 1275 York Avenue New York, NY 10021 USA and Weill Graduate School of Medical Sciences Cornell University New York, NY 10021, USA

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Kevin G. J. Pollock Scottish Centre for Infection and Environmental Health Clifton House Glasgow, G3 7LN Scotland

Allan Saul National Institutes of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD 20892 USA

Luciano Polonelli Microbiology Section Department of Pathology and Laboratory Medicine University of Parma Parma Italy

Hansjörg Schild Institute for Cell Biology Department of Immunology University of Tübingen Auf der Morgenstelle 15 72076 Tübingen Germany

Markus Radsak Institute for Cell Biology Department of Immunology University of Tübingen Auf der Morgenstelle 15 72076 Tübingen Germany

Michael P. Schmitt Division of Bacterial, Parasitic, and Allergenic Products Center for Biologics Evaluation and Research 1401 Rockville Pike, HFM 431 Rockville, MD 20852 USA

Rino Rappuoli IRIS Research Center Chiron Srl Via Fiorentina 1 53100 Siena, Italy Faiza Rharbaoui Vaccine Research Group GBF-German Research Centre for Biotechnology Division of Microbiology Mascheroder Weg 1 38124 Braunschweig Germany Paolo Ruggiero IRIS Research Center Chiron Srl Via Fiorentina 1 53100 Siena Italy

Jan Schultz Institute of Medical Virology University of Zurich 8028 Zurich Switzerland Manmohan Singh Chiron Vaccines Chiron Corp. 4560 Horton St. Emeryville, CA 94608 USA Thomas D. Szucs Hirslanden Research Seefeldstrasse 214 8008 Zurich Switzerland

List of Contributors

Tillmann S. Utesch Institute of Medical Virology University of Zurich 8028 Zurich Switzerland Jean-François Viret Berna Biotech Ltd Rehhagstrasse 79 3018, Bern Switzerland Max von Kleist Institute of Medical Virology University of Zurich 8028 Zurich Switzerland Kan Wang Department of Agronomy Iowa State University Ames, IA 50011 USA

E. G.-T. Wee Medical Research Council Human Immunology Unit Weatherall Institute of Molecular Medicine University of Oxford, The John Radcliffe Hospital Headley Way Oxford OX3 9DS UK Jerry P. Weir Division of Viral Products Center for Biologics Evaluation and Research 1401 Rockville Pike, HFM 431 Rockville, MD 20852 USA Stefan Zimmermann Institute of Medical Microbiology and Hygiene Philipps University Marburg Pilgrimstein 2 35037 Marburg Germany

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Colour Plates

Fig. 4.1 Models of programmed memory T cell generation. T lymphocytes may commit to becoming long-term memory T cells at different times during the course of activation. We propose three distinct models for programmed memory T cell development. Model A suggests that differentiation of memory T cells is a gradual process, with naï ve T cells first differentiating into effector T cells and then, stochastically, further differentiating into memory T cells. Model B suggests that brief exposure to antigen results in two distinct effector T cell populations, one committed to long-term memory development, and the other to apoptotic death. Model C, on the other hand, proposes that naï ve T cells differ in their potential to differentiate into memory T cells.

Colour Plates

Fig. 7.1 The current model for the physiological role of HSPs in cross-priming. During the course of, for example, a viral infection, pathogen-derived proteins are produced by the infected host cell. Due to the usual protein turnover, viral peptide fragments are generated by the proteasome and may be presented on MHC class I molecules. HSPs are associated with such peptides in vivo. If the virus causes necrotic death of the infected cells, the HSP–peptide complexes are released and can be taken up by APCs in a receptor-mediated fashion via CD91 and LOX-1. The APC can represent the HSP-ligated viral peptides on its MHC class I molecules, thereby enabling its recognition by naive CD8+ T cells. Moreover , released HSP activates APCs via TLR4 and TLR2, resulting in the up-regulation of costimulatory molecules and in the secretion of various proinflammatory cytokines and chemokines. This second signal enables the priming of the naive CD8+ T cells, triggering their development into effector cytotoxic T cells (CTLs). These cells are now able to kill virus-infected cells without further costimulatory signals.

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Colour Plates

Fig. 13.2 Generation of rAAV vaccines. A Life cycle of wild-type AAV. Latency is established after AAV infection of the target cells, which may involve genome integration. AAV virions are rescued after subsequent infection with helper virus, which provides the sequences needed for viral replication in trans. B Production of rAAV vaccines. Permissive cells are cotransfected with a plasmid containing the transcription unit (blue) flanked by the AAV ITRs (green) and plasmid containing the AAV genome without the ITRs. Subsequent infection of these cells with a helper adenovirus enables rescue of rAAV virions, following the packaging of the ITR-containing plasmid, along with helper virus. Helper and rAAV virions can then be separated after heating at 56 °C by centrifugation on a cesium chloride gradient. Adapted from [3].

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Fig. 13.3 Generation of recombinant herpes simplex viruses (HSV) and HSV-derived amplicons. A Recombinant HSV vectors. HSV genomic DNA is cotransfected into permissive cells along with a plasmid containing the transcription unit (red) inserted in fragment of HSV genome (gray). During mitosis, homologous recombination occurs between the HSV genome sequences within the plasmid and HSV DNA. Both wild-type and recombined viral genomes are packaged into a mixed virus particle population, from which the recombinant viruses are isolated by plaque purification. B Amplicon-derived HSV vectors. The amplicon plasmid con-

taining the transcription unit (red) and the viral cleavage and packaging site is cotransfected into permissive cells together with a helper virus genome, which contains all the regulatory and structural genes needed for viral growth, including the HSV host shutoff protein, but is defective in packaging. Immediately after transfection, the amplicon plasmid undergoes DNA replication driven by helper proteins, which results in the generation of head-to-tail plasmid concatemers. These concatemers are then cleaved into genome unit-length molecules and packaged into virus particles, which after concentration are ready for use. Adapted from [35].

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Fig. 15.1 Patterns of intestinal colonization displayed by enteric pathogens. Distinct enteric pathogens exhibit different modes of pathogenicity after interaction with the gut. This diagram illustrates the typical types of enteric infection and the level of the gut tissue associated with infection in a healthy human. EPEC enteropathogenic E. coli; ETEC enterotoxigenic E. coli.

Fig. 15.2 Interaction of Salmonella enterica with immune cells shown by confocal microscopy. A. anti- Salmonella B. anti-LAMP-1 C. Merged image. Provided by Dr Liljana Petrovska, Imperial College London.

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Fig. 16.1 Schematic representation of cells involved in immune reactions after mucosal challenge. APC, antigen-presenting cell; M, microfold epithelial cell; T, T cell; B, B cell; RBC, red blood cell.

Fig. 16.2 Goals of mucosal immunization. Successful mucosal immunization may induce humoral and cellular immune responses. Possible roles of both are shown. M, microfold epithelial cell; HEV, high endothelial venule; APC, antigen-presenting cell; the 1 symbol indicates possible sites of immune intervention.

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Fig. 16.3 Challenges for mucosal vaccination: schematic representation of key issues in mucosal vaccine development.

Fig. 21.1 Schematic depiction of mechanisms underlying immunity to tuberculosis. Upper left and middle: major steps from infection to induction of immune response ; middle and lower left: granuloma formation and breakdown; right: major T cell populations involved in protective immunity; upper right: effector mechanisms in activated macrophages.

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Fig. 21.2 Cross-priming as a major pathway of antigen presentation in tuberculosis.

Fig. 23.2 On the surface of the HIV virion, env forms a trimeric complex of gp120–gp41 dimers. The conserved surfaces facilitate the formation of a trimer. The binding of gp120 to CD4 on the T cell induces a conformational change, which exposes the coreceptor binding site. Schematic diagram reproduced with kind permission of Dr. Sattentau [8].

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Colour Plates Fig. 23.3 Schematic diagram of the likely arrangement of the HIV-1 gp120 glycoproteins in a trimeric complex. The view is from the target cell membrane. The CD4 binding pockets are indicated by black arrows, the approximate locations of the 2G12 epitopes are indicated by blue spots, and the conserved chemokine-receptor-binding regions are in red. Areas in green indicate the more variable glycosylated surfaces of gp120.

Fig. 23.4 Proposed dynamics of the T cell response to virus-specific T cells. Rare antigen-specific naïve CD8+ T cells are stimulated to divide rapidly. The frequency of effector cells (light blue) is much higher than that of those that can divide in vitro (dark blue). Persisting HIV antigen maintains the expanded T cells at a high level; without antigen these cells die by apoptosis. The population of T cells that is capable of dividing further (dark blue) maintains long-term memory and is likely to continually generate the expanded effectors.

Part I

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1 Challenges for the Vaccine Developer, including Correlates of Protection G. J. V. Nossal

1.1 Introduction

For four reasons, a book on novel vaccination strategies is particularly timely. First, the stunning advances in basic biology, and particularly in genomics, have opened up new vistas in vaccinology and have introduced a much more solid underpinning of science into a field characterized by a fair degree of empiricism. Second, the devastating impact of the HIV/AIDS pandemic has alerted the world to the imperative need for a vaccine to contain it and, as a side effect, has permitted greater publicity for other major communicable disease killers, such as tuberculosis and malaria. Third, humanitarian issues, such as immunizing all the world's children and not only the children of the rich, have taken on a new urgency after September 11, 2001. As Varmus has argued, ‘to a very great, if not measurable extent, terrorism is a manifestation of anger and resentment about the world’s inequalities’ [1]. Fourth, the quite extraordinary generosity of the Bill and Melinda Gates Foundation has permitted highly significant new sums of money to flow into vaccine research and, through the Global Alliance for Vaccines and Immunization, has raised the prospect that new and improved vaccines might promptly reach those in greatest need of them [2]. That being said, the would-be inventor of new or improved vaccines still faces formidable challenges. The expensive applied research, development work, and clinical trials that are required following the academic research phase are usually the province of industry, which has an understandable requirement to be profitable. Where the vaccine is one chiefly of interest to developing countries, novel strategies of funding and/or execution may be necessary. For example, the Gates Foundation-funded African meningitis program seeking to combat epidemics caused by Neisseria meningitidis has engaged a vaccine manufacturer in India, with its much lower labor costs, to develop a carbohydrate–protein conjugate vaccine, with the help of two manufacturers in industrialized countries with some of the raw materials and of technology transfer. Another very real challenge is the fact that most existing licensed vaccines have been developed using antibody levels generated as the key guide. Now vaccines are required where T cell immunity is essential. The correlation between measured T

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1 Challenges for the Vaccine Developer, including Correlates of Protection

cell reactivities and durable protection is frequently much more dubious. Moreover, unfortunately, animal models, particularly rodent ones, have been poor predictors of clinical efficacy. It is therefore important to explore all the mechanisms of protection that the immune system provides and to gradually build up a combinatorial image of what elements contribute to a vaccine that really works. Of course, all the correlative arguments in the world do not bypass the need for phased clinical trials. Part of the challenge will be to learn the correlates of protection from such trial results.

1.2 Mechanisms of Protection within the Immune System

The mechanisms of protection are conventionally divided into innate or primitive and adaptive or acquired, but as later chapters show, these systems are far from unrelated. Indeed, it is valuable to think of bodily defense as a complex, highly integrated series of interacting cellular and molecular processes, some depending on evolutionarily primitive recognition processes and others being much more specific and highly evolved. Moreover, because the most recent system, that which depends on the somatic generation of B and T cell repertoires, evolved in the presence of earlier systems, it is natural that functionally new processes should be ‘grafted onto’ what went before. Thus it comes as no surprise that antibodies serve their function with a high dependence on the complement system or on phagocytosis. So long as this is recognized, it is legitimate to set out the key elements of the innate and the adaptive systems. The innate immune system is evolutionarily as old as multicellular life itself, namely about two thousand million years, but its central molecular features have only recently been elucidated [3]. Table 1.1 summarizes its key elements. Probably the most important newer finding is that of the Toll-like receptors for pathogen products of various sorts. These evolutionarily old receptors form a link between the innate immune system and the adaptive system, as is fully discussed in another chapter 2. Table 1.2 deals with the more familiar mammalian adaptive immune system, which consists of a multiplicity of components. So the question is ‘Which element is the most important in protecting the host against infection?’ because that is what the vaccine developer should monitor as research on a new vaccine progresses. The matter is not simple, because various elements collaborate in vanquishing infections. Physicians have known for nearly a century that the crisis in lobar pneumonia that precedes recovery signifies a sufficiently high concentration of opsonic antibody to allow lung macrophages to engulf the invading bacteria. Without either the antibody or the macrophage, the patient would die. Yet it is clearly impractical for the researcher to seek to correlate every element of Table 1.2 with the degree of protection afforded by a vaccine. A further complexity is the fact that what leads to recovery from infection may not be identical to what is required to prevent infection or reinfection. In influenza, for example, cytotoxic T cells are probably the most important element limiting viral spread and thus speeding recovery, but antibodies in respiratory tract mucosal fluid are probably key to preventing an attack. Given these influences, it is not surprising that correlates of protection is a field that has not progressed very far.

1.3 Protection against Viruses Tab. 1.1 Some key elements of the innate defense system. . Cilia . Enzymes in mucous secretions, e. g., lysozyme . Repair mechanisms of damaged anatomical barriers, e. g. the clotting cascade or growth factor and chemokine release . Defensins . The complement cascade . Non-immunoglobulin opsonins, e. g., collectins such as mannose-binding protein or C-reactive protein, lectins, fibronectin, etc. . Recognition receptors on dendritic cells, macrophages, NK cells, and mast cells including Tolllike receptors, scavenger receptors, or integrin . Phagocytosis by polymorphonuclear leukocytes, monocytes, or macrophages . Cytokines, including interferons a, b, g and tumor necrosis factor

Tab. 1.2 Some key elements of the adaptive immune system. . B lymphocytes, their precursors and progeny, and their products – 8 different classes of antibody . T lymphocytes with a/b receptors, their precursors and progeny, their lymphokine products, and specifically CD4+ regulatory (suppressor) T cells; CD8+ cytotoxic and cytokine secretory cells; and CD1-specific CD4+ or double negative NK-1 T cells . T lymphocytes with g/d receptors, their various subsets, and their lymphokine products . Other atypical T cells . Fc receptors of various types on monocytes, macrophages, immature dendritic cells, B cells, polymorphonuclear leukocytes, NK cells, mast cells, and platelets, as well as soluble Fc receptors

The most convenient way of discussing present understanding is to deal separately with viral, bacterial, and parasitic pathogens.

1.3 Protection against Viruses

Virus infections can be relatively localized, e. g., to the respiratory or alimentary tracts (a more challenging situation for the induction of protective immunity) or they may spread systemically first via the lymph and then through the blood, where opportunities for engaging the major sites of immune induction are plentiful. Virus infections may be acute and followed either by death or by complete viral elimination; they may set up a latent infection, going underground in particular cells, only to reemerge sporadically and unpredictably, as with herpes viruses; or they can set up a chronic infection as in hepatitis B or C. Knowing the respective life histories of the viruses is important to devising appropriate vaccine research strategies. The innate immune system offers a substantial defense against viruses. In particular, the type 1 interferons, a and b, damp viral replication and thus limit their spread. NK cells become activated and, through cytotoxic activity, kill virus-infected cells. The

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1 Challenges for the Vaccine Developer, including Correlates of Protection

innate immune system also provides a variety of other potent cytokines. Macrophages have antiviral functions but can also harbor viruses and aid their spread. Adaptive immune responses come in somewhat later and usually involve both antibodies and T cells. The general rule is that antibody, with its capacity to recognize structures on the surface of a virus, plays the main role in neutralizing free virus, whereas T cells, recognizing viral peptides processed intracellularly and presented on cell surfaces, have virus-infected cells as their target. In both pathways, it is important to delineate which viral antigens are the most important. As far as antibody formation is concerned, the key molecules are those that the virus uses to gain entry into the host cell. Covering these up with antibody prevents the initiation of infection, and such antibodies are known as neutralizing antibodies. Surface glycoproteins or outer capsid proteins are the most critical viral antigens that induce neutralizing antibody. Viruses having a lipid envelope need not only to attach but also to fuse with the host cell membrane. Often, a separate fusion protein exists, which is also a neutralization target. Newly formed viruses need to be released from the infected cell. With influenza virus, for example, release is achieved via a specific molecule, neuraminidase. Although not as important as antibodies to hemagglutinin, which binds the influenza virus to the cell, antineuraminidase antibodies also participate in protection. Ancillary to these highly specific antibodies are many other antibodies that recognize the virus surface and act in an opsonic manner. Finally, antibodies to any surface structure on a virus-infected cell can lead to that cell being killed by antibody-dependent cellular cytotoxicity or by complement-dependent lysis, aiding control of infection. At the same time, many antibodies are formed to internal virus components that are irrelevant to protection. In some instances, the process of viral entry is more complex, requiring two receptors. The classical case is HIV, where the gp 120 envelope protein binds to CD4 and, subsequent to this binding, an allosteric change allows the binding of another part of gp 120 to a coreceptor, namely CCR5 or CXCR4, which are members of the chemokine receptor family. It is believed that then a further conformational change leads to insertion of a hydrophobic amino-terminal fusion peptide of gp 41 into the target cell membrane. Cross-linking via gp 41 finally results in membrane fusion and viral entry. The newly and briefly exposed antigenic determinants of gp 120, which bind to the coreceptor, appear to be more conserved than the CD4-binding epitopes and may constitute attractive vaccine candidates [4]. The T cell side of antiviral defense is entirely different. Here, any viral protein, be it surface-located, internal, or indeed nonstructural, may be presented on the surface of an infected cell or of an antigen-presenting cell that has engulfed an infected cell or portions of it. Although classically, peptides from a cell in which virus is growing are presented by MHC class I molecules, and peptides derived by endo- or phagocytosis are presented by class II molecules, there are now numerous examples of cross-presentation. Conventionally, we think of CD8+ cytotoxic T cells recognizing peptides presented by class I as the chief protectors for vanquishing a virus infection. However, the CD4+ T cells recognizing peptides presented by class II are also hugely important. They act as helpers for the production of high-affinity antibody. They also provide help for the activation of CD8+ T cells, produce cytokines with antiviral activity, and recruit phagocytic cells.

1.3 Protection against Viruses

In some ways, therefore, the vaccine researcher seeking to develop a vaccine for a disease in which T cell immunity is paramount for protection suffers from an embarrassment of riches. In looking to see if a putative vaccine has engendered a T cell response, it is truly difficult to choose among the different viral proteins, so often ‘cocktails’ are used, rendering the test less precise than it might have been. Furthermore, it is difficult to know whether to go with whole viral proteins or with known T cell epitopes from a given protein. If the latter, the investigator must face the polymorphism of the human population for both class I and class II HLA molecules and either run the test with a variety of T cell peptides or choose to learn from only a given MHC haplotype. In infections that relapse (like herpes) or that persist (like HIV), the vaccine developer has to do better than nature, which is of course quite a challenge. Success may depend on a T cell response more precise and/or more intense than that accompanying the infection. For cytopathic viruses that kill or heal, leaving protective immunity, the correlate of protection is straightforward, being neutralizing antibody. Often, a combination of animal experimentation and clinical seroepidemiology has given good information on the actual levels or titers of antibody required for protection. These then guide choice of antigen dose in the vaccine and the number of injections required. To a degree, they also dictate how often booster doses are required, as in smallpox or yellow fever, although in practice protection may well last longer than threshold serum antibody levels, presumably because of very rapid memory responses should the virus gain reentry. Memory responses obviously take a few days to become manifest, since lymphocyte multiplication and differentiation are required. They are therefore most effective in systemic infections where the pathogen takes a few days to move from the point of entry via the skin or mucus membrane into the lymph, tissues, and blood. They are less effective in purely localized mucosal infections. Things are a little more complex in a disease like influenza, which starts in respiratory epithelium but becomes more serious as it spreads more deeply. Further, in this disease the key antigens and particularly the hemagglutinin exhibit very high mutation rates. Here minor changes due to an accumulation of point mutations, and known as antigenic drift, are dealt with by a clever, arduous global process, where vaccine manufacturers, guided by World Health Organization collaborating centers, include in the vaccine the most recent circulating variants, with susceptible people being counseled to have yearly boosters. On the other hand, when genetic reassortment occurs between different viral strains, frequently a wild human virus and an influenza virus of an animal such as a pig or a chicken, preexisting immunity is frequently lacking and a pandemic can sweep the globe. These are known as shift variants. It appears that major pandemics have occurred about three times a century over the past three centuries or so. Should we be looking to correlate protection with IgA present in respiratory mucus or intestinal fluids for respiratory or intestinal infections, respectively? From a practical point of view this has not proven to be necessary, because measuring serum (mainly IgG) antibody seems to do the job. Important as IgA is, we must remember that significant quantities of IgG enter these fluids as a transudate.

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1 Challenges for the Vaccine Developer, including Correlates of Protection

Should we be measuring virus-specific T cell levels? For the ‘kill or cure’ diseases, the undoubtedly helpful T cell responses engendered by vaccines are really a bonus, and it has not proven necessary to document their numbers or persistence. As we shall see below, the situation is quite different for diseases such as HIV/AIDS or hepatitis B or C. There is a major difference between viruses that are mainly confined to body surfaces and those that become systemically disseminated. The former induce mainly mucosal immunity, which is much shorter-lived than systemic immunity. The same is true for mucosal vaccines, which tend to cause a shorter duration of protection. Still, in ‘real life’, significant serum antibody is also present and acts as a guide for the vaccine developer.

1.4 HIV/AIDS as an Example of a Persisting Virus

Many of the dilemmas facing the researcher seeking correlates of protection in a chronic virus infection are illustrated by the HIV/AIDS situation. Great early disappointment followed the realization that monomeric gp 120 protein given with adjuvant to human volunteers elicited antibodies that could neutralize laboratory-passaged HIV strains but not fresh primary isolates from patients [5]. Given the prominence of antibodies to the V3 loop and the extreme mutability of the envelope protein in this region, this really should not have been too surprising. But it is far too early to suggest that antibodies will never act protectively. Appropriate antibodies may, in times represent a useful correlate marker. We now possess a much better picture of the events that occur when HIV docks with the CD4+ cell and finally enters it [6]. When gp 120 engages CD4, a stable conformational change occurs which exposes a binding site for a coreceptor, usually CCR5 or CXCR4. This site is highly conserved, as also is the actual docking site for CD4 within the gp 120 molecule. Both sites are in recessed pockets on the inner core of the gp 120 molecule. After both CD4 and the coreceptor have engaged, the noncovalently linked gp 41 molecule changes conformation to reveal a hydrophobic fusion domain allowing viral entry into the host cell. Moreover, the gp 120–gp 41 dimers exist on the virus surface in triplets constituting a viral spike. Additional epitopes may be generated as a result of this trimerization, and what one wants is high-avidity binding to the trimer. It is not easy to elicit antibodies to these conserved elements, but some human subjects with HIV do manage to generate antibodies capable of neutralizing a broad diversity of HIV isolates. Perhaps the most informative approach has been the study of human monoclonal antibodies with this capacity [7]. The structure of one of these, b12, has been resolved to 2.7 Å and possesses a finger-like projection, which is the third hypervariable region of the heavy chain, which pokes into the recessed CD4 binding site of gp 120. Another (2G12), surprisingly, is directed against oligomannose epitopes clustered on one face of gp 120, raising the possibility of a carbohydrate vaccine. Such examples, and the clear proof that passive antibodies can protect against chimeric simian–human immunodeficiency virus (SHIV) challenge, offer the hope

1.5 Protection against Extracellular Bacteria

that eventually immunogens will be generated that are capable of eliciting antibodies with the right characteristics. This being said, overwhelmingly the most effort is being directed at vaccine approaches capable of inducing CD8+ cytotoxic T cell immunity. These are described in detail in another chapter 5. What is the evidence that HIV-specific cytotoxic T cells will be protective? The accumulation of evidence is impressive [4]. It began with the demonstration that human CD8+ T cells can suppress virus replication in autologous CD4+ T cells in vivo [8], which was soon followed by the observation that the appearance of large numbers of specific cytotoxic cells during primary infection with HIV correlated in time with a substantial fall in virus load. In the SHIV model, vaccines that elicit high CTL levels work, at least to some extent. In the opposite direction, when CD8 T cells are depleted, monkeys develop very high levels of viraemia. Further, clinical nonprogressors with long-maintained low viral loads have high CTL levels [9], as do those heavily HIV-exposed sex workers who remain uninfected [10]. In the absence of absolute proof, most investigators would rank specific cytotoxic T cells as the best surrogate marker for efficacy. At the same time, CD4+ cells should not be forgotten [11]. Although the exact mechanism is unknown, CD4+ cells are helpers for CD8+ T cell development. Of course, they are required for high-affinity antibody responses as well. Patients with low viraemia tend to have high CD4+ T cell proliferative responses to HIV antigens. Thus, it is probable that a good vaccine would induce both specific CD4+ and CD8+ responses (Table 1.3).

Tab. 1.3 Possiblecorrelates of protection in HIV/AIDS. Surrogate marker

Comments

Serum antibody

Best against actual conserved docking epitopes of virus; hard to generate. Some value against other epitopes, but mutability of virus is a problem.

Antibody in vaginal or rectal mucus

Hard to measure routinely.

CD8+ cytotoxic T cells

Which antigens and which epitopes to use for precision of measurement? Will tetramer staining prove helpful eventually?

CD4+ helper T cells

Which antigens? Which subtype?

Combination of all the above

Best hope in the long term.

1.5 Protection against Extracellular Bacteria

Strategies of host defense are different for bacteria that live extracellularly, for which antibodies are the chief agents of control, versus bacteria adapted to intracellular per-

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1 Challenges for the Vaccine Developer, including Correlates of Protection

sistence, where T cells are key. With the former type, bacteria that remain localized, e. g., Staphylococcus aureus causing impetigo or boils, are less susceptible to conquest by antibody than bacteria that invade the body, e. g., Streptococcus pneumoniae causing pneumonia, meningitis, or septicaemia. The exotoxins, which some bacteria manufacture to aid tissue destruction, allowing them to get a foothold in the host, are very important targets of immune attack. Suitably treated either chemically (to create a toxoid) or genetically (to create a nontoxic but antigenically similar analogue), such molecules become very effective vaccines. Typical examples include Clostridium tetani and Corynebacterium diphtheriae, in which the relevant toxoids are among the most effective vaccines in common use. Other bacteria, such as Clostridium perfringens or Bacillus anthracis make more than one exotoxin. For some species, such as Bordetella pertussis, it is preferable to give a combination acellular vaccine, containing not only toxoided pertussis toxin but also other molecules that contribute to virulence, such as filamentous hemagglutinin, pertactin, or fimbrial proteins. In all these examples, antibody levels give the best correlate of protection. This is not to say that T cells do not play any part in defense, but simply that, in a practical sense, measuring the appropriate antibody is the best guide for the vaccine developer. Another important group of antigens that evoke protective antibodies are the capsular polysaccharides of the bacterial cell wall. Unfortunately for the vaccine developer, genetic polymorphism in these carbohydrate epitopes, even within a single bacterial species, is common. An extreme example is Streptococcus pneumoniae, in which over 90 serologically distinct capsular polysaccharides exist, and over 20 of these serotypes are important in human disease. Furthermore, when these carbohydrate antigens are used as vaccines, they prove to be suboptimal in several ways. Infants under 1.5 years old respond poorly or not at all. Beyond this age, both children and adults mount an antibody response without T cell help, which is generally of low affinity and results in poor B cell memory. Nevertheless, a 23-valent carbohydrate-based vaccine has proven helpful in elderly individuals. A major breakthrough occurred when researchers realized that this type of vaccine could be much improved by conjugating the carbohydrate moieties to a protein carrier such as diphtheria toxoid or mutated toxin. The protein carrier provides T cell help, allowing earlier responses, affinity maturation, and good memory. The first practical fruits of this approach were vaccines against Haemophilus influenzae serogroup B, or Hib [12, 13]. There are six capsular serotypes of H. influenzae, a to f, capable of causing human disease; fortunately, serotype b is overwhelmingly the most important. When the Hib conjugate vaccine was widely deployed in industrialized countries, it was surprisingly effective and Hib meningitis was virtually eliminated – partly because nasopharyngeal carriage rates were greatly reduced and ‘herd immunity’ became manifest. Once again, for the development of this vaccine, antibodies were the obvious correlate of protection. Exactly the same principles have been applied to a pneumococcal conjugate vaccine, where it has not proven possible to include 23 serotypes, but 7-valent, 9-valent, and 11-valent vaccines have been developed by various manufacturers. The serious pathogen Neisseria meningitidis serogroup C has also yielded to this approach. A re-

1.6 Protection against Intracellular Bacteria

cent widespread deployment of this vaccine has reduced the incidence of meningococcal C meningitis in the United Kingdom by over 80 % and of deaths by > 90 % [14]. In Sub-Saharan Africa, vicious epidemics of meningococcal A meningitis sweep across the continent every few years. A carbohydrate vaccine is moderately effective in outbreak control, but the World Health Organization in conjunction with the Program for Appropriate Technology In Health (PATH) has an ambitious goal of developing and deploying a meningococcal A conjugate vaccine to be given to everyone under 21, as well as to be included in routine infant immunization. Another example of interesting research on conjugates is Salmonella typhi, for which the carbohydrate Vi antigen delivered parenterally is somewhat protective, but hopes are high that Vi-protein conjugate vaccines will work better. Much work is being directed at beating the polymorphism problem by seeking antigens that (1) are exposed at the bacterial cell surface; (2) exhibit limited or no polymorphism; (3) are important determinants of virulence; and (4) do not cross-react significantly with ‘self ’ antigens. Genome mining in those instances where the DNA sequence of the relevant bacterium has been determined is playing a major role in this research. Entirely different approaches to immunization have met with some success in diseases caused by gastrointestinal-tract pathogens. First, there is the possibility of introducing killed whole bacteria or important virulence antigens in conjunction with a mucosal adjuvant. A vaccine based on whole killed Vibrio cholerae together with purified B subunit of the cholera toxin (CTB) is licensed in some countries. Experimental oral molecular vaccines work in animal models of Helicobacter pylori disease [15]. Second, extensive investigation of live attenuated bacteria, also given orally, shows some promise. At the moment, the only licensed version of such a vaccine is the Ty21 a strain of Salmonella typhi. Serum antibodies do not represent a particularly reliable correlate of protection in these diseases, and much of the developmental research has relied on protection of volunteers against challenge administration of the relevant pathogen. Unfortunately, this is not always a good predictor of the results of phase III clinical trials.

1.6 Protection against Intracellular Bacteria

Intracellular bacteria have evolved mechanisms to foil the usually very efficient phagosome–lysosome system of macrophages and other cells and have learnt to live most of their lives either inside a phagocytic vacuole or, having learnt to escape the phagosome, within the cytoplasm of the host cell. In these hidden locations, they are protected from serum antibody, but because peptides derived from them are expressed on the surface of the infected cell, infections can be controlled by T cell immunity. It was established over a century ago that immunity to extracellular bacteria can be passively transmitted via serum [16], but it was also soon established that this did not occur with certain infections, e. g., tuberculosis. Nevertheless, solid immunity to intracellular bacteria does exist, and which can be transferred from immune to naï ve ani-

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1 Challenges for the Vaccine Developer, including Correlates of Protection

mals via lymphocyte cells [17] (which were soon shown to be T cells). Long before the difference between T and B cells was understood, a skin test to establish T cell immunity (delayed-type hypersensitivity) had been developed and widely used. The diseases caused by intracellular bacteria include some of the greatest scourges of humanity, such as tuberculosis, leprosy, and trachoma. Tuberculosis has taken on even greater significance since the HIV/AIDS pandemic, because infections that may have been well controlled flare up when T cell function wanes. The HIV/AIDS pandemic has also put the spotlight on intracellular bacteria such as Mycobacterium avium, which are relatively harmless unless T cell immunity fails. In general, infections by intracellular bacteria are chronic, and much of the pathology is associated with the immune response, e. g., granuloma formation. Usually immunity is not sterilizing, and the balance between bacterial persistence and T cell protection is somewhat labile. Both CD4+ and CD8+ T cells contribute to defense against intracellular bacteria [18], the CD8+ T cell being activated because bacteria escape into the cytosol, or because of antigens that escape, or because of cross-presentation. Various sorts of unconventional T cells also contribute to defense against intracellular bacteria. Because Stefan Kaufmann is one of the world experts in this field, please refer to his chapter 21 on tuberculosis for a discussion of this subject. Will delayed-type hypersensitivity skin tests or in-vitro measurements of T cell function be helpful to the vaccine developer in the search for, e. g., a more satisfactory vaccine against tuberculosis? Certainly, but they are not perfect correlates of protection. A person may yield a florid, highly positive Mantoux test while experiencing a flare-up and wide dissemination of tuberculosis. A negative test in a tuberculosis sufferer is in general a sign of poor prognosis (especially before the antibiotic era) but need not always be so. So the investigator also depends on the inherent plausibility of the antigen(s) chosen and on its efficacy in the most credible animal model available. Because of the chronic nature of the infections concerned, clinical testing of vaccines emerging from basic research is particularly arduous. However, adjuvant formulations and vaccination strategies (e. g., ‘prime–boost’ protocols) capable of eliciting strong CD4+ Th1 and CD8+ cytotoxic T cell responses are now available and should herald success in the long term.

1.7 Protection against Parasites

It is difficult to be dogmatic about correlates of protection in parasitic diseases, for the simple reason that there is no single licensed vaccine against any human parasitic disease. To this must be added the fact that most parasitic diseases do not themselves lead to solid immunity, Leishmania major being an exception when causing tropical sores. Nevertheless, a great deal of work has been done on animal models. I summarize here what is known about three important parasitic diseases: malaria, schistosomiasis, and leishmaniasis. Malaria is the most prevalent vector-borne disease in the world. It is caused by protozoa of 4 different species of Plasmodium, the most fatal of which is P. falciparum.

1.7 Protection against Parasites

Clearly this organism has developed powerful mechanisms to evade the host immune response, including an incredible degree of antigenic variation. Because of the chapter 22 dealing specifically with malaria, I will only mention that vaccine approaches are being directed at 4 distinct stages in the parasite's life cycle (Table 1.4). The mosquito injects a mobile sporozoite that exhibits a major circumsporozoite antigen. Antibodies to it or portions of it can block invasion of liver cells and be partially protective [19]. When the sporozoite reaches the liver cell, it develops into a shizont containing 10 000 to 30 000 merozoites. As a result, the hepatocyte presents on its surface peptides (T cell epitopes) from a number of preerythrocytic stage proteins. A CD8+ T cell attack on injected liver cells could materially decrease the total number of merozoites formed and thus be partially protective. Here, T cell immunity could correlate with protection. When the liver schizont ruptures, merozoites are released and invade erythrocytes to begin the blood stage of the cycle. A large number of merozoite surface antigens are being investigated as putative vaccine candidates, alone or, more usually, in various combinations. Here, antibody would be the effective agent. Finally, during the erythrocytic cycle, sexual stages known as gametocytes are formed. They exhibit some strong antigens, antibodies against which can inhibit sexual maturation within the mosquito after it takes a blood meal. It could well turn out that an eventual definitive malaria vaccine will contain antigens acting against all four stages. A further, more controversial, possibility is to try to neutralize a toxin, glycophosphatidylinositol, of malarial origin. An oligosaccharide from this glycan has been Tab. 1.4 Vaccine candidates from various stages of Plasmodium falciparum Stage

Antigen(s)

Desired response

Comments

Sporozoite

The major circumporozoite protein.

High affinity antibody

The most advanced vaccine candidate. Nature of adjuvant has been critical.

Liver cell schizont Various T cell epitopes, guided in part by elution of peptides from infected cells.

CD8+ cytotoxic cells

Research is most advanced for P. vivax. A promising, rather new area.

Merozoite

Proteins prominent on the surface. Proteins from internal organelles needed for invasion of erythrocytes.

High affinity antibody

An intense, competetive field. Many antigens nearing the clinic. Combinations also being tested.

Gametocytes

Antigens from surface.

High affinity antibody

The ‘unselfish’ vaccine. Does not help recipient directly but, widely used, would impair transmission.

Malarial toxin

GPI and oligosaccharides Neutralizing antibodies there from (see text).

Would be combined with other antigens.

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1 Challenges for the Vaccine Developer, including Correlates of Protection

synthesized, conjugated to a protein carrier, and used as a vaccine. It materially reduces pathology and mortality in a murine model [20]. Again, antibodies would be the correlate of protection here. Most investigators now hold the view that a final malaria vaccine should induce both T cell and B cell immunity. Schistosomiasis is the most prevalent human parasitic disease caused by a metazoan parasite. It is caused by five different species of Schistosoma, of which the most important are S. mansoni, S. hematobium, and S. japonicum. The most advanced vaccine candidate is the molecule Sm28GST [21]. Mice immunized with it consistently show reduced worm burden, impaired female worm fecundity, and low egg viability. Clinical trials are now ongoing. Evidence suggests that Th2-type CD4+ T cells correlate with protection, promoting IgE formation via IL-4 and eosinophilia via IL-5, but the matter is not straightforward because the ova of schistosomes evoke a Th2 response that aids granuloma formation and subsequent pathology. Similarly, Th1 responses induced by a radiation-attenuated S. mansoni vaccine can be protective in animals, but T cells from severely diseased humans often secrete Th1 cytokines, suggesting an involvement in pathogenesis [22]. The line between protective immunity and immunopathology is thus clearly a fine one. Immunological interest in leishmaniasis was first aroused by the realization that tropical sores, once they eventually healed, resulted in immunity to reinfection. Interest was increased by the clear-cut finding that, in a murine model, Th1 type CD4+ T cell responses cure lesions, but Th2 type immunity leads to progressive lesions and death [23]. The promastigotes of the parasite live inside macrophages. Here the Th1 cytokines, particularly IFN-g, appear to be able to activate macrophages to kill the parasites. When the infected macrophage eventually ruptures, it releases amastigotes, which have to find another macrophage to infect. Amastigote antigens can be thought of as similar to merozoite antigens; thus, antibodies could theoretically play a role here. However, the experimental evidence that immunity to leishmaniasis is T cell dependent is so voluminous that I put this notion foward with some reluctance.

1.8 Conclusions

It is perhaps a blessing that the by now quite substantial number of investigators who have been seeking to invent new and improved vaccines have not been deterred by the considerable difficulties that need to be faced after the preclinical research has reached its conclusion. I have covered a good many examples in which the correlates of protection are straightforward – substantial levels of high affinity antibodies to one or more important bacterial or viral antigens. We have seen examples in which strong experimental evidence suggests that what is needed is a substantial T cell attack, Th1 in some instances, CD8+ cytotoxic T cells in others, or perhaps both. This leaves several disease problems for which we simply do not know what the correlates of protection will be.

References

As already noted, vaccine research has always been a combination of fine basic science and enlightened empiricism. From that viewpoint, putative vaccines will simply have to be taken one by one, readied for clinical evaluation when experimental efficacy is convincing and the disease burden sufficient, and studied in humans, with the field trials probably providing the best correlates of protection. It is a grand challenge, particularly now that the position of the less privileged on our globe has been articulated and addressed. I hope that this necessarily cursory overview chapter has whetted your appetite for the many in-depth contributions in the remainder of this book.

References 1. H. E. Varmus, Lancet, 2002, Suppl. 360, s.1–s.4. 2. G. J. V. Nossal, Nat. Immunol., 2000, 1, 5–8. 3. B. Beutler, The Immunologist, 2000, 8, 123–130. 4. N. L. Letvin, D. H. Barouch, D. C. Montefiori, Annu. Rev. Immunol., 2002, 20, 73–99. 5. J. R. Mascola, S. W. Snyder, O. S. Weislow, S. M. Belay, R. B. Belshe, D. H. Schwartz, M. L. Clements, R. Dolin, B. S. Graham, G. J. Gorse et al., J. Infect. Dis., 1996, 173, 340–348. 6. R. Wyatt, J. Sodroski, Science, 1998, 280, 1184–1188. 7. M. Moulard, S. K. Phogat,Y. Shu, A. F. Labrijn, X. Xiao, J. M. Binley, M. Y. Zhang, I. A. Sidorov, C. C. Broder, J. Robinson et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 6913–6918. 8. C. M. Walker, D. J. Moody, D. P. Stites, J. A. Levy, Science, 1986, 234, 1563–1566. 9. L. Musey, J. Hughes, T. Schacker, T. Shea, L. Corey, M. J. McElraith, N. Engl. J. Med., 1997, 337, 1267–1274. 10. S. Rowland-Jones, J. Sutton, K. Ariyoshi, T. Dong, F. Gotch, S. McAdam, D. Whitby, S. Sabaly, A. Gallimore, T. Corrah et al., Nature Med., 1995, 1, 59–64. 11. A. J. McMichael, S. L. RowlandJones, Nature, 2001, 410, 980–987. 12. P. Anderson, Infect. Immun., 1983, 39, 233–238.

13. R. Schneerson, J. B. Robbins, C. Chu, A. Sutton, G. Schiffman,W. F. Vann, Prog. Allergy, 1983, 33, 144–158. 14. E. Miller, D. Salisbury, M. Ramsay, Vaccine, 2002, 20, S58–S67. 15. G. Del Giudice, A. Covacci, J. L. Telford, C. Montecucco, R. Rappuoli, Annu. Rev. Immunol., 2001, 19, 523– 563. 16. E. A. von Behring, S. Kitasato, Deutsche Med Wochenschr, 1890, 16, 1113–1114. 17. G. B. Mackaness, J. Exp. Med., 1962, 116, 381–406. 18. S. H. E. Kaufmann, E. Hug, U. VÄth, I. Müller, Infect. Immun., 1985, 48, 263–266. 19. K. A. Bojang, P. J. M. Milligan, M. Pinder, L. Vigneron, A. Alloueche, K. E. Kester, W. R. Ballou, D. J. Conway, W. H. H. Reece, P. Gothard et al., Lancet, 2001, 358, 1927–1934. 20. L. Schofield, M. Hewitt , K. Evans, M. A. Siomos, P. Seeberger, Nature, 2002, 418, 785–789. 21. A. Capron, Parasitol. Today, 1998, 14, 379–384. 22. J. K. Mwatha, G. Kimani, T. Kamau, G. G. Mbugua, J. H. Ouma, J. Mumo, A. J. Fulford, F. M. Jones, A. E. Butterworth, M. B. Roberts et al. J. Immunol., 1998, 160, 1992–1999. 23. R. M. Locksley, J. A. Louis, Curr. Opin. Immunol., 1992, 4, 413–418.

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Part II Vaccination and Immune Response

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2 Shaping Adaptive Immunity against Pathogens: The Contribution of Innate Immune Responses Stefan Ehlers and Silvia Bulfone-Paus

2.1 Introduction

A generally accepted view of adaptive immunity and, in particular, effector T cell development and function distinguishes between type 1 and type 2 responses [1]. Infection with bacteria, viruses, and fungi or stimulation of innate immune cells with components thereof, induces type 1 immunity, mediated by T helper 1 (Th1) or T cytolytic 1 (Tc1) cells. In this context, type-1 cytokines (i. e., IL-2, IL-12, IL-15, IFN-g, IFN-a, and IFN-b) are particularly involved in cell-mediated immunity to intracellular microbes. In contrast, infections by extracellular pathogens and multicellular parasites or exposure to allergens induce type 2 responses. Type-2 cytokines (i. e., IL4, IL-5, IL-6, IL-10, and IL-13) play a crucial role in promoting humoral immunity and/or immune deviation to a nonprotective response (tolerance) [2]. More than 98 % of multicellular animal species cannot produce an adaptive immune response to a pathogen [3]. Immunological memory is thus a privilege of a few vertebrate hosts, and its primary purpose is to sterilize primary infection and provide resistance to reinfection with pathogens. However, during the time it takes for clonal expansion of antigen receptor-bearing lymphocytes to occur, the primordial system of innate defense takes care of the host. Individual functions of innate immune cells are not only involved in mitigating the damaging insults of microbial or tumor growth, but are also critical in directing the differentiation processes that, after antigen encounter, ultimately lead to T and B effector cells. Full-fledged adaptive immunity, in turn, is the result of cells of the adaptive immune system calling upon cells of the innate immune system, either to provide professional phagocytes to engulf and destroy pathogens (type 1 immunity) or to release toxic mediators so as to establish environments that are inhospitable to helminth or arthropod invaders (type 2 immunity). As a consequence, the innate and adaptive immune systems should be viewed as two interdependent parts of a single integrated immune system. Several novel vaccination strategies strive to exploit innate pathways of immune activation to manipulate, at will, quantitative and qualitative aspects of the acquired immune response. The purposes of this chapter are (1) to delineate some of the me-

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2 Shaping Adaptive Immunity against Pathogens: The Contribution of Innate Immune Responses

chanisms that trigger the activation of innate immune cells during infection and (2) to describe the mediators and dissect the major molecular pathways regulating the differentiation of effector functions of adaptive immunity, while (3) highlighting implications for the development of novel vaccination strategies.

2.2 Activation of Innate Immunity: Sensing the Enemy

Innate defenses are primarily aimed at recognizing foreign structures and eliminating them. Since infectious pathogens have often evolved to subvert these mechanisms, specialized cells of the innate immune system are also capable of presenting digested antigen to T cells in a context and environment appropriate for optimal effector cell development. Activation of innate immunity to provide this context may be conceptually divided into two stages (Figure 2.1): (1) a phase of antigen sensing during which a combination of surface receptors detects the presence of non-self structures on invading microorganisms (‘detection phase’) and (2) a phase of translating this sensory information into a language understood by the cells of the adaptive immune system, e. g., chemokines and cytokines (‘transmission phase’) [4].

Fig. 2.1 Overall view of innate immunity shaping adaptive immune responses. Cells of the innate immune system (DC = dendritic cells; MC = mast cells) detect the presenceof foreign structures, such as pathogen-associated molecular patterns, by surfaceexpressed pattern-recognition receptors,and translate this sensory input into a language (cytokines, chemokines) understood by cells of the adaptive immune system, skewing the response to either T helper 1 (Th1)/T cytolytic 1 (Tc1) or Th2/Tc2-type immunity.

2.2 Activation of Innate Immunity: Sensing the Enemy

2.2.1 Pathogen-associated Molecular Patterns

The costimulatory signals required for differentiation of adaptive immune responses are exclusively turned on when cells of the innate defense system interact with pathogen-associated molecular patterns (PAMPs, i. e., highly conserved structures that are necessary for the survival of microorganisms). Many pattern-recognition receptors primarily serve the purpose of enhancing phagocytosis. For example, the macrophage mannose receptor (recognizing terminal mannose and fucose residues on microbial cell walls) or the macrophage scavenger receptor (recognizing polyanionic ligands such as double stranded RNA, lipopolysaccharide, and lipoteichoic acid) are mainly involved in clearing pathogens from the site of invasion [5, 6]. Discrimination between different classes of microorganisms, however, is afforded by the Toll-like receptors 1 through 9 [7, 8]. Since PAMPs have multiple effects on the signals generated by cells of the innate immune system and may therefore be useful as natural adjuvants in driving adaptive responses, they are described here in detail (Table 2.1).

Tab. 2.1 Recognition of pathogen structures via surface receptors on innate immune cells. Pattern-recognition receptors on innate immune cells

Pathogen-associated molecular patterns or similar insults

TLR1/TLR2

Triacylated lipopeptides

TLR2

Lipoproteins, lipoteichoic acid, glycolipids, modulin, arabinose-capped lipoarabinomannan, GPI-anchored molecules from parasites

TLR2/TLR6

Diacylated lipopeptides, zymosan

TLR3

Double-stranded RNA

TLR4

Lipopolysaccharides

TLR5

Flagellin

TLR7 and TLR8

Imidazoquinoline Derivatives

TLR9

Unmethylated CpG-motif oligonucleotides

Complement receptors

Activated components of complement coating microbial surface

Scavenger receptors

Polyanionic compounds, e. g., lipoteichoic acid, double-stranded RNA, lipopolysaccharide

Mannose–fucose receptor

Terminal mannose and fucose on microbial glycoproteins/ glycolipids

CD14

Monomeric lipopolysaccharides

CD48

Fimbrial protein FimH on enterobacteria

CD91

Heat-shock proteins 70, 90, gp 96; calreticulin

IgE/FceR-crosslink

Parasitic lectins

unknown

Allergens

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Peptidoglycan (PG) is a component of all bacterial cells. PG chains consist of alternating b-MurNAc(1 ? 4)GlcNAc disaccharide units linked via b-(1 ? 4) glycosidic bonds [9]. Peptide sidechains, which are attached to the carboxyl group of muramic acid residues, are primarily composed of five amino acids (L-Ala-D-Glu-X-D-Ala-DAla, where X stands for L-Lys or diaminopimelic acid). Certain variations both in peptide and glycan structure exist [10], depending on the microorganism. PG was reported to stimulate macrophages and DCs via TLR2 [11]. A core constituent of PG, muramyl dipeptide (MurNAc-L-Ala-D-isoGln), however, is not recognized by TLR2, but does activate cells via the intracellular sensor protein NOD2 [12]. Lipopeptides and lipoproteins are components of both Gram-positive and Gram-negative cell walls and may be viewed as functional substituents of PG. A 19-kDa lipoprotein derived from M. tuberculosis activates cells via TLR2 [13]. Mycoplasma macrophage-activating lipopeptide-2 (MALP-2) requires the cooperation of TLR2 and TLR6 to initiate proinflammatory responses, whereas certain bacterial lipopeptides require dimerization of TLR2 with TLR1 [14–16]. Since MALP-2 is diacylated, and bacterial lipopeptides are triacylated, distinct structural requirements seem to dictate the dimerization partner for TLR2 [17]. Lipoteichoic acid (LTA) is an amphiphilic, negatively charged glycolipid found in most Gram-positive bacteria. Appropriate purification of LTA from Staphylococcus aureus, or chemical synthesis of LTA, have revealed LTA to be a potent and exclusive TLR2 stimulus [18]. Structural requirements include a lipid anchor and d-Ala substituents, as l-Ala derivatives reduce the activity by a factor of 100 [19]. Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and is composed of polysaccharides attached to a lipid portion (lipoid A). Lipoid A is responsible for all biological activities ascribed to LPS, and the spatial conformation of lipoid A – in part determined by the number and length of acyl chains – determines biological activity on a given cell type [20, 21]. LPS recognition by TLR4 requires several accessory molecules. LPS is first bound to the LPS-binding protein LBP, which transfers LPS monomers to CD14 [22]. Exactly how CD14 facilitates LPS recognition is unclear, as is the exact role of MD-2, another component of the LPS-recognition complex [23]. Ultimately, LPS interacts with TLR4 and initiates both MyD88-dependent and -independent intracellular signaling cascades [24]. Some forms of LPS (e. g., from Porphyromonas gingivitidis or Leptospira interrogans) seem to be recognized by TLR2 [25, 26]. The structural requirements for this interaction are not clear, other than that this recognition is greatly enhanced in the presence of TLR1. Flagellin is the protein subunit of bacterial flagella. It is recognized by TLR5, possibly via highly conserved N- and C-terminal ends that form its hydrophobic core [27, 28]. TLR5 is expressed on the basolateral, but not the apical, surface of epithelium. Therefore, flagellin of invading pathogens, but not commensal bacteria, induces intestinal epithelial cells to mount an inflammatory response [29]. Unmethylated CpG DNA motifs are prevalent in bacterial but not vertebrate genomic DNA and are recognized by host cells bearing TLR9 [30]. TLR9 is not on the cell surface but is present in vesicles that fuse with lysosomes [31]. Some CpG motifs are very powerful activators of NK cells and induce IFN-a production by DC2 cells; other motifs are potent B cell activators, triggering cell cycle progression and antibody secretion

2.2 Activation of Innate Immunity: Sensing the Enemy

[32]. Nonspecific activation of innate responses afforded by CpG can increase resistance in animal models of intracellular infection and sepsis [33]. The ability of CpG to act as a potent adjuvant was confirmed in studies using both model antigens such as hen egg lysozyme or ovalbumin, as well as tumor antigens and antigens from infectious agents, including hepatitis B, HIV, and influenza virus or Leishmania major [32, 34, 35]. In fact, CpG is a stronger Th1-like adjuvant for inducing B and T cell responses than the gold standard, Freund’s complete adjuvant, as measured by its ability to drive the differentiation of cytolytic Tcells and IFN-g secreting cells [36]. We should note that optimal CpG motifs for activation differ between human beings and mice [37, 38]. Lipoarabinomannans (LAMs) are lipoglycans restricted to the genus Mycobacterium. The carbohydrate backbone is comprised of a D-mannan core and a D-arabinan domain. At its reducing end, the mannan core is terminated by a glycosyl-phosphatidylinositol anchor, and the arabinan domain is capped by either mannosyl (ManLAM) or phosphoinositide residues (PILAM) [39]. PILAMs prevalent in rapidly growing mycobacterial species (M. smegmatis) activate mammalian cells in a TLR2-dependent manner to secrete TNF and IL-12. In contrast, ManLAM is not an agonist for TLR2 [40, 41]. A small mycobacterial glycolipid, phosphatidyliositol dimannoside (PIM) also activates cells in a TLR2-dependent manner [42]. Double-stranded RNA (dsRNA) is produced by most viruses during their infectious cycle. DsRNA induces protein kinase R activation via TLR3 [43]. TLR3 is expressed predominantly on myeloid DCs, making them the prime targets for initiating innate responses against viruses [44]. TLR3-deficient mice also show a reduced response to the viral RNA mimic polyinosine-polycytidylic acid (poly I:C) [43]. Glycosylphosphatidylinositol-anchored molecules (GPI) are the predominant antigens present on the plasma membrane of protozoa such as Trypanosoma cruzi and Plasmodium falciparum [45]. GPI from T. cruzi are recognized by TLR2 and potently induce nitric oxide, TNF, and IL-12 in murine macrophages [46]. The PI moiety is mostly composed of unsaturated fatty acids, which are essential for optimal biological activity [47]. FimH is a subunit protein of type 1 fimbrial adhesins present in many enterobacterial species, e. g., Escherichia coli and Salmonella enterica. It can bind directly to a GPI-anchored receptor present also on the surface of mast cells (CD48). Ligation of CD48 by FimH-expressing bacteria results in bacterial uptake into caveolar chambers, promotes bacterial survival inside the cytosol, and has been linked to the release of inflammatory mediators by mast cells [48]. Heat shock proteins (HSPs) are released from necrotic cells during tissue injury or lysis of infected cells and can form stable complexes with peptides. Some of them, e. g., HSP60, can be recognized by TLR4 [49]; others, e. g., gp96, HSP90, and HSP70, interact with antigen-presenting cells via a common receptor, CD91 [50]. HSP-chaperoned peptides enter the macrophage/dendritic cells through CD91 and are processed and presented by the MHC class I and MHC class II molecules, resulting in stimulation of CD4 and CD8 T cells. HSP-DC interaction through CD91 leads to maturation of DCs and secretion of an array of proinflammatory cytokines [51]. Immunization of mammals with HSP-peptide complexes elicits potent responses against the HSP-chaperoned peptides [52].

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Parasite-derived glycans and lectins, e. g., from Schistosoma mansoni eggs or filarial nematodes, trigger responses by TLR-independent mechanisms and are known for their potent induction of Th2 responses [53, 54]. One well characterized lectin from S. mansoni eggs, termed IPSE, leads to cross-linking of IgE on basophils which release IL-4 and IL-13, thus driving a strong Th2 response [55, 56]. All currently known PAMPs are derived from prokaryotic, fungal, viral, or protozoan pathogens, and which receptors are required for recognition of ‘patterns’ expressed by multicellular eukaryotic parasites or allergens is still unknown. Whether as-yet undefined molecular patterns are at all necessary to skew adaptive immunity towards a type 2 pattern after encounter with these different types of insults remains to be determined. It is possible that a different type of signal, such as the absence of PAMPs – similar to the lack of self-MHC I for NK-cell activation – is a stimulus in itself for triggering type 2 immunity [57]. 2.2.2 Host Cellular Sensors

At the immunological level, innate defenses rely mostly on granulocytes, mast cells (MC), macrophages, dendritic cells (DC), natural killer (NK), NKT, and gdT cells. These cells serve as a bridge between PAMPs and the antigen-specific cells of adaptive immunity, translating the sensory input of pattern-recognition receptors into soluble mediators that communicate with T and B cells via specific cytokine/chemokine receptors. Although many cell types contribute in various ways to linking innate and adaptive responses, we shall focus in this chapter – for the purpose of clarity, and because the two prototypic immune responses (type 1 and type 2) are regulated differently by them – on the two pivotally involved cell types, DCs and MCs. 2.2.2.1 Dendritic Cells The key cell type, and indeed the most potent type of any antigen-presenting cell, which is able both to sense a foreign insult and to orchestrate the cells of the adaptive immune system, is the dendritic cell [58]. Like other cell types within the immune system, immature (im) DCs are continuously produced from hematopoietic stem cells within the bone marrow. FLT-3 ligand and, to a lesser extent, granulocytemacrophage colony-stimulating-factor (GM-CSF) are the key growth and differentiation factors in vivo [59]. Immature DCs have long been considered ‘sentinels’ of the immune system, since they are specialized, relatively long-lived phagocytic cells, which are resident in most tissues, where they survey the local environment for pathogens [60]. Pattern-recognition receptors, in particular TLRs, allow DCs to recognize microbial antigens and mature into highly effective antigen-presenting cells, undergoing changes that enable them to activate pathogen-specific lymphocytes that they encounter in the lymph nodes [24]. Maturing DCs lose the ability to take up and process antigen, but they up-regulate surface expression of MHC class I and II, as well as of costimulatory and adhesion molecules such as CD86, CD80, CD40, and CD54. Mature DCs express proinflam-

2.2 Activation of Innate Immunity: Sensing the Enemy

matory cytokines like IL-6, IL-12, IL-18, IL-23, and IL-27 and are able to detect the expression of cytokines by infected cells, thus integrating different signals of danger. Another consequence of imDC stimulation is the up-regulation of CCR7 expression and down-regulation of CCR5 expression, which allows DCs to migrate to regional lymph nodes [61]. There, mature DCs prime naive T cells and, depending on the predominating maturation signals, differentially induce Th1 versus Th2 development [62]. Bacterial CpG DNA, double-stranded viral RNA, LPS, as well as CD40 ligand and IFN-g induce immature DCs to produce IL-12 and promote Th1 immune responses. In contrast, anti-inflammatory molecules such as IL-10, TGF-b, corticosteroids, and prostaglandin E2 promote Th2 or regulatory T cell immune responses [63]. The ability of DCs to prime greatly diverse immune responses is the result of different DC subsets and their unlimited plasticity [63]. In addition to immature DCs, stem cells also give rise to two types of DC precursors (pre-DCs) – monocytes (preDC1) and plasmacytoid cells (pre-DC2) – during hematopoiesis [58, 63]. Functionally, immature DCs and pre-DCs differ since pre-DCs are more directly involved in innate immunity against microbes. Pre-DC1 phagocytose and kill various bacteria and fungi, and pre-DC2s play a major role in the early antiviral innate immune response by producing large amounts of IFN-ab upon viral infection [58, 63, 64]. Therefore, these cells were also called ‘natural interferon-producing cells’ (NIPCs). Unlike other innate immune cells, such as neutrophils, eosinophils, and basophils, which die after performing their functions, pre-DC1 and pre-DC2 differentiate into DCs. When placed in culture with GM-CSF and IL-4, or after bacterial uptake, pre-DC1 differentiate into immature myeloid DCs. Upon CD40 ligand-induced activation, DC1 produce large amounts of IL-12 and prime Th1 and cytotoxic T cell immune responses. In contrast, pre-DC2 differentiate into immature DC2 in the presence of IL-3 or CD40 ligand, and mature DC2 primarily induce Th2 immune responses [65, 66]. Interestingly, when immature DC2 cells become infected by a virus, they take on the typical morphology of mature DCs and up-regulate costimulatory molecules, but acquire the distinct ability to differentiate naïve CD4+ T cells into producers of IFN-g and IL-10 [67, 68]. 2.2.2.2 Mast Cells Mast cells (MC) are derived from CD34+ stem cells. They initiate their differentiation in the bone marrow under the influence of stem cell factor and interleukin-3. Their final maturation takes place in connective tissues [69]. Within minutes following activation, mast cells extrude granule-associated substances such as histamine. Within hours, mast cell activation is followed by de novo synthesis of numerous cytokines (TNF-a, interleukins 1–10, IL-12, IL-13, IL-15, IL16, IL-18, IL-25, and GM-CSF) and chemokines (CCL2–5, CCL8, CCL11, IL-8) [70, 71]. Mast cells are long-lived and therefore able to respond repeatedly to the same stimulus [72]. In addition to their well-established central role in the pathogenesis of allergic disorders, MCs are now also appreciated as key effector cells in the induction of protective immune responses to bacteria [72–74]. This notion is supported by several inde-

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pendent lines of evidence. MCs are found at the portals of entry of many infectious agents, i. e., within mucosal surfaces of the gastrointestinal and respiratory tracts, or in the skin close to peripheral nerves and often associated with blood vessels [75, 76]. MCs are equipped with a large variety and number of receptors to detect bacteria or signs of bacterial infection, including Toll-like receptors (TLRs), CD48, and complement receptors [77–80]. Furthermore, genetically MC-deficient animals, such as KitW/KitW-v mice, exhibit insufficient host defense responses in various models of bacterial infection, e. g., in cecal ligation and puncture-induced acute septic peritonitis [73, 81]. Finally, protection from bacterial infections in mice can be improved by enhancing MC numbers and/or function [82, 83]. Mast cells may exert many different effects on inflammatory processes: (1) in early stages, mast cells can be activated via TLRs in the peripheral tissues through interaction with microbes; (2) the release of cytokines, such as IL-4 and IL-13, may influence DC maturation, and as a consequence, biases T cell differentiation to a Th2/Tc2 phenotype; (3) mast cell-derived chemokines may recruit distinct subsets of inflammatory cells to the site, amplifying a predominantly type-2 response; and (4) activated mast cells can migrate to secondary lymphoid organs, where they can directly influence the development of T cell immune responses via the release of cytokines and the expression of costimulatory molecules [84, 85]. 2.2.3 Nonpeptide MHC Ligands Triggering Invariant T-cell Receptors

NKT cells are a subset of T cells that express a heavily-biased T cell receptor repertoire as well as the NK cell receptor CD161 and are deficient in immunological memory. In contrast to conventional T cells, NKT cells recognize glycolipid antigens presented by the nonpolymorphic MHC class I-like molecule CD1 d [86]. Although they are not strictly necessary for the generation of a polarized Th cell response, they can modulate the latter by secreting large amounts of IL-4, IL-10, IL-13, as well as IFN-g or TNF upon T cell receptor engagement [87, 88]. A glycosphingolipid originally isolated from the sea sponge Agelas mauritianus, a-galactosylceramide (a-GalCer), is a potent inducer of NKT activity [89]. Optimal stimulation of NKT cells requires direct contact between NKT cells and DC through CD40–CD40–ligand interactions and IL-12 production by DC [90]. Most studies indicate that a-GalCer enhances Th2 polarization, but Th1 differentiation may also be affected. In vivo activation of NKTcells with a-GalCer ameliorates the diseases caused by Cryptococcus neoformans or encephalomyocarditis virus and inhibits development of the intrahepatocytic stages of Plasmodium yoelii and Plasmodium berghei in mice [91–93]. a-GalCer potentiates protective immune responses induced by malaria vaccines [94].

2.3 Translating Innate Immune Activation into Regulatory Circuits

2.3 Translating Innate Immune Activation into Regulatory Circuits : Molecular Pathways Shaping Adaptive Immunity 2.3.1 TLR-initiated Signaling Cascades

Upon recognition of their cognate ligands, TLRs induce the expression of a variety of host defense genes. These include inflammatory cytokines and chemokines, antimicrobial peptides, costimulatory molecules, MHC molecules, and other effectors necessary to arm the host cells against the invading microorganism and to prime adaptive immunity. After ligation of TLRs, receptor dimerization occurs, triggering recruitment of the adapter MyD88 to the receptor complex. Association of the TIR (Toll/IL-1R) domain of MyD88 with the TIR part of TLRs facilitates association of the death domain of MyD88 with the IL-1R-associated kinase (IRAK) [95, 96]. IRAK then autophosphorylates, dissociates from the receptor complex, and interacts with TNF-receptor-associated factor 6 (TRAF6) [97]. TRAF6 leads to activation of MAP kinases and, by recruiting ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), activates AP-1 and NF-kB [98, 99]. In MyD88-deficient mice, all TLR-9-initiated signals are abolished [100]. In contrast, TLR4-initiated signals in these mice are transduced by a different adaptor pathway (TIRAP), leading to up-regulation of some costimulatory molecules on DCs, but not to the induction of IL-12 [101]. In addition, IFN-inducible protein 10 (IP-10, CXCL-10) is induced even in MyD88-deficient macrophages in response to LPS [102]. Since IRF-3 is required for IP-10 gene transcription, it is likely that IRF-3 activation contributes to MyD88-independent pathways of TLR-mediated cell activation. Further regulatory mechanisms and signaling pathways downstream of TLRs exist, such as the Rac1–PI3K–AKT pathway activated by TLR2 [103]. Much remains to be learnt concerning the bifurcation of intracellular pathways that differentially determine type 1 vs. type 2 cytokine secretion during infection. In a mycobacterial infection model, TNF production was predominantly ERK-dependent, but independent of p38 MAP kinase activation, whereas IL-10 secretion was predominantly p-38-dependent, but independent of ERK signaling [104]. The use of small molecules as inhibitors of these putatively divergent pathways is an attractive novel option to selectively skew adaptive immune responses. At present, there is little evidence that specific TLRs differentially induce Th1 vs. Th2 responses. However, certain subsets of DCs respond differently to different PAMPs because they express different TLRs. Thus, plasmacytoid DCs express TLR7 and TLR9, whereas myeloid DCs express many of the remaining TLRs, in particular TLR3, but not TLR7 and TLR9 [105–107]. This may provide a means of fine-tuning the adaptive response by targeting only certain specialized DCs. TLR2-dependent activation of mast cells was reported to preferentially induce TNF, IL-4, IL-5, IL-6, and IL-13, whereas TLR4 ligation led to TNF, IL-1b, IL-6, and IL-13, but not IL-4 or IL-5 secretion. In the same study, TLR2-mediated mast cell activation led to degranulation, but TLR4-mediated activation did not [108].

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All MyD88-dependent signals favor Th1 responses. In the absence of MyD88, no Th1 differentiation occurs, whereas Th2 development is unaffected by MyD88 deficiency [109]. It is unclear whether Th2 differentiation is determined by specific, asyet unknown intracellular signals, or whether it is simply the default pathway. Surprisingly, human patients with a complete, recessive IRAK-4 deficiency seemed to suffer only from pyogenic bacterial infections, but were apparently resistant to other ubiquitous microorganisms such as Mycobacterium avium, the latter being clinically highly relevant in patients with hereditary defects in the IL-12/IFN-g signaling pathways [110, 111]. The fact that significant protective immunity against common pathogens is afforded even in the absence of critical receptors and/or major signaling pathways indicates that there is substantial plasticity and room for compensatory mechanisms in immune cell activation [110, 112]. 2.3.2 Molecules Involved in Recruiting Effector Cells

Correct and efficient priming of adaptive responses, and indeed, the overall type of immunity itself, heavily depend on the rapid recruitment of professional antigenpresenting cells together with T and B lymphocytes. Thus, molecules coordinating cellular movement from the blood stream into either inflammatory sites or sites of antigen presentation, such as lymph nodes, play a critical role in directing the priming process (Figure 2.2). Conversely, differential regulation of surface receptors capable of detecting gradients of chemoattractants is a crucial factor determining the cellular environment in which priming of immune responses takes place [113]. 2.3.2.1 Defensins Defensins are 2–6-kDa, cationic, microbicidal peptides that are classified, on the basis of their size and pattern of disulfide bonding, into a, b, and y (extant only in primates) categories. a-Defensins are particularly abundant in neutrophils, whereas b-defensins are more often produced by epithelial cells lining for instance the skin, the lung, and the genitourinary tract [114]. Defensins are produced constitutively or in response to microbial products. Defensins can kill or inactivate a wide spectrum of bacteria, fungi, or viruses by disrupting the microbial membrane [115, 116]. However, defensins are not only direct effector molecules of innate antimicrobial immunity. They can also enhance phagocytosis [117, 118], induce the activation and degranulation of mast cells [119, 120], and stimulate bronchial epithelial cells to augment IL-8 production [121]. In this way, defensins are involved in recruiting and activating neutrophils into inflammatory sites. Because some defensins can enhance TNF and IL-1 production – while reducing IL-10 secretion – in monocytes, they may play a role in amplifying the local inflammatory response [122]. Human neutrophil-derived a-defensins 1 (HNP1) and HNP2 are chemotactic for human monocytes and T cells [123, 124]. They are selectively chemotactic for resting CD4/CD45RA and CD8 T cells, whereas human b-defensin 2 (hBD2) is chemotactic for immature DCs and CD45RO memory T cells by interacting with human CCR6 [125, 126]. HBD3 and hBD4 are also chemotactic for monocytes via an as yet unidenti-

2.3 Translating Innate Immune Activation into Regulatory Circuits

Fig. 2.2 Major chemokine pathways linking innate and adaptive immune responses. Cells of the innate immune system, triggered by structures on microbes, release chemokines, thereby recruiting different type of cells during the initial phase of the immune response. Type 1 responses are dominated by cells bearing CCR5 and CXCR3 (underlined) and are further amplified by IFN-g derived from NK and Th1 cells up-regulating expression of CXCR3binding chemokines. Type 2 responses are dominated by cells bearing CCR3 (italics) and are further modulated by IL-4 and IL13 derived from Th2 cells and mast cells. DC (dendritic cell), MC (mast cell), imDC (immature dendritic cell), MF (macrophage), NK (natural killer cell), PMN (polymorphonuclear granulocyte), eos (eosinophil granulocyte), baso (basophil granulocyte), EndC (endothelial cell), EpC (epithelial cell), PAMP (pathogen-associated molecular patterns).

fied receptor [127, 128]. Defensins may also be involved in the maturation of immature DCs either directly or indirectly, by inducing the production of TNF and IL-1 [116]. Defensins therefore appear to be important potentiators of T cell priming. Indeed, a mixture of HNP1–3, when administered intranasally simultaneously with ovalbumin into C57BL/6 mice, enhanced the production of OVA-specific serum IgG antibody and the generation of IFNg, IL-5, IL-6, and IL-10 secreting OVA-specific CD4+ T cells [129]. Intraperitoneal injection of HNP1–3, together with keyhole limpet hemocyanin or B-cell lymphoma idiotype Ag, into mice not only augmented the levels of Ag-specific IgG, but also enhanced the resistance of immunized mice to tumor challenge [130]. In addition, when murine (m) mBD2 and mBD3 were used in a DNA vaccine approach together with B cell lymphoma epitopes, mice generated potent humoral immune responses and developed antitumor immunity [131].

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2.3.2.2 Chemokines Chemokines are a superfamily of small heparin-binding cytokines that induce directed migration of various types of leukocytes through interaction with a group of seven transmembrane G protein-coupled receptors [132]. TLR activation induces the release of chemokines from tissue macrophages and resident DCs. For example, both TLR2 and TLR4 ligation result in the expression of CCL3, CCL4, and CCL5. TLR2 signaling, however, preferentially leads to secretion of IL-8, while TLR4 signaling leads to CXCL10 production [133]. IL-8 recruits neutrophils to the site of chemokine production, whereas CXCL10 guides activated T cells into infected tissues. Thus, the discrimination of the pathogen by TLRs and the subsequent production of a distinct subset of chemokines is one of the earliest points at which the immune system tailors its response to specific pathogens. CCL3 and CCL4 are also crucial for the initial influx of CCR5+ NK cells into infected tissues, as demonstrated in murine models of cytomegalovirus and Toxoplasma gondii infection [134, 135]. These NK cells represent an important early source of IFN-g, providing an environment favorable for Th1 differentiation. IFN-g in turn induces the expression of the chemokines CXCL9, CXCL10, and CXCL11, instigating the recruitment of activated CXCR3+ T cells and NK cells into the tissue and amplifying the protective response [136]. CXCL10 may also be induced in the absence of IFN-g, after TLR ligation, and is also important for the initial influx of CXCR3+ NK cells and T cells. In terms of modulating humoral immunity, CCL3 treatment stimulated strong antigen-specific serum IgG and IGM responses, and CCL4 administration promoted higher serum IgA and IgE responses, as well as higher titers of antigen-specific mucosal secretory IgA levels in mice. These chemokines may therefore differentially enhance serum and mucosal humoral immune responses [137]. ImDCs express several chemokine receptors, including CCR1, CCR5, and CCR6 [138, 139]. These are important for recruiting imDCs in response to early pathogeninduced release of CCL3, CCL4, and CCL20 and for keeping them in the tissue. After TLR-mediated activation, however, DCs down-modulate the expression of these chemokine receptors and up-regulate the expression of CCR7, allowing them to respond to CCL19 and CCL21 expressed in lymphatic tissues [61]. This serves as a stimulus for DCs to leave the tissue and ultimately migrate into the T cell-rich regions of lymph nodes, where priming of T cells occurs. Generally speaking, Th1-dominated responses are associated with the recruitment of CCR5- or CXCR3- bearing cells, whereas Th2-dominated responses are more closely dependent on the influx of CCR3-expressing cells [140]. Mast cell granule-derived serine proteases also play a major role in recruiting cells to the site of infection [141]. Chymase, a serine protease found in human mast cells, induces monocyte and neutrophil migration directly [142]. Murine mast cell protease-6, also known as tryptase, induces neutrophil extravasation indirectly by enhancing endothelial cell IL-8 production [143]. In addition, histamine is a major mediator synthesized by mast cells. Vasodilation and inflammation are among the prime consequences of it being released from the granules in which it is stored. Histamine promotes the expression of Th2 polarizing (e. g., IL-6 and IL-10) cytokines from hu-

2.3 Translating Innate Immune Activation into Regulatory Circuits

man lung macrophages [144]. Histamine also inhibits IL-12 production and augments IL-10 secretion of maturing monocyte-derived DC, resulting in a change from Th1 to Th2 in their T cell-polarizing function [145]. In addition, activated human plasmacytoid DCs respond to histamine by a marked down-regulation of IFN-a and TNF [146]. Most importantly, mast cell granules contain preformed TNF, which is a major mediator of neutrophil influx into sites of mast cell activation. Upon appropriate stimulation, mast cells are also capable of releasing – without degranulation – CCL20, a potent chemoattractant for imDC and T cells [147]. 2.3.3 Molecules Involved in T and B Cell Differentiation

Cytokines provide the most direct link between innate and adaptive immunity, because they regulate the communication between dendritic cells, lymphocytes, and other host cells in the course of an immune response [148, 149]. The local milieu of cytokines determines the commitment of T cells to a Th1/Tc1 or Th2/Tc2 type, as well as the immunoglobulin class switch in B cells, and drives the overall immune response towards either tolerance or immunity (Figure 2.3).

Fig. 2.3 Major cytokine pathways linking innate and adaptive immune responses. Cells of the immune system, triggered by structures on microbes, release cytokines that skew T cell development into a Th1/Tc1 or a Th2/Tc2 pathway. Feedback loops involving IFN-g (type 1 immunity) or IL-10, TGF-b, IL-4, and IL-13 (type 2 immunity) are indicated. DC (dendritic cell), MC (mast cell), Treg (regulatory, CD4+CD25+ T cell), Tmem (memory T cell).

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2.3.3.1 Th1-inducing Cytokines Type I interferons (IFNs) were among the first cytokines identified, originally because of their antiviral activity and their effects on the cells of the innate response. IFNs have recently been shown to play an important role in connecting innate and adaptive immunity by acting on T cells and dendritic cells [4]. The term IFN-a/b defines a large group of closely related cytokines, which exert their activity by binding to a common receptor, the IFN-ab receptor. These cytokines include IFN-a, -b, -d, -t, -o and -k [150]. Although virtually any cell type can express IFN-a/b following viral infection, NIPCs produce 200–1000 times more IFN than other blood cells after microbial infection or after stimulation with bacterial components such as LPS and bacterial DNA [66, 151, 152]. IFN-a/b produced by NIPCs and macrophages can enhance the efficiency of the innate immune response by activating natural killer and other host reactive cells. Type I IFNs provide important signals for the differentiation and activation of DCs. In particular, IFN-a/b promotes the rapid differentiation of GM-CSF-treated human monocytes into DCs with enhanced T cell stimulatory capacity [4, 153]. Importantly, IFN-a/b plus GM-CSF-matured DCs induced a potent primary antibody response when pulsed with antigen and injected into SCID mice reconstituted with autologous peripheral blood lymphocytes [154]. In addition, IFN-a/b treatment of imDCs induces phenotypic and functional maturation of these cells, as shown by their expression of DC markers and their migratory response to chemokines [155]. The effects of IFN-a/b on DC maturation imply that they can exert an adjuvant activity. Indeed, experiments in mice revealed that (1) type I IFNs are extremely potent adjuvants when injected into immuncompetent mice with a reference antigen, (2) IFNs act directly on DCs, and (3) the natural adjuvant activity associated with infectious agents is mediated by IFN-a/b [149]. Interferon-gamma (IFN-g) is a potent activator of the antimicrobial functions of phagocytes and plays an important role in resistance to many pathogenic bacteria, fungi, and intracellular parasites [156]. It is mainly produced by Th1 cells and natural killer cells, but other T cell subsets such as NKT cells, CD8+ T cells, and gdT cells are also capable of secreting IFN-g. Furthermore, macrophages, dendritic cells, and B cells are able to produce IFN-g when appropriately stimulated [157]. Both type I and type II interferons activate natural killer (NK) cells to kill virus-infected cells and release cytokines. Interleukin-18 (IL-18) is a proinflammatory cytokine that belongs to the IL-1 cytokine family, due to its structure, receptor family, and signal transduction pathways [158, 159]. Similar to IL-1b, IL-18 is synthesized as a precursor requiring caspase-1 for cleavage into an active IL-18 molecule. Produced mainly by antigen-presenting cells, it induces the production of Th1 cytokines, e. g., IFN-g, and enhances cellmediated cytotoxicity in the presence of IL-12 [160, 161]. Interestingly, IL-18 in the absence of IL-12 induces the production of Th2-related cytokines from T and NK cells and basophils/mast cells. Therefore, IL-18 should be seen as a cytokine that enhances innate immunity and both Th1- and Th2-driven immune responses [162]. Interleukin-15 (IL-15) and IL-2 both utilize the IL-2Rb and the IL-2Rg chain as receptor structures; therefore, the two cytokines induce similar biological activities,

2.3 Translating Innate Immune Activation into Regulatory Circuits

such as stimulating NK, T, and B cells to proliferate, secrete cytokines, become cytotoxic, or produce antibodies [163]. Nevertheless, there are major differences between these two cytokines in terms of their cellular origins and the mechanisms controlling their synthesis and secretion. In contrast to the predominantly T cell-restricted expression of IL-2 transcripts, wide-spread constitutive expression of IL-15 mRNA occurs in a variety of tissues, including placenta, skeletal muscle, kidney, lung, heart, fibroblasts, epithelial cells, and activated monocytes. However, IL-15 protein is produced by only a limited number of cells, such as activated macrophages and epithelial cells [163]. Findings obtained from gene-targeted mice deficient in individual components of the IL-2/IL-15 system demonstrate distinct roles of IL-15 and IL-2 for the activation of the innate immune system [164, 165]. IL-15 is of pivotal importance for maintaining NK cell homeostasis, for promoting the differentiation of gdT cells, and for inducing CD8+ memory T cells [163]. IL-15 also promotes monocyte differentiation into Langerhans' cells [166]. Furthermore, some of the effects induced by IFN-a on the differentiation and/or activation of DCs seem to be mediated by IL-15, which is produced by DCs in response to IFN-a [167, 168]. In addition, IL-15 is a potent inhibitor of apoptosis [169–171]. Phagocytosis and chemokine production of neutrophils are strongly enhanced by IL-15 [172–174]Ë and expression of IL-15 in macrophages is upregulated in response to numerous signals that trigger innate immune responses, such as LPS, mycobacteria, or Toxoplasma gondii [175]. Most importantly, exogenous IL-15 improves host defense against and/or clearance of Salmonella [176], Plasmodium falciparum [177], and Cryptococcus neoformans [178]. Interleukin-12 (IL-12) is a heterodimeric pro-inflammatory cytokine formed of a 35-kDa light chain (p35) and a 40-kDa heavy chain (p40), which is produced mainly by DCs and phagocytes (monocytes/macrophages and neutrophils) in response to pathogens during infection [179–181]. In synergy with TNF and IL-18, IL-12 can induce the production of large amounts of IFN-g by NK cells, a crucial event in the early phase of the innate immune response [182, 183]. IL-12 acts not only on NK cells, but also on NKT cells and T cells, inducing proliferation, cytotoxic activity, and the differentiation of Th1 cells [182]. T cells and NK cells, in turn, enhance the production of IL-12 through IFN-g giving rise to positive feedback during Th1 immune responses. Surprisingly, both IL-4 and IL-13 are also potent IL-12 inducers [184]. Recent data argue against an absolute requirement of IL-12 for Th1 responses, since two new members of the family of IL-12p40 related heterodimeric cytokines, IL-23 and IL-27, are also involved in the maturation of Th1 responses [185, 186]. Interleukin-23 (IL-23) is a heterodimer, comprising IL-12p40 and the recently cloned IL-23-specific p19 subunit, that binds to a receptor complex composed of the IL-12Rb1 subunit and a private IL-23R [183, 185]. IL-12 and IL-23 both promote cellular immunity by inducing IFN-g production and proliferative responses in target cells. IL-23, which is produced predominantly by macrophages and dendritic cells, differs from IL-12 in the T cell subsets that it targets. Whereas IL-12 acts on naïve and effector CD4+ T cells, IL-23 preferentially acts on memory CD4+ T cells. Recent studies of IL-23 receptor expression and IL-23 overexpression in transgenic mice re-

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vealed that IL-23 plays a crucial role in autoimmune inflammation in the brain by a direct effect on macrophage function [187]. Interleukin-27 (IL-27) is a newly discovered heterodimeric cytokine that consists of an IL-12p40-related protein, EBI-3 (Epstein–Barr virus-induced gene 3), and p28, an IL-12p35-related polypeptide [183, 186]. IL-27 is a strong inducer of IFN-g production, particularly in synergy with IL-12 and IL-18. Mice deficient for EBI-3 have markedly decreased numbers of invariant NKT cells, which, in addition, produce significantly less IL-4 and IFN-g than normal mice [87]. Thus, EBI-3 also plays a critical regulatory role in the induction of Th2-type immune responses. IL-27 is produced by activated antigen-presenting cells and is mostly involved in the Th1 commitment of naive CD4+ T cells, mediated by IL-27R-dependent up-regulation of the major signal-transducing IL-12Rb2 chain [188]. In contrast, IL-23 (like IL-15) is critically important for the maintenance of immunological memory [183, 186], since IL-23R is expressed on memory, but not on naïve T cells [185, 189]. Interleukin-6 (IL-6) is a potent differentiation factor for B and T cells. IL-6 produced upon TLR ligation in DCs is a necessary requirement for T cell activation whenever CD4+CD25 regulatory T cells are present [190]. Indeed, IL-6-deficient mice were severely compromised in their induction of OVA-specific immune responses as long as CD4+CD25+ cells were present, but not when the latter were removed. Therefore, innate immune recognition by TLRs seems to control the activation of adaptive immune responses by at least two distinct mechanisms: the induction of costimulatory molecules on DCs and the production of IL-6, which renders pathogen-specific T cells refractory to the suppressive activity of CD4+CD25+ regulatory T cells. Since release of suppression is necessary for efficient T cell priming during vaccination, IL-6 is an attractive candidate for an immunopotentiating adjuvant. Indeed, when recombinant human IL-6 was used as an adjuvant in a subunit vaccine against tuberculosis consisting of the culture filtrate proteins of Mycobacterium tuberculosis emulsified in the adjuvant dimethyl-dioctadecylammonium bromide, an increased Th1 response characterized by enhanced IFN-g production and cell proliferation was observed [191]. Moreover, a specific local IgA response to heterologous antigen was increased in the lungs of mice when genes for murine IL-6 were coexpressed in recombinant vaccinia virus and inoculated intranasally [148]. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is by far the hematopoietic growth factor most widely used to augment immune responses [192]. It was initially defined as a survival and growth factor for hematopoietic progenitor cells and as a differentiation and activating factor for granulocytic and monocytic cells [193–195]. GM-CSF plays a crucial role in vitro for the differentiation of dendritic cells from precursor cells from bone marrow and peripheral blood [196]. GM-CSF is secreted by several different cell types, including fibroblasts, endothelial cells, and smooth muscle cells, after stimulation by proinflammatory cytokines such as IL-1 and TNF-a [197, 198]. LPS stimulates GM-CSF secretion from monocytes or macrophages, which could further potentiate the cellular response to LPS. It has been known since the early 1970s that endotoxin administration to mice raises the serum levels of GM-CSF [199]. The injection of GM-CSF into mice leads to increased numbers of neutrophils and cycling peritoneal macrophages [200]. It was

2.3 Translating Innate Immune Activation into Regulatory Circuits

therefore proposed that GM-CSF blockade may have some antiinflammatory effects and be employed for host protection. The immunomodulatory effects, which include the activation and augmentation of many of the functions of neutrophils, monocytes, macrophages, and dendritic cells, make this cytokine a candidate for a natural vaccine adjuvant [201, 202]. Indeed, the use of soluble GM-CSF (fusion protein), as well as gene-transduced, plasmid DNA, in vaccination-based therapies has proven to be relevant for the induction of cellular as well as humoral immune responses [203–205] therefore enhancing host defenses against a broad spectrum of invading organisms. 2.3.3.2 Th2-inducing Cytokines Interleukin-4 (IL-4) is one of a number of cytokines produced by activated mast cells and plays a predominant role in many immune and inflammatory reactions. Two biological activities have been defined in detail: IL-4 induces selective IgG1 and IgE isotype switching in B cells and promotes the differentiation of naïve CD4 T cells into a subset of T helper cells that express IL-4 as well as IL-5, IL-10, and IL-13 [206]. These cytokines and Ig isotypes are hallmarks of a Th2 response and play a protective role in immunity to extracellular pathogens [2]. Interleukin-13 (IL-13) belongs to the Th2 cytokine family and is structurally related to IL-4, belonging to the same a-helix protein family [207, 208]. The production of IL-4 and IL-13 is restricted to activated T lymphocytes (in particular, Th2 cells), mast cells, basophils, natural killer cells, and dendritic cells [208, 209]. Both cytokines are functionally active on a wide variety of cell types such as macrophages, NK cells, eosinophils, and mast cells. IL-4 and IL-13 act synergistically to promote DC maturation induced by bacterial products or proinflammatory mediators (such as TNF or CD40 ligand), as evaluated by up-regulation of MHC class II and costimulatory molecules [210]. Both cytokines show important immunosuppressive and antiinflammatory activities, which include the inhibition of proinflammatory cytokines (like IL-1, IL-6, IL-10, IL-12, and TNF-a) and chemokines (like IL-8, CCL3–5, and CCL11). In mice, a coordinate regulatory sequence element, situated in the intergenic region between the genes for IL-4 and IL-13, is critical for the optimal expression of type 2 cytokines. Mice deficient in this regulator had normal IL-4 producing mast cells, but their capacity to develop Th2 cells was completely absent in vitro and in vivo [211]. Initially, IL-13 was thought to be functionally redundant with IL-4 as a predominant antiinflammatory factor secreted during type-2 T cell responses. However, IL-13 possesses several additional properties that distinguish it from IL-4 [212]; for example, IL-13 plays a key protective role in the expulsion of helminths from the gut and, unlike IL-4, is the major contributor to granulomatous pathology in schistosomiasis [213, 214]. Therapeutic administration of IL-13 inhibitors to mice successfully prevents allergic responses and Schistosoma egg-induced lung pathology [215]. Agonist IL-13-treatment of mice, by inducing mastocytosis, eosinophilia, IgE synthesis, and mucus production, protects against ectoparasites and gastrointestinal worms and suppresses inflammation induced by Th1 cytokines [216]. Future studies are required to understand if this scenario holds true in humans.

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Interleukin-10 (IL-10) was initially described as a mouse Th2 cell product, but is now known to be produced by a wide range of cell types, including T cells, B cells, keratinocytes, monocytes/macrophages, and dendritic cells. It activates B lymphocytes and NK cells and has an inhibitory effect on T cell and macrophage activation [217]. IL-10 reduces the production of proinflammatory cytokines such as TNF-a, IL-1, IL-6, IL-12, IFN-g, and GM-CSF, but also of chemokines like IL-8 and CCL3. Furthermore, IL-10 down-regulates the production of molecules important in triggering specific immunity such as MHC class II and costimulatory molecules, e.g., CD86, thereby inhibiting antigen-presenting cell maturation [218]. Some bacterial pathogens, like mycobacteria, Bordetella pertussis, or Yersinia enterocolitica, have developed mechanisms for modulating cytokine production by host cells. These bacteria can induce the production of antiinflammtory cytokines, such as IL-10 and TGF-b, which dampen the immune response [219–221]. These immunosuppressive cytokines may also contribute to the generation of so-called regulatory T cells which down-regulate immune activation. It is noteworthy that IL-10 also is a potent stimulator of NK cell activity [222]. Cytotoxic lysis of infected cells may contribute to the clearance of pathogens and facilitate antigen acquisition from dead cells for cross-priming by activated dendritic cells, providing a link between the innate and the adaptive immune responses [223, 224]. The transforming growth factor (TGF)-b family comprises three closely related cytokines with potent immunoregulatory proteins that affect the process of inflammation and repair both positively and negatively [225]. TGF-b regulates processes such as angiogenesis, chemotaxis, fibroblast proliferation, and the controlled synthesis and degradation of matrix proteins, e. g., collagen and fibronectin [226]. Many cell types express TGF-b constitutively and produce more when stimulated. These include macrophages, T and B cells, platelets, and fibroblasts. Importantly, TGF-b is also produced by regulatory T cells, which down-modulate the function of Th1 and other immune cells. TGF-b inhibits immune functions of T and B cells by reducing TNF-a and IL-1 production in peripheral blood monocytes and directly inhibits DC maturation. In a model of murine malaria, low amounts of active TGF-b production by splenic mononuclear cells were associated with a lethal outcome, and anti-TGF-b treatment transformed a normal resolving infection into a lethal one characterized by a hyperproduction of proinflammatory cytokines [227].

2.4 Implications for Vaccine Development

Inactivated pathogens and highly purified recombinant proteins or peptides without adjuvants do not elicit efficient Th cell responses. Some novel chemical adjuvants (such as ISCOMS or SBAS4) can enhance antigen-specific T cell responses, but the magnitude is often still not sufficient [228, 229]. Therefore, it is imperative to optimize strategies that will enhance antigen presentation by inducing a favorable costimulatory milieu. Biological adjuvants do this by activating TLRs.

2.4 Implications for Vaccine Development

Live carrier vaccines in particular have the advantage of bringing a variety of strongly activating PAMPs with them. For example, the widely used tuberculosis vaccine strain, Mycobacterium bovis BCG, is a powerful inducer of T cell responses because it contains PG, LAM, lipoproteins, lipopeptides, and CpG motifs as TLR agonists, and because it can persist for some time within antigen-presenting cells, providing long-lasting T cell priming [230]. However, the particular combination of PAMPs in a given bacterial strain may also favor some undesired responses because of a significant cross-regulation of PAMPs interacting simultaneously with different pattern recognition receptors. For instance, ligation of the mannose receptor (with ManLAM, present on certain mycobacterial strains) at the same time as stimulation of TLRs (e. g., TLR4 with LPS) reduces the production of IL-12 in DCs compared to DCs stimulated with the TLR agonist only [231]. This may effectively diminish the adjuvant Th1-inducing potency of, for example, a BCG carrier vaccine. Similar mechanisms may be operative in other bacterial carrier systems, although the precise ligands and receptors involved are probably different. As a consequence, the combination of highly purified recombinant molecules (PAMPs, chemokines, and cytokines) may prove to be superior to bacterial vaccine strains, because their individual dosage and composition can be modified to fit the particular requirements of the vaccinee. In a rational approach, an optimal adjuvant should attract professional antigenpresenting cells, and this may be best achieved by mimicking the damage signals normally provided by microbial components. Thus, modified – i. e., detoxified – forms of LPS, lipopeptides, dsRNA, or CpG DNA will induce an array of chemokines, and empirical studies are now needed to precisely determine the composition of the ‘cocktail’ necessary to recruit a defined set of inflammatory cells (e. g., only imDC but no neutrophils). Adjuvants should then facilitate the entry of antigens inside the cell, e. g., by enhancing the expression of CD14, scavenger receptors, mannose receptor, etc. An optimal adjuvant should also provide signals leading to both DC maturation and T cell costimulation. Depending on the type of immune response desired, the adjuvant should induce a heavily biased cytokine environment, directing either Th1 or Th2 development. Again, a mixture of chemokines and/or cytokines, together with selected and partially modified PAMPs, may best accomplish this. The current challenge is to identify the distinct signaling pathways operative in inducing this biased repertoire of cytokines and to provide exactly the right quantity and quality of stimuli to achieve the desired skewing of adaptive responses [232]. Because many chemokines and cytokines are pleiotropic, significant interference and cross-regulation events may lead to side-effects and less-than-expected performance of such adjuvant cocktails, and thorough in vivo investigation of their safety and efficacy is needed before they can be used in humans. Finally, it is possible that no mixture of vectors or adjuvants can provide all the stimuli in the correct combination necessary to elicit optimal cellular responses. Therefore, prime–boost scenarios using different presenting/adjuvant strategies with the same antigen consecutively, may result in the strongest elicitation of antibody, lymphoproliferative, and cytotoxic T cell responses [233]. In this way, vaccination protocols would make use of the full armamentarium that the innate immune system itself deploys to shape an optimal adaptive immune response.

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190. Pasare C, Medzhitov R, Toll pathwaydependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 2003, 299, 1033–1036. 191. Leal IS, Florido M, Andersen P, Appelberg R, Interleukin-6 regulates the phenotype of the immune response to a tuberculosis subunit vaccine. Immunology 2001, 103, 375–381. 192. Hamilton JA, GM-CSF in inflammation and autoimmunity. Trends Immunol. 2002, 23, 403–408. 193. Burgess AW, Metcalf D, The nature and action of granulocyte-macrophage colony stimulating factors. Blood 1980, 56, 947–958. 194. Handman E, Burgess AW, Stimulation by granulocyte-macrophage colony-stimulating factor of Leishmania tropica killing by macrophages. J. Immunol. 1979, 122, 1134–1137. 195. Gamble JR, Elliott MJ, Jaipargas E, Lopez AF,Vadas MA, Regulation of human monocyte adherence by granulocyte-macrophage colony-stimulating factor. Proc. Natl. Acad. Sci. USA 1989, 86, 7169–7173. 196. Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, Muramatsu S, Steinman RM, Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colonystimulating factor. J. Exp. Med. 1992, 176, 1693–1702. 197. Zucali JR, Dinarello CA, Oblon DJ, Gross MA, Anderson L, Weiner RS, Interleukin 1 stimulates fibroblasts to produce granulocyte-macrophage colony-stimulating activity and prostaglandin E2. J. Clin. Invest. 1986, 77, 1857– 1863. 198. Leizer T, Cebon J, Layton JE, Hamilton JA, Cytokine regulation of colonystimulating factor production in cultured human synovial fibroblasts. I. Induction of GM-CSF and G-CSF production by interleukin-1 and tumor necrosis factor. Blood 1990, 76, 1989–1996. 199. Sheridan JW, Metcalf D, Studies on the bone marrow colony stimulating factor (CSF): relation of tissue CSF to serum CSF. J. Cell Physiol 1972, 80, 129–140.

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226. Govinden R, Bhoola KD, Genealogy, expression, and cellular function of transforming growth factor-beta. Pharmacol. Ther. 2003, 98, 257–265. 227. Omer FM, Kurtzhals JA, Riley EM, Maintaining the immunological balance in parasitic infections: a role for TGF-beta? Parasitol. Today 2000, 16, 18– 23. 228. Cox JC, Coulter AR, Adjuvants: a classification and review of their modes of action. Vaccine 1997, 15, 248–256. 229. Moingeon P, Haensler J, Lindberg A, Towards the rational design of Th1 adjuvants. Vaccine 2001, 19, 4363–4372. 230. Hess J, Schaible U, Raupach B, Kaufmann SH, Exploiting the immune system: toward new vaccines against intracellular bacteria. Adv. Immunol. 2000, 75, 1–88. 231. Nigou J, Zelle-Rieser C, Gilleron M, Thurnher M, Puzo G, Mannosylated lipoarabinomannans inhibit IL-12 production by human dendritic cells: evidence for a negative signal delivered through the mannose receptor. J. Immunol. 2001, 166, 7477–7485. 232. Moingeon P, Strategies for designing vaccines eliciting Th1 responses in humans. J. Biotechnol. 2002, 98, 189–198. 233. Ramshaw IA, Ramsay AJ, The primeboost strategy: exciting prospects for improved vaccination. Immunol. Today 2000, 21, 163–165.

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3 Adjuvant-induced Th2- and Th1-dominated Immune Responses in Vaccination James M. Brewer and Kevin G. J. Pollock

3.1 Introduction

Currently, no effective vaccines are available against diseases such as HIV [1], malaria [2], and tuberculosis [3]. Although drug treatments have been developed, these conditions continue to cause substantial morbidity and mortality worldwide, mainly because the treatments are expensive and typically of long duration. These factors combine to reduce patient compliance and, in the absence of drug monitoring, they lead to low effectiveness and may even result in multi-drug resistance. Clearly, an effective vaccine against any of these diseases would make a considerable contribution to world health. The development of vaccines has mainly focused on the identification of antigens that elicit the appropriate specific immune response to generate immunity. With increasing progress in vaccine research, this process has become increasingly refined, moving from whole organisms to extracts, toxoids, or single proteins isolated from pathogens or expressed by recombinant DNA techniques. With progress in our understanding of the structures of the T and B lymphocyte antigen receptors and their ligands, this research has ultimately moved to the smallest unit of antigen capable of eliciting adaptive immunity, the T or B cell epitope. The drive behind this increasing refinement has been the promise of vaccines with increased effectiveness, improved safety profiles, and greater ease of manufacture. However, one unwanted side effect as the refinement of these vaccines improved was a concomitant reduction of their immunogenicity. In experimental situations, this lack of immunogenicity can be easily remedied by formulation of the antigen with a vaccine adjuvant. Adjuvants have been defined as ‘agents that act nonspecifically to increase the specific immune response or responses to an antigen’ [4]. Essentially this means that adjuvants can act to increase the specific immune response to an administered antigen and should also act with a wide range of antigens. Janeway also called adjuvants ‘the immunologist’s dirty little secret' in recognition of the fact that, although a great deal of effort has gone into the design of vaccine antigens, the study of adjuvants has been largely empirical, and the mechanisms of how they exert their activity are far less studied or understood

52

3 Adjuvant-induced Th2- and Th1-dominated Immune Responses in Vaccination Tab. 3.1 Examples of Th1/Th2 induction by vaccine adjuvants . Arrows indicate adjuvant-induced changes in Th1/Th2 associated responses. Adjuvant

Antigen

Mouse strain

Subset

Criteria

Reference

FCA

OVA

129/Sv x C57BL/6) F2 BALB/c

Th1

: IFNg ; IL-5 : IFNg ; IL-5

[9]

OVA

Th1

[10]

Lipid A derivatives (MPL)

Leishmanial Ag (LeIF)

BALB/c

Th1

: IFNg ; IL-4

[11]

E. coli LT

Tetanus toxin epitope

BALB/c

Th1

: IFNg

[12]

ISCOMs

OVA

Th1

: IL-12 : IFNg : IgG2a : IFNg

[13]

HSV-2 Ag

BALB/c C57BL/6 BALB/c

OVA

BALB/c

Th1

[15]

HSV-2 Ag

BALB/c

Th1

: IgG2a : IFNg : IgG2a : IFNg

Alum/IL-12

HIV gp120

BALB/c

Th1

: IgG2a : IFNg

[16]

CpG

Tumor peptide Autoantigen (PDC)

Syngeneic B6 SJL/J

Th1 Th1

: IFN g : IFN g ; IL-4

[17] [18]

Microparticles

Mucin peptide

C57BL/6

Th1

[19]

M. tuberculosis 38 kDa Ag

C57BL/6

Th1

: IFN g ; IL-4 : IFN g

OVA

Th2

: IL-5 ; IFN g : IL-4 : IgE

[9]

OVA

(129/Sv x C57BL/6) F2 BALB/c

Influenza

BALB/c

Th2

[22]

HSV Ag

BALB/c

Th2

: IL-5 : IL-6 : IL-4 : IL-5

Cholera Toxin

OVA

BALB/c

Th2

: IL-4 ; IFN g

[24]

L3

Diphtheria Toxoid

Outbred NMRI Th2 mice

: IgG1 ; IgG2a

[25]

NISV

Alum

MF-59

Th1

Th2

[14]

[14]

[20]

[21]

[23]

3.2 The Two-Signal Model of Adjuvant-induced Immune Activation

[5]. One of the confounding issues in investigating the mechanisms of action of vaccine adjuvants has been the diverse range of unrelated substances that have adjuvant activity (Table 3.1). These include oil emulsions, natural and synthetic surfactants, mineral gels, bacterial derivatives, and some completely esoteric substances such as breadcrumbs and tapioca [6–8]. Trying to devise a unifying theory to explain this phenomenon is clearly not an easy task.

3.2 The Two-Signal Model of Adjuvant-induced Immune Activation

Induction of antigen-specific responses to protein antigens requires the activation of T helper cells. The signaling requirements for this activation have been described based on two distinct signals that are generated in the T cell and induced by APCs (Figure 3.1). Signal 1 is the cognate signal delivered to T cells by peptide/ MHC class II complexes on the surface of APCs [26]. As mentioned above, this interaction, which engenders antigen specificity on the APC-T cell interaction, has occupied a great deal of vaccine development activity. However, Signal 1 is usually inadequate for effective vaccination without the presence of a second signal, Signal 2 [26, 27]. Several studies have demonstrated that Signal 2 is induced in T cells by costimulatory molecules or cytokines expressed by APCs and is essential for T cell activation leading to immunity. In the absence of effective provision of Signal 2, T cells enter a state of functional hyporesponsiveness or anergy [27]. In terms of vaccine development, it appears that both whole organisms and adjuvants can program APCs to express costimulatory molecules to induce Signal 2 in T cells [28]. Although this model may explain the lack of immunogenicity of peptide vaccines,

Anergy

Antigen Signal 1 Signal 2 Adjuvant

APC

Activation

T cell

Fig. 3.1 A two-signal model of T cell activation. Signal 1 is provided through the interaction between the TcR with cognate peptide presented in the context of MHC molecules. Provision of Signal 1 alone results in induction of specific hyporesponsiveness or anergy in T cells [27]. A second costimulatory signal can act together with Signal 1, resulting in T cell activation. Both Signals 1 and 2 are stimulated in T cells through interaction with APCs. Although treatment of DCs with antigen alone may only be able to provide stimulus for Signal 1, the addition of an adjuvant may activate APCs, increasing the provision of both signals resulting in T cell activation [28].

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3 Adjuvant-induced Th2- and Th1-dominated Immune Responses in Vaccination

it fails to explain how vaccine adjuvants or whole organisms can actually program the ability to induce Signal 2. Several in vitro and in vivo studies have demonstrated that dendritic cells (DCs) are the most effective APC population in inducing activation and proliferation of naïve T cells. DCs are strategically situated in diverse tissues such as the epidermis and gastrointestinal mucosa, where the potential for invasion by pathogens is high [29, 30]. Advances in the culture and isolation of DCs have enhanced our understanding of their biology and demonstrated the existence of subsets of DCs, some of which represent discrete stages of differentiation [31, 32], while others belong to distinct lineages [33, 34]. Nonlymphoid DCs found in tissues are specialized for high antigen capture [35, 36]. However, for optimal T cell activation, these cells must be activated, to allow migration to T cell areas of lymph nodes and to acquire increased antigen presentation and costimulatory activity [29, 30]. The term ‘Signal 0’ has been coined to describe the activation event in DCs, and several endogenous ligands have been described that can stimulate Signal 0 [37], including proinflammatory cytokines and T cell-derived signals [29]. However, several exogenous agents also activate DCs [29, 38]. Significantly, many of these exogenous agents with the capacity to activate DCs also possess adjuvant activity [39, 40], raising the possibility that adjuvants may have a common function in activating DCs. A mechanism whereby exogenous agents could activate the immune system was proposed by Janeway [5]. He hypothesized that a series of pathogen-associated molecular patterns (PAMPs) were recognized by a set of, at the time, uncharacterized pattern-recognition receptors (PRRs). Further investigations have indeed revealed a set of evolutionarily conserved motifs expressed by some bacteria and viruses that are recognized by members of a series of mammalian receptors, called toll-like receptors (TLRs) [38, 41]. These highly conserved receptors are expressed on DCs and macrophages and mediate DC activation upon encountering the appropriate microbial stimulation (Figure 3.2). However, given that TLRs are germline encoded, one of the major arguments against PRR-PAMP interactions regulating the decision by the immune system to activate is the potentially huge number of pathogen-derived ligands that would need to be recognized [42]. Clearly, most of this research has been performed in relation to how the immune system responds to invasion by pathogens, where an evolutionary pressure exists to allow the immune system to recognize infectious non-self. If the set of ligands recognized by TLRs were to be expanded to include all adjuvants, ranging from inorganic salts to synthetic particles to plant glycosides, the range of receptors required would be unfeasibly enormous. Unsurprisingly, given the lack of any evolutionary pressure, results suggest that such an innate recognition system does not apply to nonmicrobial adjuvants [43, 44]. However, this does raise the issue of how DCs are activated in response to these adjuvants. Given the massive heterogeneity of agents with adjuvant activity, it seems more likely that adjuvants may stimulate the production of endogenously synthesized factors, which could then stimulate the activation of DCs. One of the common activities of adjuvants is the ability, in varying degrees, to stimulate local inflammation at the injection site. Indeed, it was formerly thought that the induction of local reactions

3.2 The Two-Signal Model of Adjuvant-induced Immune Activation

flagellin TLR5

LPS

CpG Mature DC TLR9 T

TLR4 Signal 0 TLR3 dsRNA

T

TLR2

proteolipids

Immature DC

Tissue

T

Lymph Node

Fig. 3.2 Recognition of PAMPs by PRRs. Direct activation of DCs has been observed with a number of microbial products (PAMPs) that are thought to act by directly binding pattern-recognition receptors (PRRs) expressed on DCs [5]. Many PAMPs have adjuvant activity, presumably as a result of this interaction [39]. Receptors for a number of PAMPs have been defined as belonging to the Toll-like receptor (TLR) family. Ligation of TLRs generates a hypothetical activation signal, Signal 0, in immature DCs, resulting in changes in endocytosis, migration, presentation, and costimulation associated with mature DCs.

following vaccination is essential for the induction of an effective immune response [45]. More recently, studies have demonstrated that necrotic cells possess ‘natural’ adjuvant activity through the ability to activate DCs. Thus, it is possible that the induction of local tissue injury and cellular necrosis observed following the administration of adjuvants might consequently induce DC activation [42]. This would tend to support a ‘danger’-based system of immune recognition of adjuvants that induce local inflammation. Significantly, one prediction from this model would be an inability to separate the degree of tissue damage induced by an adjuvant from the magnitude of the immune response it can produce. Such a relationship would clearly have significant implications for the development of safe and effective vaccine adjuvants [7]. However, several previous studies have attempted, with success, in separating adjuvant activity from toxicity, indicating that this relationship is more complex than predicted from the danger model [46–48]. Interestingly, studies with the oil emulsion adjuvant, MF-59 in vivo have demonstrated that macrophages rather than DCs are the main cell type involved in clearance of the oil depot in the tissue. However, further analysis of MF-59 induced reactions demonstrated that in the paracortical T cell areas of the draining lymph node, DCs were the predominant cell type carrying adjuvant [49]. The authors proposed that, following uptake, adjuvant-induced macrophage death was responsible for the transfer of antigen from macrophages to DCs in vivo [50]. This suggests that, rather than causing gross tissue toxicity, the ability of some adjuvants to activate immune responses may be related to their ability to

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3 Adjuvant-induced Th2- and Th1-dominated Immune Responses in Vaccination

induce cell death in specific types of cells, such as macrophages, involved in their removal.

3.3 Th1 and Th2 Induction by Vaccine Adjuvants

Aluminum compounds were originally identified as adjuvants over 70 years ago [51] and for much of this time they were unique in their widespread application to human vaccines. This was generally in the form of vaccines adsorbed to either preformed aluminum oxyhydroxide, more commonly known as aluminum hydroxide, or aluminum hydroxyphosphate (aluminum phosphate) gels, in order to overcome problems with the heterogeneity of protein aluminate precipitates [52]. The application of these adjuvants, collectively referred to here as alum, has been largely limited to certain bacterial and viral vaccines where protection against infection is known to be dependent on the generation of neutralizing antibodies [53]. When used appropriately, alum gels have enjoyed a good safety profile, which is undoubtedly the major factor in ensuring their continued use. However, alum is unlikely to fulfill the adjuvant requirements of many modern vaccines, due to the restricted range of immune responses induced by this adjuvant. Early studies demonstrated that, although alum adjuvants are very effective in promoting the expansion of humoral immune responses, including IgE production [54], they fail to stimulate cell mediated immune (CMI) responses such as delayed type hypersensitivity (DTH) [55]. More recently, the control of cellular and humoral immune responses has been attributed to mutually antagonistic subsets of CD4+ T helper (Th) lymphocytes referred to as Th1 and Th2 cells, respectively [56, 57]. Subsequent studies have demonstrated that, although alum can stimulate Th2 type responses and the production of cytokines such as IL-4 and IL-5 as well as B cell production of IgG1 and IgE, it fails to stimulate Th1 responses such as IFN g production and B cell IgG2 a secretion [6, 58]. This poses a significant problem to the continued development of vaccines, particularly given that the effectiveness of vaccines against the three diseases causing most global mortality – HIV, tuberculosis and malaria – are entirely or partially dependent on the generation of Th1 type immunity [1–3, 59]. In experimental situations, this problem can be overcome by employing one of the wide range of experimental adjuvants, in particular Freund’s complete adjuvant (FCA; [60]), that are known to induce antigen-specific Th1 responses (Table 3.1). However, the use of FCA causes inflammation, induration, or necrosis with disseminated granulomas being reported in the lungs, liver, kidneys, heart, lymph nodes, and skeletal muscles of rabbits or rats after s.c. or i.v. injection [61]. Therefore, for use in humans, adjuvants such as FCA are unacceptably toxic, inducing chronic granuloma formation and tissue destruction. In fact, if FCA is accidentally injected into humans, the affected area often has to be surgically removed to prevent longterm abscesses that result. Clearly, the lack of a clinically applicable adjuvant that induces Th1 responses poses a significant obstacle to the effective clinical application of vaccines against these diseases. Many of the developmental adjuvants listed in

3.4 Antigen Dose Effects

Table 3.1 have aimed to fulfill this role, although as yet none of them has succeeded. One reason underlying this lack of success is our poor understanding of what features of an adjuvant can lead to preferential Th1 or Th2 induction. Clearly understanding these mechanisms would facilitate the rational development of new and more effective adjuvants.

3.4 Antigen Dose Effects

Several studies have indicated that adjuvants may function by targeting antigen to APCs. This is particularly true of particulate adjuvant formulations that have been shown to engender quantitatively more effective uptake of antigen by phagocytic APCs than comparable levels of soluble antigens [62–64]. Although macrophages have been widely used as APCs in these models, recently particulate antigen has also been demonstrated to have a similar effect on DCs [44]. Furthermore, the increase in antigen internalization observed is reflected in the levels of peptide/MHC complexes detected on the surface of these cells, thus effectively increasing the dose of antigen administered [44, 64]. Antigen dose has been demonstrated to have significant effects of the generation of Th1 versus Th2 responses to antigens [65]. Historically, high doses of allergen have been used for immunotherapy and more recently, they have been associated with the preferential induction of Th1 cytokine production by CD4+ T cells from allergic donors [66]. In contrast, in vitro incubation with lower doses of antigen has been associated with Th2 responses [66]. In vitro studies with TcR transgenic CD4+ cells have also demonstrated similar effects of high and low antigen doses on the polarization of naïve T cells [67, 68]. However, other studies have demonstrated that ‘very’ high doses of antigens return the polarizing effect towards Th2 responses [68, 69]. The effect of antigen dose on Th cell cytokine production has been proposed to be mediated through alteration of the density of T cell epitopes on antigen-presenting cells that subsequently influence the magnitude of Signal 1 supplied to T cells [68]. From these studies it would then be hypothesized that low Signal 1 intensity may lead to Th2 responses, whereas high intensity signals may lead to Th1 responses. Similarly, studies with altered peptide ligands have shown that peptides that are weak T cell ligands stimulate Th2 type responses compared with strong T cell ligands [68]. These studies then indicate that particulate antigens or allergens would alter the effective dose of antigen administered and perhaps the subsequent Th1/Th2 response. Indeed many studies have associated the administration of particulate antigens with Th1 induction (Table 3.1). For example, HayGlass and colleagues [63] demonstrated that high molecular weight glutaraldehyde-polymerized ovalbumin (OVA) induces a strong Th1-like response in vivo in contrast to equivalent doses of soluble antigen alone that induced neither a Th1 or Th2 biased response. However, without a clearly defined dose and response pattern for all antigens, the generality of this mechanism in controlling Th1 and Th2 responses in vivo clearly remains ques-

57

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3 Adjuvant-induced Th2- and Th1-dominated Immune Responses in Vaccination

tionable. Significantly, it has been observed that the form of the antigen used in these studies is an important issue, because dose–response effects for soluble versus particulate antigens are quite distinct [68].

3.5 The Three-signal Model of Adjuvant-induced Immune Activation

Much of what we understand about the regulation of Th1 and Th2 responses has been derived from studies in vitro. These studies have clearly demonstrated the overriding effects of exogenous cytokines, in particular IL-12 and IL-4, in the preferential generation of Th1 and Th2 cells, respectively, from monospecific precursor cells [56, 57, 70]. Further analyses of the role of APC-derived cytokines in T cell activation have identified a third requirement, Signal 3, for optimal activation of CD4 or CD8 cells. This third signal is associated with the presence of proinflammatory cytokines such as TNF, IL-1 and IL-12 produced by APCs [71, 72]. Significantly, these cytokines have been implicated in regulating the phenotype of developing T cell responses. Therefore, as well as being involved in optimal T cell activation, Signal 3 has been suggested to act as a polarizing signal in regulating the development of Th1 or Th2 responses (Figure 3.3) [37]. In vitro studies have demonstrated that the proinflammatory cytokine IL-6 is associated with stimulating the preferential expansion of Th2 cells. This appears to be mediated first by signaling via a suppressor of cytokine signaling, (SOCS)-1, to inhibit IFNg production and therefore Th1 differentiation, and second, by activating nuclear factor of activated T cells (NFAT), leading to production of IL-4 by naï ve T cells and selective differentiation into Th2 cells [73]. However, in vivo studies with knockout mice do not support this role for IL-6 in Th2 induction. IL-6 deficient mice produced increased Th2 (IL-4, IL-5, and IL-10) and Th1 (IFNg) cytokine responses to antigens prepared in alum compared with wild-type mice, suggesting that IL-6 acted

Activation

Antigen Signal 1 Signal 2 Signal 3

Adjuvant

Th1 or Th2? APC

T cell

Fig. 3.3 A three-signal model of T cell activation. In addition to Signals 1 and 2, Signal 3 has been proposedto regulate the differentiation of T cells into Th1 and Th2 phenotypes. As with the other signals, this signal is probably induced in T cells through interactions with APCs [37]. Environmental instruction, for example by adjuvants, may determine the ability of APCs to direct T cell differentiation.

3.5 The Three-signal Model of Adjuvant-induced Immune Activation

to inhibit cytokine responses generally [74]. Similarly, La Flamme and colleagues found that the Th2 response associated with injection of eggs from Schistosoma mansoni was similar in wild-type or IL-6-deficient mice [75]. Furthermore, although IL-6deficient mice injected with antigen in FCA had unaltered Th1 responses, Th2 responses were increased, suggesting that, in a Th1 polarizing condition, IL-6 may actually act to inhibit Th2 induction [74]. The role of IL-1 and TNF in polarizing Th1 and Th2 responses also remains unclear. Early studies demonstrated that IL-1 had differential effects on cloned Th1 and Th2 cells, with only Th2 clones expressing IL-1R and requiring IL-1 as a growth factor [76]. Similarly, antigen-specific T cell proliferation by lymph node cells isolated from mice primed in vivo with antigen prepared in alum but not FCA could be inhibited by IL-1-neutralising antibodies [58]. In vivo studies have demonstrated that, although both IL-1 and TNF have limited adjuvant activity, they do not appear to cause clear polarization of the resulting T helper cell response [77]. Similarly, immunization of IL-1RI knockout mice with KLH precipitated with alum supplemented with Corynebacterium parvum in FCA did not result in significant changes in the Th1/Th2 response compared with wild-type mice [78]. Previous studies demonstrated that TNFR1-deficient mice have normal Th1/Th2 profiles in response to immunization with antigen prepared in alum. However, following immunization with the same antigen prepared in FCA, TNFR1-deficient mice had greatly increased Th1 responses compared with wild-type mice, suggesting that TNF may regulate adjuvant-induced Th1 but not Th2 responses via TNFR1-mediated signaling [74]. Of the proinflammatory cytokines capable of delivering signal 3 to T cells, only IL-12 has a clear effect on polarization of the developing T cell response both in vitro and in vivo [79]. IL-12 is produced by DCs macrophages and B lymphocytes and stimulates production of several cytokines, in particular, IFNg from both T and NK cells, thus driving Th1 development [80]. Early studies demonstrated that administration of exogenous IL-12 had adjuvant activity in several models, including vaccination against Leishmania infection, where Th1 induction is essential for resistance [81]. One mechanistic explanation for this observation involves the previously described ability of IL-4 to down-regulate IL-12 receptor b2 chain (IL-12R b2) expression on T cells, therefore inhibiting the signaling pathway leading to Th1 induction [82]. Studies have shown that the effects of IL-4 on IL-12Rb2 expression can be inhibited during the early stages (before 24 h) of the developing immune responses by IL-12 itself [83, 84] (Figure 3.4). IL-12 plays an important role in the activity of many vaccine adjuvants. Studies in knockout mice demonstrate that IL-12 mediates Th1 induction by FCA [85], although some residual IL-12-independent IFN g production was noted in these mice. Similarly, both ISCOM and NISV adjuvant activity is dependent on IL-12 [13, 86]. In contrast, the activity of the mucosal adjuvant CT has been shown to be independent of IL-12 [87], although this may be due to the adjuvant activity of CT being largely mediated through Th2 cells [88]. Determining how IL-12 maintains Th1 cells and consequently cell-mediated immunity will provide new insights into controlling the immune response and thus may influence the design of new vaccines and immunotherapies.

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A.

IL-12Rβ β2

-

IL-12Rβ β1

IL-12Rβ β1

ThP γc

IL-12

IL-12Rβ β2

IL-12Rβ β1

ThP γc

ThP

Th2

IL-4

IL-4

IL-4Rα α

IL-4

B.

IL-12Rβ β1

IL-4Rα α

IL-4

IL-12

IL-12Rβ β2

-

IL-12Rβ β1

IL-12Rβ β2

IL-12Rβ β1

IFNγγ

ThP

γc

Th1

IFNγγ

IL-4Rα α

IL-4

Fig. 3.4 Regulation of IL-12R expression by IL-4, IL-12, and IFNg. A. IL-4 rapidly down-regulates IL-12Rb2 on naive T helper cells (ThP), rendering them unresponsive to further IL-12 effects and ultimately resulting in Th2 generation [82]. B. Early exposure to IL-12 can inhibit the effects of IL-4 on IL-12Rb2 expression, allowing IL-12 effects to override, resulting in Th1 development [83]. Endogenous or exogenous IFN g further inhibits the effect of IL-4 and facilitates polarization to Th1[82].

As mentioned above, APC activation, including proinflammatory cytokine production in response to pathogen-associated molecules, is predominantly regulated by direct recognition via TLRs [38, 41, 89]. Signaling through TLRs is mediated through a common intracellular domain on the TLR, similar to that found in the IL-1 receptor, called a TIR domain. Signaling via this pathway involves the adaptor protein MyD88 and results in activation of NF-kB transcription factors and MAP kinases. Interestingly, microarray analysis indicates that the same core set of NF-kBdependent genes are activated in DCs by diverse pathogens such as influenza, E. coli, and Candida albicans and, significantly, their components such as dsRNA, LPS, and mannan had similar effects to whole pathogens [38, 90]. Activation of NFkB then acts to induce the production of proinflammatory cytokines such as IL-1, TNF and IL-12. The general tendency of proinflammatory cytokines to promote Th1 induction may reflect the differing transcription requirements for the production of Th1 and Th2 signature cytokines. In Th1 cells, IFN g production has been shown to be regulated by both NFAT and NF-kB transcription factors [70]. In contrast, NF- kB signaling does not appear to be associated with Th2 cell IL-4 production, although studies have shown that IL-1-induced NF- kB activation can facilitate the proliferation of Th2 cells [91]. It therefore appears that signaling through TLRs

3.6 Th2 Induction by Adjuvants

would result in a Th1 biased response either directly or via induction of proinflammatory cytokine production by APCs. This hypothesis has been tested in MyD88deficient mice, which fail to signal through their TLRs. In these mice, Th1 induction by FCA is significantly reduced, while Th2 responses to antigen prepared in alum is unaffected in comparison with wild-type mice [43]. Again, this reinforces the position made above, that, although it makes sense for the immune response to have evolved to recognize pathogen-associated motifs, it makes no sense to suggest that it would recognize an inorganic mineral gel. However, it still begs the question, ‘How are Th2 responses induced?’

3.6 Th2 Induction by Adjuvants

As mentioned above, much of what we understand regarding the regulation of the induction of Th2 responses has been derived in vitro. These studies have demonstrated the central role played by IL-4 in controlling Th2 responses [56] and have revealed the transcriptional events leading to induction of Th2 cells from monospecific precursors in considerable detail [57, 70]. Despite the detail with which we understand Th2 differentiation in vitro, it is less clear how these events occur in tissues – this is mainly due to the far greater level of complexity of the developing immune system in vivo. This is a particular problem in Th2 responses, since the major source of IL-4 in vivo is Th2 cells themselves, which produce IL-4 as part of an autocrine growth loop [56]. This anomaly has piqued interest in identifying a population of cells that could act as ‘prime movers’ to produce an initial source of IL-4 to subsequently support Th2 responses. As described above, the development of mice with disrupted cytokine genes has made a significant contribution to the study of factors involved in regulation of Th1 and Th2 immune responses in vivo [57]. Although some studies have been largely confirmatory, for example, those on the role of IL-12 in Th1 cell differentiation [85], clear evidence to confirm the role of IL-4 in induction of Th2 responses in vivo has been less forthcoming. For example, disruption of IL-4 or IL-4 mediated signaling has been shown to result in a reduced ability to produce Th2-type responses upon infection with Leishmania major or Nippostrongylus brasiliensis [92, 93]. By extrapolation from these studies, we may anticipate that alum may have no, or reduced, adjuvant activity in IL-4-deficient mice. However, alum not only continues to have effective adjuvant activity in these mice, but it also induces Th2 cells producing IL-5 at similar levels as in wild-type mice [9]. Similarly, mice immunized with rabbit anti-mouse IgD develop normal IL-4-producing Th2 cells in the absence of IL-4 signaling [94]. The Th2 response induced by alum in the absence of IL-4 could be due to compensatory effects of IL-13, which shares a common signaling pathway with IL-4 via binding to IL-4Ra chain and subsequent phosphorylation of signal transducer and activator of transcription (Stat) 6 [95–97]. However, results in Stat 6 or IL-4Ra deficient mice demonstrated that alum could induce levels of antigen-specific IL-4 and IL-5 production that were at least equivalent to those found in wild-type mice [21]. Although

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these results clearly indicate that alum can stimulate Th2 development independently of either IL-4 or IL-13, it remains unclear how Th2 responses to a variety of stimuli, including alum adjuvants, can be induced in vivo. Interestingly, although Th2 cytokine responses were normal in IL-4Ra and Stat 6 deficient mice these studies clearly showed that alum could induce Th1 responses under these circumstances [21]. This suggests that, in normal immunocompetent animals, alum-induced IL-4, rather than inducing Th2 associated responses, primarily functions to inhibit the development of Th1-type responses. A likely mechanism for this inhibition, involving IL-4-mediated down-regulation of IL-12Rb2 expression and consequently IL-12 mediated signaling, is described above (Figure 3.4). Significantly, previous studies have shown that coadsorption of IL-12 on alum gel elicits a switch in the alum-induced response from Th2 to Th1 [16, 98]. This suggests that adsorption of IL-12 onto alum not only acts to positively promote Th1 responses, but can also counteract the actions of IL-4 in inhibiting Th1 induction, presumably through IL-12 inhibition of IL-4 mediated IL-12Rb2 down-regulation (Figure 3.4). Indeed, the studies in IL-4 and IL-4 signaling knockout mice suggest that the latter mechanism may be more important in inducing Th1 responses. These observations illustrate that adjuvants such as alum that are currently in use have the potential to be improved and consequently, that understanding the mechanism of action of alum would facilitate the rational design of new, safe adjuvants. The studies described above also imply that looking for an early source of IL-4 in vivo, although not relevant to the induction of Th2 responses, is clearly important in the inhibition of Th1 induction. Several sources of early IL-4 production have been proposed, including cells of the adaptive and innate immune responses. These include, gdT cells, NK1.1+CD4+ T cells, NK1.1-CD4+ T cells, eosinophils, and cells of the mast cell/basophil lineage (reviewed in [56]). Therefore, it is possible that adjuvants in general, and alum in particular, may be able to influence the Th1/Th2 balance of an immune response through effects on any of these cell populations. In fact, histological analysis of injection sites after administration of antigen prepared in adjuvant has revealed that, unlike Freund’s adjuvant which induces a predominantly macrophage infiltrate, the antigen depot induced by alum adjuvant contains significant levels of eosinophils as well as macrophages [99]. However, it is not clear from studies of adjuvants or pathogen infection models, exactly how cellular events at the site of injection can regulate the development of T cell responses in the local lymph nodes. Obstacles to these effects are, first, that cytokines have generally very short effector ranges in vivo [100], and second, that unlike memory or effector T cells that are already Th1/ Th2 committed, naïve T cells lack the ability to migrate into tissues [101]. In this context it is significant that CCR2-deficient mice, which fail to recruit monocytes to the site of M. tuberculosis infection, are incapable of mounting a Th1 response in vivo [102]. This suggests that cells migrating to the site of infection, under the stimulus of CCR2 signaling, may be able to carry information to the draining lymph nodes to instruct the developing T cell response – a event that has been called an ‘innate bridge’ [102]. After injection of alum, it is therefore possible that the cells that could be affected by and respond to early IL-4 production may not necessarily be T cells themselves but an intermediary cell acting as an innate bridge [37] (Figure 3.5).

3.7 Differential Activation of DCs

Th1

IL-12

IL-12 Th1 inducing stimuli

ThP DC1 CD40-CD40L

IL-10

IL-10

ThP Th2 inducing stimuli

DC2 CD40-CD40L

Tissue

Th2 Lymph Node

Fig. 3.5 Environmental conditioning of DCs and recall in the lymph node. How environmental conditions in the periphery are communicated to the lymph node to regulate T cell development remains unclear, although it is highly likely that DCs play a significant role. Studies have demonstrated that Th1- or Th2-inducing stimuli can induce DCs to produce distinct cytokine profiles and may result in the DCs being conditioned to become Th1-inducing (DC1) or Th2-inducing (DC2) [37, 89]. It has subsequently been proposed that in the lymph node, away from these environmental conditions, DCs recall their experience by reproducing DC1 or DC2 cytokine profiles upon stimulation via CD40-CD40L interactions with T cells [107].

3.7 Differential Activation of DCs

DCs are highly likely to be involved in forming an innate bridge through their recognition of exogenous agents in tissues and subsequent migration to lymph nodes to activate naïve T cells. For DCs to fulfill this role, their differentiation from common precursors into Th1- or Th2-inducing APCs would have to show plasticity [37, 89]. Although several studies have isolated Th1-inducing (DC1) and Th2-inducing DCs (DC2) from lymphoid tissue ex vivo, these cells appear to belong to distinct lineages rather than arise from a common precursor [33, 34]. Nevertheless, further studies have demonstrated that endogenous agents such as IL-12 and/or IFNg can program precursor cells to differentiate into DC1 [32, 103, 104], whereas treatment with IL-4 [103], IL-10 [105], or PgE2 [104] can cause differentiation into DC2. Significantly, exogenous microbial stimuli have also been demonstrated to polarize DCs into DC1 or DC2 [106]. This differential activation of DCs depends on the

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microbial stimulus to induce production of either IL-12 (DC1) or IL-10 (DC2) by the DC [107]. DC1-inducing stimuli include a variety of agents that are known TLR ligands [38, 106]. Similarly, many microbial agents have been defined that induce maturation towards DC2. These include ES-62 produced by the filarial nematode Acanthocheilonema viteae [31], soluble egg antigen (SEA) from Schistosoma mansoni, Cholera toxin [106], and heat-killed yeasts [107]. However, in contrast to DC1-inducing stimuli, receptors for these microbial agents have not been clearly defined. This has led speculation that DC2-inducing receptors do not exist and that perhaps induction of Th2 responses occurs due to other mechanisms entirely independent of conventional PAMP/PRR interactions [38]. More recently, this proposal has been challenged by the observation that a lyso-phosphatidylserine unique to S. mansoni eggs and adult worms can interact with TLR2, resulting in Th2 polarization [108]. One temporal limitation of the ‘innate bridge’ concept is that, after encounter with exogenous agents in the periphery, the stimulus to produce polarizing cytokines is lost during transit into the lymphatics. One solution to this problem was suggested by the group of Reis e Sousa, who propose that the experience of interactions with pathogens in the periphery is recalled by DC in the draining lymph node after CD40CD154 interactions with T cells. This interaction causes the DC to secrete high levels of IL-12 or IL-10, depending on the experience of the DC in the periphery (Figure 3.5) [107]. These results suggest a model in which all DCs, irrespective of subset, can behave as flexible APCs. Further studies in this area should therefore allow the rational manipulation of DCs to induce appropriate Th cell responses by using existing or new adjuvants.

3.8 Inappropriate Th1/Th2 Responses to Vaccines

Modulation of the immune response for protection against infectious organisms has resulted in unintentional disease exacerbation, most notably in clinical trials of a pediatric RSV vaccine in the 1960s [109]. However, in murine models with parasite pathogens such as Schistosoma and Leishmania, the consequences of inappropriate Th1 or Th2 induction have been apparent. Th1 polarization with SEA from S. mansoni emulsified in FCA produced a vigorous Th1-mediated inflammatory response, but this resulted in unforeseen exacerbation of immunopathology compared with immunization with SEA alone and ultimately resulted in death [110]. Similarly, in Leishmania infection, where it has been recognized that Th1 responses are protective and Th2 responses exacerbate disease, only use of appropriate Th1-inducing adjuvants can lead to protection [93, 111]. One of the most effective adjuvants for Th1 induction in this model is IL-12 [98]; however, it is clear that IL-12 may also participate in a severe inflammatory response, resulting in unwanted tissue damage [112]. Thus, caution should be exercised when attempting to design vaccines, which must be protective as well as safe.

3.10 Human Th1 Vaccines

3.9 Human Th2 vaccines

To a large extent, the development of new-generation Th2 vaccines has focused on improving the safety, efficacy, and public acceptability of existing vaccines. MF-59 stimulates potent Th2 responses to a variety of vaccine antigens, with concomitant production of IL-4 and IL-5 [23]. Furthermore, this adjuvant has been combined with subunit influenza antigens and is a more effective adjuvant than alum [113]. Importantly, the addition of MF-59 to subunit influenza vaccines significantly enhances the immune response in elderly patients without causing clinically important changes in the safety profile of the influenza vaccine [113, 114]. As a result of this impressive safety profile, MF-59 has also recently been clinically tested in pediatric vaccines [115]. Other adjuvants used for Th2 potentiation in clinical trials include chitosan, which stimulates mucosal immunity to a formulation of the nontoxic mutant of diphtheria toxin, with subsequent enhancement of local and systemic antibody after nasal challenge [116]. Nevertheless, alum remains the main adjuvant approved for human use. Furthermore, the rational application of immunomodulators to adjuvants such as alum that are in current clinical use suggests potential strategies to generate more effective Th2 [117] or indeed Th1 [16] responses.

3.10 Human Th1 Vaccines

Few vaccines induce effective Th1 responses in humans. Those vaccines that do, for example, BCG [118] and whole cell pertussis vaccines [119], have been associated with local or systemic toxicity. Due to its ability to induce Th1 responses and its continued use in humans, BCG vaccines have been exploited as vaccine carriers, for example, in HIV/AIDS, tuberculosis, and malaria vaccines [120]. This approach uses recombinant BCG expressing the vaccine to generate immunity as part of the immune response to the organism. A simpler approach has been to incorporate BCG as an adjuvant as part of the vaccine [121]. However, although this approach appears to be capable of inducing Leishmania-specific Th1 responses, the duration of these responses was limited and the vaccine failed to protect volunteers [121]. This suggests that providing long-term Th1 immunity may require antigen persistence together with a persisting Th1 stimulus such as IL-12 [122]. However, as mentioned above, administration of IL-12 to humans is not without problems in itself, as clinical trials in cancer immunotherapy have shown [123]. Nevertheless, research into various TLR ligands continues to suggest novel adjuvants for Th1 responses that may find use in human vaccines. For example, bacterial lipoproteins (BLPs) such as the 19 kDa secreted lipoprotein from M. tuberculosis and synthetic lipopeptides comprising the active portion of these ligands are effective adjuvants in vitro [124]. These compounds appear to work via TLR2 on APCs to induce costimulatory molecule expression and inflammatory cytokine secretion [124]. Sig-

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nificantly, BLPs have been found to be safe in human trials, although they were unable to stimulate sufficient HIV-specific immunity in a therapeutic vaccine trial [125]. Nevertheless, further clinical trials have demonstrated that the ability of BLPs to stimulate Th1 responses can be enhanced by the addition of alum and QS21 as coadjuvants [126]. As with IL-12, an unwanted consequence of the adjuvant activity of TLR ligands may be the resulting immunopathology that could occur, although the clinical data above argue against this. However, as with other Th1 adjuvants, questions of efficacy remain and the transition from laboratory to clinical vaccination remains elusive. Nevertheless, recent insights into immune mechanisms will continue to pave the way for more rational design of Th1 adjuvants.

3.11 Conclusion

There is clearly a major demand for vaccines capable of eliciting strong Th1-type, cellular immune responses for effective vaccination against, amongst other diseases, HIV/AIDS, tuberculosis, and malaria. Although vaccines based on live, attenuated pathogens will continue to have an impact in the future of vaccination, regulatory conditions dictate that most vaccines will preferably be based on nonliving or even nonreplicating vaccines. Furthermore, the increasing prevalence of immunosuppressed individuals makes the use of live vaccines less likely, due to their potential virulence. For nonliving vaccines to be effective inducers of Th1 responses, formulation with an adjuvant is essential. The types of adjuvant that induce potent Th1 responses are likely to be intimately connected with the induction of inflammatory responses, with consequent safety concerns. Furthermore, the maintenance of Th1 immunity may require persistence of inflammation, as has been seen in Leishmania vaccines, which raises further safety implications. It is possible that environmental exposure to infectious agents after vaccination could provide this long-term stimulus, although not if the complete eradication of these diseases is an objective. Clearly, the demand for new, safe, and effective Th1-stimulating adjuvants has never been so central to successful vaccination. Continuing to explore the immunological mechanisms that regulate Th1 immunity is the only possible route to identifying potential candidates to fulfill this demand in a rational and informed fashion.

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79. Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Ann Rev Immunol 1995, 13, 251–276. 80. Scott P. IL-12: Initiation cytokine for cell-mediated immunity. Science 1993, 260, 496–497. 81. Afonso LCC, Scharton TM,Vieira LQ, et al. The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 1994, 263, 235–237. 82. Gollob JA, Kawasaki H, Ritz J. Interferon-gamma and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression. Eur J Immunol 1997, 27, 647– 652. 83. Ouyang W, Ranganath SH,Weindel K, et al. Inhibition of Th1 development mediated by GATA-3 through an IL-4independent mechanism. Immunity 1998, 9, 745–755. 84. Himmelrich H, Parra-Lopez C, Tacchini-Cottier F, et al. The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major downregulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12. J Immunol 1998, 161, 6156–6163. 85. Magram J, Connaughton SE, Warrier RR, et al. IL-12-deficient mice are defective in IFN gamma production and type 1 cytokine responses. Immunity 1996, 4, 471–481. 86. Brewer JM, Tetley L, Richmond J, et al. Lipid vesicle size determines the Th1 or Th2 response to entrapped antigen. J Immunol 1998, 161, 4000–4007. 87. Grdic D, Smith R, Donachie A, et al. The mucosal adjuvant effects of cholera toxin and immune-stimulating complexes differ in their requirement for IL-12, indicating different pathways of action. Eur J Immunol 1999, 29, 1774– 1784. 88. Lycke N. The mechanism of cholera toxin adjuvanticity. Res Immunol 1997, 148, 504–520. 89. Reis e Sousa C. Dendritic cells as sen-

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4 Memory Alexander Ploss and Eric G. Pamer

4.1 Introduction

Immunologic memory, manifested as specific resistance to a second encounter with a pathogen, has been appreciated for more than 2000 years. In his book The History of the Peleponnesian War, Thucydides describes how a plague – the true pathogen is unknown – swept through Athens in 431 BCE [1]. Thucydides made several observations on the plague, including the fact that those who survived an infection were ‘never attacked twice – never at least fatally’. In other words, the few individuals surviving a first episode of plague did not develop disease when similar epidemics recurred. Although many similar examples of immunity following survival of infection were appreciated over the millennia, intentional exploitation of the mammalian immune system to prevent disease is a relatively recent activity. Between 1718 and 1721, Lady Mary Wortley Montagu, the wife of the British Ambassador to Constantinople, described in her letters a local practice among the Turks for preventing smallpox [2]. Turkish women infected healthy individuals intravenously with a small inoculum of matter collected from smallpox lesions, which caused a mild form of the disease, but rendered the person immune to severe smallpox. Almost 80 years later, in 1796, the British physician Edward Jenner reported successful protection of humans against smallpox infection by vaccination with cowpox virus [3]. Vaccination, a term derived from vacca (Latin for cow), became widespread upon confirmation of Jenner's results [4]. Nearly a century later, Louis Pasteur, while attempting to demonstrate the bacterial cause of chicken cholera, discovered that bacteria grown in culture lose their virulence but maintain the ability to elicit protective immunity. His discovery that chickens immunized with attenuated bacteria developed only mild disease but became resistant to infection with fully virulent bacteria of the same species validated Jenner's concept of immunization [5]. Thus, in the late 19th century, immunization with attenuated vaccines was developed for a number of diseases and applied in Europe and America. However, many vaccines, particularly those focusing on syphilis, tuberculosis, and salmonellosis, were unsuccessful. Neither Pasteur nor Robert Koch, who outlined

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the parameters required to characterize an infectious disease [6], could explain mechanisms of infectious disease and immune defense. Groundbreaking work by Emil von Behring and Shibasaburo Kitasato led to the discovery of antibodies, opening the field of ‘humoral’ immunity. Serum transfer from animals infected with diphtheria into healthy animals and subsequent infection with the pathogen made recipient animals resistant to the normally lethal inoculum. This method of conferring ‘passive immunity’ provided the first real opportunity to define immune mechanisms. The nature of the antitoxin antibody was recognized by von Behring and Kitasato to be diphtheria-specific, because it could not confer protection to any other disease [7]. The intense focus on serum-mediated immunity relegated the demonstration by Metchnikoff of cell-mediated antimicrobial defense to the back burner, and it took many decades for this aspect of protective immunity to gain a position at center stage [8]. Indeed, a series of seminal papers by Murphy, including experiments demonstrating the ‘role of lymphoid tissue in the resistance to experimental tuberculosis in mice’ [9], remained widely unrecognized [10]. Not until the 1940s did Medawar and Gibson redirect attention to cell-mediated inflammatory processes [11]. Medawar and Gibson observed that skin graft rejection correlates with cellular infiltration [12, 13]. The pioneering experiments of Landsteiner and Chase showed that resistance to tuberculosis could be established by passive transfer of mononuclear cells [14, 15]. Additional studies by Mackaness and colleagues demonstrated the critical role of cellular immunity in certain bacterial infections [16]. Subsequently, Gowans demonstrated that adaptive immunity is mediated by recirculating, long-lived lymphocytes [17, 18]. The thymic origin of these lymphocytes, which were therefore termed T cells, was recognized, and they were shown to play a central role in mediating resistance to intracellular pathogens [19, 20]. The role of CD4+ and CD8+ T lymphocyte subsets in adoptive protection against intracellular infection was elucidated shortly thereafter [21–23]. Both CD4+ and CD8+ T cells provide long-term protective immunity against infections, and much has been learned in the last decade about T cellmediated immunity. This chapter reviews our current understanding of immunologic memory, focusing on more recent advances.

4.2 Characteristics of Memory Cells

Memory T and B lymphocytes mediate long-term protective immunity to infectious pathogens. A characteristic feature of memory cells is their long-term survival in the absence of reexposure to the pathogen. Additional characteristics of memory T cells are their increased frequency and enhanced responsiveness to antigen. Thus, upon reexposure to antigen following secondary infection, memory cells expand more rapidly and vigorously and acquire effector functions faster than naïve T cells [24–26]. Indeed, lower concentrations of antigen [27] and less costimulation [28] are required for memory T cell activation. The physiologic basis for these hallmarks of immune memory has become clearer in recent years.

4.3 CD8+ T Cell Memory

4.3 CD8+ T Cell Memory 4.3.1 Phenotyping Memory CD8+ T Cells

Memory T cell subsets are commonly distinguished from naïve cells on the basis of surface marker expression. A number of surface molecules associated with memory and naïve CD8+ T cells are involved in cell adhesion and chemotaxis, consistent with distinct trafficking of different T cell populations. The expression of the adhesion molecules CD44 and CD62L has been used to define memory cells. Although CD44lo and CD62Lhi are naive cells, they become CD44hi and CD62Llo upon activation. To make matters more complicated, however, CD62L is reexpressed on a fraction of memory cells, and expression of CD44 varies among different mouse strains [29]. Recent studies have demonstrated that memory CD8+ T cells can be divided into effector memory (CD62Llow CCR7-) and central memory (CD62Lhi CCR7+) populations, on the basis of L-selectin (CD62L) and CC-chemokine receptor 7 (CCR7) expression [30, 31]. The expression of tyrosine phosphatase CD45 isoforms is also used to distinguish between T cell differentiation stages. The CD45RB isoform is generally detected on naive cells, but memory cells express the CD45RA and CD45RO isoforms. However, because CD45 isoform expression on memory versus naïve T cells is complex and can change with nonspecific activation and cytokine treatment, CD45 is a relatively unreliable marker for memory T cell definition [32]. The IL-2 receptor a chain (CD25) and CD69 are rapidly expressed by T cells upon encountering cognate antigen, but their expression is not sustained on memory T cells. Some memory T cells, however, express the IL-2 receptor b beta chain (CD122), a component common to both IL-2 and IL-15 receptors, the latter cytokine being essential for memory CD8+ T cell maintenance [33, 34]. Ly-6C has also been used as a marker to distinguish effector from memory T cells. Low and intermediate expression of this marker is characteristic of effector T cells, but it is highly expressed on memory T cells [27, 35–37]. More recently, classification of human virus-specific T cells into naïve, effector, and memory cells on the basis of CD27 and CD28 expression was described [38]. Human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and hepatitis C virus (HCV) specific T cells, although similar during primary infection, give rise to distinct memory T cell populations during chronic infection, which are characterized by differential expression of various surface antigens and effector molecules. Naïve CD8+ T cells have CD27+ CD28+ and express CCR7. Early and intermediate effector T cells downregulate CD28, CCR7, and CD45RA and express cytotoxic factors such as perforin, granzyme A, and GMP-17 (a marker for cytotoxic granules and lysosomes). Memory cells appear to lose the CD28, CD27, and CCR7 markers and maintain CD45 expression at low levels. They exhibit a greater cytotoxic potential than nonprimed cells, and their telomeres are shortened. In this study, T cell populations responding to persistent viral infections were more diverse

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than expected. EBV and HCV promoted the development of early effector memory T cells, but HIV induced intermediate and CMV induced terminally differentiated memory T cells. In aggregate, recent studies demonstrate the remarkable diversity within memory T cell populations and illustrate the importance of integrating phenotypic and functional characteristics when assessing memory T cells. 4.3.2 Enhanced Responsiveness of Memory CD8 + T cells: Potential Mechanisms

Enhanced sensitivity of memory T cells for cognate antigen likely results at least in part from more rapid signal transduction. Priming of naïve T cells leads to association of the tyrosine kinase Lck with the CD8 coreceptor, thereby enhancing TCR signaling. The association between Lck and CD8 is maintained in memory cells and contributes to the lower stringency for activation of effector and memory cells [39]. Additional qualitative differences have been reported further downstream in the signaling cascade, involving the mitogen-activated protein (MAP) kinases ERK1 and 2 (extracellular signal-regulated kinase), which activate transcription factors essential for T cell activation. In contrast to naïve T cells, memory cells show an increased ability to phosphorylate ERK1 and ERK2, thereby accelerating signal propagation [40]. In addition to enhancing the rate of T cell activation, regulation of signaling molecules can also influence activation-induced differentiation of memory cells. For example, transgenic mice expressing a partially calcium-independent mutant of the calcium/calmodulin kinase II (CaMKII) gB show an increase in the number of T cells in secondary lymphoid organs with a memory phenotype, suggesting that this kinase is important for the development of memory [41]. Lymphocyte differentiation is associated with programmed alterations in gene expression, which is largely regulated by structural changes in chromatin [42]. Distinct gene expression profiles have been reported for memory cells [40]. For example, mRNA levels for several cytokines including RANTES [43], interferon-g (IFN-g), and cytotoxic molecules such as perforin and granzyme B [44–48] are elevated in memory T cells. Transcripts for these genes are rapidly translated in memory T cells following TCR stimulation, circumventing the time required for transcriptional activation. 4.3.3 Generation of Memory CD8+ T Cells

Studies of CD8+ T cell memory have been greatly facilitated by the increasing spectrum of mice with targeted genetic deletions. For example, mice deficient in CD27, a member of the TRAF-linked tumor necrosis factor (TNF) receptor family that plays a costimulatory role during T cell priming, have a markedly diminished virus-specific memory CD8+ T cell response [49]. On the other hand, mice lacking the CD28 costimulatory molecule, although deprived of CD86-mediated helper signals, mount nearly normal memory CD8+ T cell responses in response to lymphocytic choriomeningitis virus (LCMV) or Listeria monocytogenes infection [50, 51], supporting the

4.3 CD8+ T Cell Memory

notion that the costimulatory networks contain built-in redundancies that can compensate for the absence of CD28. However, costimulatory requirements for memory T cell generation can vary significantly, because different pathogens create distinct inflammatory milieus. For example, induction of long-term immunity to influenza virus infection, in contrast to LCMV and Listeria monocytogenes infection, requires CD28 signaling [52]. 4-1BB, a TNF receptor family member, is another costimulatory molecule that has an impact on memory T cell formation. Mice lacking 4-1BB ligand have diminished late expansion of virus-specific effector and memory CD8+ T cells, suggesting that 4-1BB signals play a role in sustaining T cell activation and memory generation [53, 54]. TNFR-associated factor 2 (TRAF2) is an adapter protein, which associates with the cytoplasmic tail of several TNF receptor family members including CD27, CD40, OX40, and 4-1BB and thereby links signals to downstream pathways. Hence, the absence of TRAF2 is likely to abrogate signaling through multiple costimulatory pathways. However, studies using dominant negative forms of TRAF2 demonstrated that in, response to influenza virus infection, only secondary CD4+ and CD8+ T cell expansion is decreased, while the primary expansion is not affected [55]. Cytokines are also implicated in the generation of antigen-specific memory T cells. Infection of IL-15- or IL-15Ra-deficient mice with LCMV generates a potent primary response, but the memory pool decreases gradually over time, leading to impaired secondary CD8+ T cell expansion [56]. This demonstrates differential IL-15 requirements for naïve and memory CD8+ T cells [33]. In the absence of IL-15 signaling, the memory response is also diminished following vesicular stomatitis virus (VSV) infection; however, the primary CD8+ T cell expansion also appeared to be impaired [57]. These studies suggest that in some circumstances IL-15 may play a role in the generation of memory CD8 T cells. CD4+ T cells have also been implicated in the generation of CD8+ T cell memory; however, their importance varies with the type of infection. In the setting of LCMV infection, CD4+ T cells are not necessary for primary CD8+ T cell responses, but they are indispensable for long term CD8+ T cell memory [58]. Remarkably, CD4+ T cells contribute to CD8+ T cell memory at the time of initial priming, because activation of memory CD8+ T cells occurs independently of CD4+ T cell help. In contrast to what occurs during viral infections, generation of memory CD8+ T cells during bacterial infections is less dependent on CD4+ T cells. For example, although mice lacking the class II transactivator (CIITA) have diminished numbers of CD4+ T cells, they generate memory CD8+ T cells following infection with Listeria monocytogenes [59]. Consistent with this result, CD4+ T cell-depleted, CD4-/-, and MHC class II-/mice infected with Listeria monocytogenes mount protective recall responses following secondary challenge [60]. An important mechanism by which CD4+ T cells provide help to CD8+ T cell is through CD40-CD154 interactions [61, 62]. Antigen-presenting cells (APCs) were originally thought to be the mediators for these signals [63]. Recent data demonstrates, however, that activated CD8+ T cells that express CD40 may receive direct help for CD4+ T cells expressing CD154 [64]. Administration of agonistic anti-CD40 monoclonal antibodies greatly enhances the protective potential of adoptively trans-

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ferred L. monocytogenes-specific CD8 T cells, providing further support for the role of CD40 in memory T cell generation and/or maintenance [65]. Signals transmitted from CD4+ T cells are not uniformly stimulatory. In recent years, CD4+ CD25+ T regulatory cells have been implicated in the control of autoimmunity [66]. During infection with Listeria monocytogenes, CD4+ CD25+ T regulatory cells can suppress memory CD8+ T cell expansion [67]. Thus, CD4+ T cells play both positive and negative roles in the control of CD8+ T cell memory. Primary infection with L. monocytogenes induces memory T cells specific for multiple distinct peptides. CD8+ T cells restricted by the nonclassical MHC class Ib molecule H2-M3 and specific for N-formyl methionine peptides expand more rapidly than MHC class Ia-restricted CD8+ T cells during a primary infection. However, the memory responses of H2-M3- versus MHC class Ia-restricted T cells are dramatically different. H2-M3-restricted memory T cells express activation markers upon reencountering antigen [68] but do not proliferate, but MHC class Ia-restricted T cells undergo explosive expansion [69]. The underlying mechanism for this disparity remains unclear but may reflect differences in thymic selection of these two CD8+ T cell types [70]. 4.3.4 Maintaining CD8+ T Cell Memory

Most infections are eliminated by the mammalian host, yet memory T cells persist afterwards for many years. Determining the mechanisms that enable long-term memory T cell persistence in the absence of antigen has been a great challenge. Pioneering studies with adoptively transferred, antigen-specific memory T cells demonstrated the ability of these cells to survive in MHC-deficient mice [71, 72]. These observations provide strong evidence that, once the differentiation program is initiated, memory CD8+ T cells can persist in the absence of antigen. However, immunological T cell memory that is maintained without MHC contact may become functionally impaired. For example, memory CD4+ T cells specific for the male (HY) antigen show defective responses upon rechallenge and also alter their surface phenotype expression if they are kept in MHC deficient hosts [73]. If maintenance of memory T cells is MHC- and antigen-independent, what regulates their persistence? Recent studies have demonstrated that IL-15 is crucial for invivo maintenance of memory CD8+ T cells [74]. Both primary and memory CD8+ T cell expansion is diminished in IL-15- or IL-15-receptor-deficient mice [57], and maintenance of memory T cell populations following immunization is markedly attenuated in the absence of IL-15 signaling [33, 56, 75, 76]. The gradual decline in memory CD8+ T cell numbers in the absence of IL-15 results from decreased proliferation by memory cells [33]. Interestingly, IL-7 can compensate for the missing survival signals in the absence of IL-15. When IL-7 is overexpressed in IL-15-/- mice, memory CD8+ T cells are generated [75], but removal of IL-7 and IL-15 completely inhibits the homeostatic proliferation of CD8+ memory T cells [76]. These findings emphasize the joint contribution of these two cytokines to promoting CD8+ memory T cell proliferation. Surprisingly, IL-2, a factor best known for its ability to stimulate T cell proliferation, appears to play a negative role in memory T cell survival [34].

4.3 CD8+ T Cell Memory

In comparison with effector CD8+ T cells, memory CD8+ T cells express high levels of bcl-2 mRNA and protein, explaining their relative resistance to apoptosis [44, 77]. One effect of IL-15 is to increase bcl-2 levels in memory T cells, providing a second IL-15-mediated mechanism for memory CD8+ T cell maintenance [78]. Cap structures that protect telomeres from degradation and terminal fusion play an essential role in stabilizing chromosome ends [79]. Telomeres tend to progressively shorten when cells proliferate, eventually resulting in death of the proliferating cell. Telomere length, however, can be maintained in cells with activated telomerase, which adds DNA to telomeres with each cell division [80]. Naïve as well as memory T cells undergo massive proliferation following antigen encounter. To compensate for telomere shortening, telomerase is more active in memory than naïve or effector T cells [81], potentially contributing to their long-term survival. 4.3.5 Models of CD8+ T cell Memory Generation

Although some aspects of memory T cell maintenance have been characterized, the mechanisms that generate memory CD8+ T cells are less clear. Several models for memory generation have been proposed and are outlined in this section. The recent introduction of methods that precisely quantify antigen-specific T cells during immune responses has provided immunologists with a framework upon which to build these models. The linear differentiation model of memory T cell formation proposes progressive differenttiation of T cells from naïve to effector to memory cells (Figure 4.1). Following initial stimulation, an activation program drives naïve T cell differentiation into effector cells [82], followed by further differentiation of a subset of effector T cells into memory T cells [83]. Early support for this model came from TCR repertoire analyses of effector and memory T cells. The TCR repertoire of primary CD8+ T cell responses is similar to that of memory CD8+ T cells, suggesting that memory cells are stochastically chosen from the population of effector CD8+ T cells activated during the primary infection [84, 85]. Although linear differentiation of memory T cells was supported by adoptive transfer studies with T cell receptor (TCR) transgenic (tg) CD8+ T cells specific for the male H-Y antigen [86], the most direct evidence in support of this model came from studies using a clever system for genetically marking T cells that had acquired effector function [87]. In this study, memory T cells were derived from effector T cells. When memory cells form during the course of an immune response (Figure 4.1) remains controversial. One recent study demonstrated that T cells require several weeks from the time of antigen encounter to differentiate into memory T cells [40]. Other studies showed that rechallenge of mice with a high inoculum of antigen 5 days after primary immunization resulted in a memory-like CD8+ T cell response, suggesting that memory T cells may already be present during the primary immune response [88]. A variation of the linear differentiation model proposes that different memory T cell subsets develop at different times following T cell priming. According to this model, primed effector T cells can, in the absence of further stimuli, develop into

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Model B: Early generation of memory cells

Model C: Pre-programmed memory cell potential

antigen & inflammation

1 week

2 weeks

naïve cell

memory cell

effector cell

memory precursor

3 weeks

Fig. 4.1 Models of programmed memory T cell generation. T lymphocytes may commit to becoming long-term memory T cells at different times during the course of activation. We propose three distinct models for programmed memory T cell development. Model A suggests that differentiation of memory T cells is a gradual process, with naïve T cells first differentiating into effector T cells and then, stochastically, further differentiating into memory T cells. Model B suggests that brief exposure to antigen results in two distinct effector T cell populations, one committed to long-term memory development, and the other to apoptotic death. Model C, on the other hand, proposes that naïve T cells differ in their potential to differentiate into memory T cells (see colour plates page XXXIII).

CCR7+ CD62L+ central memory cells, which home to lymph nodes and lack effector functions. In contrast, effector memory cells, which are CCR7- CD62L-, derive from effector cells that have undergone more prolonged stimulation with antigen. This subset of memory cells produces high levels of effector cytokines, maintains cytolytic activity [30], and traffics to peripheral tissues [31]. Some evidence suggests that effector memory cells can convert to central memory cells upon antigen clearance [89]. Evidence in support of this model comes from the analysis of T cell priming by live versus killed bacterial vaccines. Live bacteria, which induce substantial inflammation, result in large effector memory T cell populations, but killed bacteria, which induce an attenuated inflammatory response, give rise to T cells with a central memory phenotype [59].

4.3 CD8+ T Cell Memory

Another model for memory T cell development is the decreasing-potential model, which is based on the progressive loss of proliferative capacity [90, 91]. The underlying idea of this model is that successful memory T cell generation requires a short duration of antigenic exposure during T cell priming. According to this model, larger numbers of functional memory T cells should be produced when effector T cells are briefly exposed to antigen, thereby avoiding antigen-driven apoptosis (AICD). Support for this model comes from situations of overwhelming antigen dose or during chronic infections, such as with HIV or hepatitis-C, where antigen-specific T cells either disappear or become dysfunctional [92–94]. A variant of the decreasing potential model suggests that the timing of Tcell priming during an immune response determines which cells enter the memory lineage [95]. The circulation of lymphocytes between the blood and lymphatic system is a rather slow process, with turnover times of 12 to 24 hours. Thus, the time that it takes for individual lymphocytes to encounter antigen presented in secondary lymphoid organs can vary significantly. Since antigen presentation is transient [96], cells recruited later encounter antigen for a shorter time period than cells that are initially recruited. Brief exposure to antigen would be sufficient to induce their proliferation and differentiation into effector cells; however, the signal would be inadequate to trigger death pathways. Further studies are required to provide experimental support for this model. The third model for memory T cell generation is the instructive model, which posits that effector and memory T cell differentiation diverges during T cell priming. According to this model, memory T cells directly differentiate from naïve T cells without passing through the effector stage. Although experimental support for this model is sparse, the notion that naïve T cells might take their cues from the inflammatory and costimulatory context and undergo differentiation into either effector or memory T cells remains plausible. T cell programming is a relatively new concept that should be incorporated into models of memory T cell generation (Figure 4.2). Early studies demonstrated that

A.

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B.

3 4 5 6 7 8 9 10 11 12 Days Following Infection

Bacterial infection

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Innate Immune Response

3 4 5 6 7 8 9 10 11 12 Days Following Infection

Adaptive T cell Response

Fig. 4.2 Antigen-independent proliferation and memory generation. A. Upon infection, innate immune mechanisms initiate pathogen clearance and provide stimuli that enhance T cell priming. Following the contraction phase, a memory lymphocyte population remains. B. CD8+ T cell expansion, contraction, and memory formation do not require progressive infection or prolonged inflammation. After successful priming,T lymphocytes undergo a programmed, antigen-independent expansion.

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CD8+ T cells specific for different epitopes undergo coordinate expansion and contraction in response to infection, despite substantial differences in antigen expression levels and stability of peptide–MHC complexes [97]. One explanation for this finding is the relatively short duration of effective in vivo antigen presentation, which results from down-regulation by the nascent T cell response [96]. Although feedback inhibition limits T cell priming to the very early phase of the infection, the brief period of priming is sufficient to initiate a program of antigen-independent T cell expansion [82, 83]. Remarkably, the process of T cell expansion, contraction [98, 99], and memory formation occur in the absence of both antigen and inflammation.

4.4 CD4+ T Cell Memory

Although CD8+ T cell memory has been extensively investigated, less is known about the generation and maintenance of memory CD4+ T cells. One explanation for this disparity is the difficulty of directly identifying antigen-specific CD4+ T cells during the course of an immune response. One recent study, however, has compared CD4+ and CD8+ memory T cell responses to LCMV and found that virus-specific memory CD4+ T cells decline in frequency over time while memory CD8+ T cells are maintained at high frequencies [100]. In this section we concentrate on recent studies focusing on memory CD4+ T cells. 4.4.1 Differentiation of Effector and Memory CD4+ T Cells

Naïve CD4+ T cells, upon in vivo priming, differentiate into either Th1 or Th2 effector cells. Although the mechanisms leading to Th1 or Th2 differentiation remain incompletely understood, the innate inflammatory response elicited by the invading pathogen plays a major role in this process. Many viruses and bacteria induce IL-12 and interferon-g (IFN-g) secretion, driving the differentiation of naïve CD4+ T cells into Th1 cells. In contrast, Th2 immunity, which is induced by intestinal helminth infection, is associated with innate inflammatory responses that produce IL-4, driving naïve CD4+ T cell differentiation into Th2 cells. Although the Th1/Th2 lineage decision has important consequences for the primary immune response, long-term memory T cells maintain the phenotype induced at the time of priming. Thus, although memory CD4 T cells do not produce cytokines in the absence of stimulation, upon reencountering antigen they rapidly reexpress the cytokines elicited during their primary activation. Several studies have demonstrated that epigenetic chromatin remodeling during the primary T cell response accounts for specific cytokine expression profiles of reactivated memory T cells [101].

4.4 CD4+ T Cell Memory

4.4.2 Phenotype of Memory CD4+ T Cells

Memory CD4+ T cells have a lower threshold for activation than naïve T cells. Indeed, memory CD4+ T cells respond more rapidly to antigen, with enhanced cytokine secretion and greater proliferation in response to low antigen doses [102]. In addition to greater responsiveness to antigen, memory CD4+ T cells are also present at higher frequencies in immune animals [103]. Although cytokine production by naïve CD4+ T cells is restricted to IL-2 and IL-3, memory CD4+ T cells secrete a greater variety of cytokines [104]. Adoptive transfer of in-vitro generated antigen-specific Th1 and Th2 cells leads to long-lived memory cells, which produce a pattern of cytokines closely related to that of the originally transferred cells [104]. As mentioned in the previous section, epigenetic imprinting accounts for the fidelity of memory T cell cytokine production. 4.4.3 Memory Generation and Maintenance

Mechanisms for CD4+ memory T cell generation have not been defined. Although models similar to those outlined for CD8+ T cell memory formation may be applicable to CD4+ T cell memory generation, existing data most strongly support linear differentiation from naïve to effector to memory CD4+ T cells. In-vitro studies, for example, demonstrated that effector CD4+ T cells can differentiate into memory cells without the requirement for cell division [105]. However, the factors involved in the transition from effector to memory T cells remain unclear. Remarkably, CD4+ T cell memory is efficiently generated in the absence of IL-2, IL-4, or IFN-g [32]. As with CD8+ T cells, initial antigen stimulation is sufficient to initiate programmed expansion in CD4+ T cells [106], and extended antigenic stimulation is not required for differentiation into memory cells or for long-term persistence. Indeed, memory CD4+ T cells persist in the absence of both antigen and MHC class II molecules [72]. The cytokine requirements for long-term memory CD4+ T cell survival are distinct from those described for memory CD8+ T cells. For example, while memory CD8+ T cells decrease in frequency in the absence of IL-15, memory CD4+ T cells are maintained independently of IL-15. Consistent with this finding, CD122 is expressed at low levels on memory CD4+ T cells [107]. In addition, memory CD4+ T cells undergo homeostatic proliferation in the absence of both IL-7 and IL-15 [76], and CD4+ T cells from mice deficient in the common cytokine receptor g–chain, which is shared by receptors for IL-2, -4, -7, -9, and -15, can generate long-lived CD4+ memory T cells [108]. Differences in CD8+ and CD4+ T cell memory maintenance have been detected following viral infection: virus-specific CD4+ memory T cells gradually decline, while CD8+ memory T cells remain stable. Although many factors have been excluded as essential to CD4+ T cell memory formation, the underlying mechanisms that drive differentiation of effector into memory T cells remain mysterious.

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4.4.4 Trafficking of Memory CD4+ T Cells

CD8+ as well as CD4+ T cells differentiate in memory subsets with distinct effector functions and migration capacities [30]. The differences in homing and trafficking of CD4+ T cells can be attributed to their expression of the selectin CD62L and the chemokine receptor CCR7. Central memory (CCR7+, CD62L+) CD4+ T cells preferentially home to lymphoid tissues, whereas effector memory (CCR7-, CD62L-) recirculate in peripheral tissues long after immunization. The localization of central and effector memory CD4+ T cells in different organs has been directly visualized by immunohistology on whole body sections [109].

4.5 B cell Memory 4.5.1 Generation of B Cell Memory

Similar to memory T cells, memory B cells respond rapidly to secondary exposure to antigen, proliferating and producing high affinity antibodies. B cells require accessory signals provided by helper T cells for full differentiation and are also activated by certain microbial stimuli. There are two distinct developmental pathways for antigen-specific B cells [110]. B-1 and marginal zone B cells proliferate and differentiate into antibody-secreting plasma cells, thereby providing the most rapid humoral response to antigen. Although many plasma cells have a life span limited to a few days or weeks, some develop into long-lived cells that mediate long-term humoral immunity. Some antigen-activated B cells migrate into follicles where they proliferate and form germinal centers (GCs), an important site of antibody secretion and affinity maturation. B cells expressing high affinity immunoglobulins receive positive signals from T helper cells, leading to B cell proliferation, survival, and maturation. Lymphotoxin (LT) is important for development of peripheral lymphoid organs, since mice lacking lymphotoxin-a (LT-a) receptor have no lymph nodes or Peyer’s patches and fail to form germinal centers. These structural deficits are linked to defective isotype-switching following primary or secondary immunization with T celldependent antigens [111]. The important role of germinal centers in memory B cell formation was demonstrated in adoptive transfer experiments [112]. B cells from LT-a-/- donors were capable of giving rise to a memory population, whereas a memory IgG B cell response could not be elicited in LT-a-/- hosts. These studies clearly support previous observations, which point out the important role of germinal center structures for memory B cell development [113–115]. Some of the factors determining the lineage decision between plasma cells and memory B cells have been identified [116–118] and include cell surface receptors, cytokines, and transcriptional regulators. Stimulation through CD40-CD154, IL-4, expression of PAX5 and BCL-6 inhibit plasma cell differentiation. On the other hand,

4.5 B cell Memory

absence of CD40, IL-3, IL-6, and IL-10 and the commensurate degradation of BCL-6 and induction of Blimp-1 (B-lymphocyte-induced maturation protein 1) promote plasma cell differentiation. CD4+ T cells are crucial for the generation of long-term humoral immunity. Direct antigen-specific T–B cell interactions have been visualized with adoptive transfer systems [119], and the molecular requirements for CD4+ T cell help were identified recently. One important participant in this process is the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), which was originally identified as altered in X-linked lymphoproliferative syndrome (XLP). SAP controls signaling via the family of SLAM surface receptors, including CD84, CD150 (SLAM), CD229, and CD244 and hence plays a fundamental role in T cell function [120]. Mice deficient in the SAP gene (SAP-/-) mount strong antibody responses during an acute viral infection, but they fail to generate long-lived virus-specific plasma and memory B cells [121]. Adoptive transfer experiments demonstrated that SAP expression by CD4+ T cells, but not B cells, is essential for generation of long-term humoral immunity. 4.5.2 Maintenance of B Cell Memory

It has been proposed that long-term humoral immunity is maintained by memory B cells, which continuously differentiate into short-lived plasma cells [122, 123]. Recently, however, long-lived plasma cells were shown to survive for prolonged periods of time and maintain humoral immunity in the absence of memory B cells [124]. Maintenance of humoral immunity appears to be antigen independent, because memory B cells persist in the absence of antigen [125]. Some experiments support the hypothesis that survival of long-lived plasma cells is determined by microenvironment rather than intrinsic factors. In-vitro experiments, for example, show that plasma cells isolated from tonsils undergo apoptosis unless rescued by stromal cells [126]. Further support for this concept comes from the observation that plasma cell maintenance is restricted by the capacity of the splenic red pulp to provide `space' [127], suggesting that plasma cell homeostasis is regulated by competition for specific survival niches [128]. Nerve growth factor (NGF) regulates neuronal cell development and survival [129]. The finding that patients with autoimmune disorders often have elevated NGF plasma levels [130, 131] and that NGF receptor shares structural similarities with some cytokine receptors suggested a role for this cytokine in the immune system. B cells can produce NGF under basal conditions and constitutively express NGF receptors. It turns out that NGF acts as an autocrine survival factor for memory B cells and that its neutralization completely inhibits humoral memory responses [132].

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4.6 Conclusions

The journey to deciphering memory T and B cell development continues to be a great scientific adventure, and many unanswered questions remain that will require additional clever models and experiments. The motivation for moving forward, however, is substantial. Delineating basic mechanisms is important, and applying the knowledge gained from an enhanced understanding of immunologic memory to the development of vaccines against both infectious diseases and malignancies will be especially satisfying.

Acknowledgements

We thank Drs. Phillip Wong, Heather van Epps, Amariliz Rivera, Adrian Davies, and Maria Lara-Tejero for useful discussions and critically reading the manuscript. We also thank Holger Dormann and Sarah Amsellem for technical assistance.

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5 T Cell-based Vaccines Katharina M. Huster, Kristen M. Kerksiek, and Dirk H. Busch

Summary

Most vaccines currently in use focus on the priming of B cells. For many infections, however, protective immunity is mediated predominantly by T cells. To develop effective vaccines against diseases such as HIV, malaria, and tuberculosis, it is therefore necessary to obtain more information about the mechanisms required for in vivo induction of antigen-specific effector T cells and the generation of long-living memory T cell populations. In this chapter we summarize recent advances in the analysis of antigen-specific T cell responses and T cell memory and discuss the importance of distinct T cell parameters and characteristics for the development of vaccines intended to induce T cell-mediated immunity.

5.1 Introduction

The goal of vaccine development is the induction of effective protective immunity. Naturally occurring protective immunity, which can develop after primary exposure to infectious agents and protects against reinfection with the same pathogen, was recognized already in ancient Greek times; as early as 430 BC, during the Peloponnesian War, Thucydides recorded that people who had recovered from the plague could care for infected individuals without getting the disease a second time [1]. With vaccination, the immune system is exposed to antigen in a noninfectious or mildly infectious way, with the goal of protection against subsequent infection. Vaccination has been attempted since the 11th century, when the Chinese inoculated children by inhalation of scab from the smallpox pustules of mildly infected people or by rubbing the dried material into a scratch. However, general acceptance of this technique was understandably limited, due to the high risk of accidentally inducing lethal infection with human smallpox material. Jenner overcame this problem by using a cross-reactive bovine analog of smallpox, vaccinia or cowpox (vacca = cow). This vaccination strategy was so successful that the WHO declared the eradication of smallpox in 1980.

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Although the phenomenon of protective immunity has been recognized for more than 2500 years, the mechanisms necessary to achieve and maintain pathogen-specific immunity are still not well understood. It is known that subpopulations of B cells, which make up the humoral part of the adaptive immune system, as well as T cells, composing the cellular part of the adaptive immune system, can become memory cells, which respond faster and more effectively to a second – or after vaccination, the ‘real’ – infection. Most vaccines are designed to initiate a humoral, or B cellmediated, response. B cells reexposed to their specific antigen undergo changes resulting in the secretion of higher affinity antibodies, and the persistence of these cells over years ensures enduring antigen-specific protection [2]. Antibody responses to vaccination have been studied extensively over the past decades, facilitated by the ease of determining specific antibody serum levels ex vivo. These studies have revealed that qualitative (e. g., neutralizing capacity, affinity, antibody isotype) and quantitative (e. g., serum concentration) parameters of antigen-specific antibodies can be directly correlated with the efficacy of protective immunity against distinct pathogens [3]. Some of these parameters have been successfully translated into clinical applications designed to assess the efficacy of vaccination, monitor the maintenance of protective antigen-specific antibodies, and determine the necessary timepoints for effective booster vaccination. B cell-based vaccination strategies do not, however, provide effective immunity against all pathogens; numerous intracellular infectious agents, such as viruses and many bacteria and parasites, can circumvent the effects of neutralizing antibodies. Control and elimination of infection by these pathogens is mediated by other effector arms of the immune system, particularly by T cells. The cellular compartment of the adaptive immune system can be divided into two main subsets: T helper cells and cytotoxic T cells are defined by their coreceptors CD4 and CD8, respectively, and use their specific T cell receptor (TCR) to recognize peptide antigen in association with MHC molecules. CD4+ T cells recognize endosome-derived antigens on MHC class II molecules, and CD8+ T cells detect peptides in the context of MHC I molecules, which usually present antigen derived from the cytosolic compartment. Furthermore, T cells help B cells in the induction of antibody responses, and recent work has demonstrated that CD4+ T cell help is crucial for the development of CD8+ T cell-mediated protective immunity. With the recent development of better tools for the analysis of antigen-specific T cell responses directly ex vivo, we can now characterize the functional changes and mechanisms required for effective generation of T cell-mediated protective immunity. These tools also allow us to directly monitor the effects of different vaccination strategies on the T cell compartment, an advance that provides enormous support for the development of new vaccines with strong induction of memory T cells. In this chapter we give an overview of T cell-based vaccination strategies, with emphasis on how their success can be monitored directly ex vivo. Our discussion focuses largely on the value of distinct T cell parameters (such as phenotypical and functional T cell subpopulations and T cell avidity) as markers for the success and quality of protective immunity induced by various vaccination strategies.

5.2 Ex-vivo Detection of Antigen-specific T Cells

5.2 Ex-vivo Detection of Antigen-specific T Cells

It is indisputable that improvement of vaccination strategies often requires stronger in vivo induction of antigen-specific effector and memory T cells or, for therapeutic vaccination, modulation of already existing antigen-specific T cell populations. Indeed, many vaccination protocols and reagents have been developed over the past decades with the specific goal of improving T cell immunogenicity. However, improvement of T cell-based vaccination is dependent on accurate monitoring of the success of T cell vaccination and correlation of vaccine-induced T cell status with the quality of protection. The presence and frequencies of antigen-specific T cells in vivo can be detected by ‘function-dependent’ and ‘function-independent’ methods. Until recently, the limiting dilution assay (LDA) was the only technique available for determination of antigen-specific T cell frequencies [4]. LDA is based on sequential dilution and in vitro expansion of cell samples in 96-well plates, followed by functional assays (such as proliferation or cytotoxicity assays) to ascertain the number of wells containing antigenspecific cell lines or clones. Recent studies using more advanced methods for ex vivo T cell analysis demonstrated that the numbers obtained by LDA assays often substantially underestimated the true in vivo T cell frequencies [5–7]. The development of assays that require only very brief in vitro restimulation, such as the enzymelinked immunospot assay (ELISPOT) [8] and intracellular cytokine staining [5], considerably improved determination of T cell frequencies (Figures 5.1 and 5.2). In these assay systems, T cells responding specifically to antigen are detected through their ability to rapidly produce effector cytokines such as IFNg, TNFa, IL-2, or IL-4. Cytokine capture is achieved by using an affinity matrix, which can be established extracellularly (e. g., by using nitrocellulose, ELISPOT) or on the cell surface (a socalled cytokine capture assay, CCA [9]). Another method, intracellular cytokine staining (ICS), is based on accumulation of cytokines within the cell by blocking the secretion apparatus (e. g., in the presence of brefeldin A or monensin) and subsequent intracellular staining for the cytokine of interest (Figure 5.1). All these assays have proven to be very sensitive, allowing ex vivo detection of antigen-specific T cell populations with frequencies as low as 0.02 %. One advantage of the ELISPOT assay is that relatively little cell material is needed in comparison to flow cytometry-based detection systems (CCA, ICS). However, flow cytometry allows simultaneous staining of different cytokines and surface antigens, providing a more precise phenotypic analysis of the responding cell populations. Multiparameter ELISPOT assays have recently been developed to compensate for some of the limitations of this method [10]. The CCA keeps cells alive, and its combination with cell separation techniques such as FACS or sorting with paramagnetic beads allows purification of antigen-specific T cells for further analysis of the cell population. All these assays detect antigen-specific T cells based on an effector function – rapid production of cytokines in response to in vitro restimulation with antigen. Only T cells capable of responding with the readout effector function under the chosen in vitro restimulation conditions are detected. Because antigen-specific T cell populations under in vivo conditions seem to

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Figure 5.1 Methods for ex vivo detection of antigen-specific T cells. Left: Function-dependent T cell assays. Enzyme-linked immunospot assay (ELISPOT) capture the secreted cytokine on the surface of a membrane; cytokine-spots are visualized by enzyme/ substrate reactions. Cytokine-capture assays (CCA) are based on the same principle, but secreted cytokines are captured on the cell surface and detected with fluorescence-conjugated antibodies; the stained T cell remains alive. Intracellular cytokine staining (ICS) involves accumulation of cytokines within the stimulated cell and detection with fluorescence-conjugated antibodies following membrane fixation and permeabilization. Right: Function-independent T cell assays. MHC multimers bind with higher avidity to T cells than MHC monomers and can be used to directly stain epitope-specific T cell populations. To avoid strong T cell stimulation through surface-bound MHC multimers, modified reagents (Streptamers) can be removed from the cell surface after competitive disruption (staining and removal at 4 °C) (see colour plates page XXXIV).

⇒ disassembly of MHC multimers

5.2 Ex-vivo Detection of Antigen-specific T Cells

be quite heterogeneous, especially in respect to their functional phenotypes, it is unlikely that function-dependent T cell detection assays will be able to detect the entire population. For this reason, T cell detection methods that are independent of effector functions and do not require in vitro incubation, the so-called MHC multimer techniques, have been developed [7, 11, 12]. The basic principle of the MHC multimer is to use the natural T cell receptor ligand, the MHC–epitope complex, as a staining probe (Figures 5.1 and 5.2). Because of the low affinity of TCR–MHC interactions, it is not possible to achieve stable binding by using monomeric MHC–epitope complexes. Multimerization, however, increases the relative binding avidity of the reagent to surface TCRs to a degree that allows epitope-specific binding to T cells. MHC multimer reagents conjugated with fluorochromes can be used for identification of T cells by flow cytometry, with staining characterized by high specificity and sensitivity [13]. The most successful system for ex vivo T cell staining has been the multimerization of MHC–peptide complexes to tetrameric molecules by specifically biotinylating a sequence tag fused to the C-terminus of the MHC heavy chain, followed by oligomerization of the MHCs with streptavidin, which has four biotin binding sites [7, 12]. The increase in relative binding avidity to specific TCRs achieved by MHC multimerization is sufficient to enable detection of antigen-specific T cells within a wide range of physiological binding strengths for their ligand. Even ‘low–affinity’ T cells can be stained with MHC tetramers, and several studies have demonstrated that MHC tetramer staining allows identification of more that 99 % of a given epitope-specific cell population [7]. Dimerization of MHC molecules, usually generated as Ig-fusion proteins, also allows epitope-specific T cell staining, but ‘low–affinity’ T cells might not be detected as well as with MHC tetramers [14]. The MHC multimer has revolutionized T cell research over the past few years. MHC class I multimer reagents have been used for extensive phenotypic characterizations of antigen-specific T cell populations in both animal models and humans. MHC class II multimer reagents are more difficult to generate, and the epitope sequences have often not been precisely determined. However, several recent studies have demonstrated that the general approach of MHC multimer staining can be used effectively for detecting T helper cells [15, 16]. As long as MHC multimer staining is performed at 4 °C, T cells can be identified and purified without altering their original phenotype. However, since MHC multimer reagents represent the natural ligand bound to the TCR, placement of purified MHC multimer-stained T cell populations into in vitro cell culture results in functional alterations such as TCR internalization, activation, overstimulation, and cell death [17–20]. This intrinsic shortcoming of conventional MHC multimer staining substantially limits the applications of the technology for further analysis of ex vivo purified T cells as well as for clinical medicine. To address the problem, a modified MHC multimer technology has been developed [20]; so-called MHC streptamers (Figure 5.1) allow removal of surface-bound MHC multimer reagents after cell staining and purification, conserving the phenotypical and functional status of isolated cell populations. This very promising approach might further broaden the applications of MHC multimer technologies for ex vivo T cell analysis.

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5.3 In vivo Kinetics of Antigen-specific T Cell Responses 3 Fig. 5.2 Direct ex vivo analysis of antigen-specific CD8 + and CD4+ T cells. C57BL/6 mice were infected twice with Listeria monocytogenes expressing the model antigen Ovalbumin. Splenocytes were harvested 5 days after recall infection, followed by intracellular cytokine staining or MHC multimer staining. Dot plots show lymphocyte staining for lineage markers (y-axis) and cytokine or MHC multimer (x-axis); percentages of double-positive cells are indicated. (a) Splenocytes were incubated in the absence (left) or presence (right) of 10 –6 M SIINFEKL peptide for 5 hours; for the last 3 hours, Brefeldin A (BfA) was added. Intracellular staining for IFN g is shown. (b) Same procedure as in (a) but intracellular staining for TNF a. (c) Splenocytes were restimulated in vitro with the MHC class II-restricted Listeria epitope LLO 190–202 in the presenceof BfA as describedin (a) and subsequently stained for intracellular IFN g (left) or TNF a (right). (d) On the same splenocytes as in (a-c), H2-K b/SIINFEKL multimer staining was performed (right; left is unstained control). Note that different frequencies of antigen-specific T cells are detected when using different methods. Differences between frequencies staining for intracellular IFNg and TNF a are observed in the CD8 + T cell compartment (a, b) but not in the CD4+ T cell compartment (c).

5.3 In vivo Kinetics of Antigen-specific T Cell Responses

The development of MHC multimer technologies has enabled analysis of antigenspecific T cell responses in far more detail than previously possible. Originating from a very small precursor population of naive T cells [21, 22], clonally expanding antigen-specific T cells are first detectable 3–5 days after primary antigen exposure and usually reach their maximal size of expansion after 7–8 days [7, 23]. During this proliferation phase, activated T cells differentiate and mature into effector T cells and are detectable in both lymphoid and nonlymphoid tissues throughout the body, with some accumulation close to the site of antigen presence. After the effector phase, many of the expanded T cells are rapidly deleted by apoptosis (contraction phase), and only a small number of antigen-specific cells are maintained as memory T cells. Whereas effector T cells are capable of conferring protection against an infection early after priming, memory T cells have the exclusive task of maintaining T cellmediated protective immunity for longer periods of time. Interestingly, in comparisons of different antigen-challenge systems, T cell responses to immunodominant or subdominant epitopes, or CD8+ vs. CD4+ T cell responses, and the general kinetics of T cell expansion and contraction during primary immune responses are remarkably similar [7]. This observation has led to the recent finding that early instructive events during the initial priming period induce cellular programs of differentiation, expansion, and contraction that are remarkably independent of antigen prevalence and other environmental factors [24–26]. These experimental results are very important for our understanding of how to design and improve T cell-based vaccines, because they suggest that optimization of antigen delivery to antigen-presenting cells

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(APC) and in vivo T cell priming conditions during the early vaccination period will be more effective than efforts to prolong the time of antigen presence or to provide T cell growth/survival factors later during the immune response. If the primary immune response supports effective generation of antigen-specific memory T cells, a secondary challenge with the same antigen results in a much more rapid immune response [27]. Kinetics of recall responses therefore differ from those of the primary antigen challenge; memory cells exhibit effector function within a few hours after activation and expand to a maximal population size (burst size), which is often significantly larger than that of the primary response, 2–3 days earlier than antigen-specific cells reacting to the first antigen exposure. However, when comparing different antigens, delivery systems, and cell subtypes, recall immune responses are far more heterogenous than primary responses. Determination of the mechanisms resulting in these differences is of major importance for the design of more effective T cell-based vaccines. Reimmunization procedures (booster vaccinations) are often needed to elevate frequencies of protective cell populations above a threshold required for measurable protection against the real pathogen [28]. Thus, although an ideal T cell vaccine should be capable of mimicking the effect of any naturally occurring infections, inducing long-lasting and effective protective immunity after a single application most currently available vaccination strategies are dependent on boost vaccinations. There are several possible explanations for the diverse outcomes of immunization. For example, the presence of antigen-specific memory T cells with immediate early effector function might inhibit development the local microenvironmental conditions required for further T cell activation and expansion, a scenario that would particularly affect vaccination strategies employing live attenuated vector systems. The efficacy of live vectors clearly depends on initial replication within tissues, which is necessary for expression of sufficient antigen and induction of inflammatory responses [29]. Clearance of the vector before it can reach lymphoid tissue, which is believed to be the unique site for T cell priming and expansion [30], can inhibit the development of a recall response. Another factor that might dramatically affect vaccine efficacy is the delivery of antigen to subpopulations of APC, specifically to dendritic cells (DC), which are believed to be absolutely essential for initiation of T cell responses [31]. Antigen-specific T cells compete for epitope recognition, especially when the number of APC is very low [32–34]. The kinetics of epitope generation and presentation can also favor the activation and expansion of T cells specific for a distinct epitope. These differences might not become apparent during a primary response, when the time window for T cell priming is relatively large, but they could substantially shape the quality of recall responses. In addition, the in vivo life span of antigen-presenting DC appears to be negatively regulated through the effector functions of antigen-specific T cells, particularly those with cytotoxic activity [35]. Thus, a successful immunization strategy needs not only to deliver antigen efficiently to distinct APC, it must also reach sufficient numbers of APC to overcome the counterproductive negative feedback mechanisms of memory T cells. As discussed later in this chapter, an attractive approach to the enhancement of T cell immunogenicity during primary and recall im-

5.4 Effector Function and Subtypes of Effector T Cells

mune responses might be selective delivery of survival factors to APC, prolonging their in vivo life span in the presence of high frequencies of cytotoxic memory T cell.

5.4 Effector Function and Subtypes of Effector T Cells

T cells exert their antimicrobial activity through a variety of effector functions. These can generally be divided into cell–cell contact-dependent (e. g., killing of infected cells via Fas-FasL) and cell contact-independent (e. g., activation of macrophages through secreted IFNg) functions. Subtypes of T cells are characterized by distinct effector functions, and the in vivo activation and differentiation requirements of these T cell subsets might differ substantially. Mediation of the best protective immunity against different types of infection is likely to require induction of T cell subtypes with specific effector function patterns. Thus, a thorough understanding of the nature of protective T cell populations is crucial for the design of vaccines optimized for specific diseases. (A detailed discussion of differences in disease- and pathogen-specific vaccine requirements is beyond the scope of this brief overview of T cell-based vaccines; you can find more detailed information about vaccine development for specific diseases in the other chapters of this book.) CD4+ T cells, also known as T helper (TH) cells, can be divided into at least three subsets (TH0, TH1, and TH2) based on their cytokine secretion patterns [36]. TH0 cells are naive CD4+ cells that have not yet differentiated into a functional TH subtype. The type and amount of antigen and the APC subset encountered during the first antigen exposure play critical roles in the further differentiation of TH0 cells into either a TH1 or TH2 phenotype [37, 38]. In the presence of IL-4, naive CD4+ precursors preferentially differentiate into TH2 cells that secrete cytokines such as IL-4, IL5, IL-10, and IL-13. In contrast, production of IL-12 and/or IL-18 by APC results in differentiation of CD4+ precursors into TH1 cells that secrete TNFa and IFNg. It is generally believed that several rounds of cell division are required for completion of this differentiation process [39]. Some recent reports indicate that proliferation is not always a prerequisite for naive TH cell differentiation, but this does not appear to be a general phenomenon [40]. The close connection between proliferation and differentiation is not apparent for memory TH cells, which are usually characterized by rapid and vigorous effector cytokine production long before cell division occurs. Once initial polarization towards a TH1 or TH2 phenotype has been established, the process is self-perpetuating: TH1 cells secrete cytokines that further enhance TH1 responses and down-regulate TH2 responses, and vice versa. Another population of TH cells, the regulatory T cell subset (Treg), is characterized by immunomodulation of other T lymphocytes. Treg are best known for their capacity to inhibit proliferation of ‘conventional’ T cells [41]. The main functional role of Treg during an immune response is probably prevention of overzealous clonal expansion of antigen-specific T cells and proliferation of potentially autoreactive T cells out of the pool of polyclonal pathogen-specific T cells. Different types of Treg have been described, most of which are characterized by overexpression of the transcription factor

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Foxp3 [42, 43] and surface expression of CD25 (alpha chain of the IL-2 receptor) [44]. Although some Treg seem to recognize foreign epitopes, similar to conventional T cells, it is likely that the majority detect self-peptides presented by MHC class II molecules. Treg subtypes also differ in the mechanisms of their regulator function: some inhibit cell proliferation through a poorly understood cell–cell contact-dependent pathway, while others regulate via secretion of IL-10 and TGF-b. Knowledge of Treg cell activation and inactivation in vivo is highly relevant for T cell-based vaccines because these populations might be interesting targets for modulation of T cell responses. Preliminary studies indicate, for example, that the efficacy of Toll-like receptor ligands such as CpG-DNA and LPS as adjuvants is due in part to release of factors from activated APCs that temporarily suppress Treg functions [45]. Treg cells appear to influence in vivo reexpansion of memory T cells even more than T cell priming [46], indicating that they should be taken into consideration when addressing improvement of booster vaccinations. The majority of CD8+ T cells are cytotoxic T cells, which lyse infected target cells through expression of Fas ligand on their cell surface or by the release of effector molecules such as perforin and granzymes from large secretory granules. Directly bactericidal substances (e. g., granulysin), which are released from the same type of granules, have been identified in the human system [47]. Most CD8+ effector cells can be defined as Tc1 cells, which secrete TH1-like cytokines (TNFa, IFNg). As described for the TH cell system, CD8+ T cell populations with other differentiation patterns exist [48], but these cell subtypes are not as well characterized as those in the CD4+ T cell compartment. Nevertheless, some CD8+ T cells clearly show a TH2-like phenotype (Tc2 cells), and others have characteristics of regulatory T cells (CD8+ suppressor cells) [49, 50]. Different subpopulations of effector T cells interact with distinct target cell populations. For example, cytokines secreted by TH1 or Tc1 cells activate macrophages, enabling them to kill intracellular microorganisms more efficiently. TH2 cells, on the other hand, influence the production of antibodies with different Ig subclasses by B cells. As described above, the development of function-based methods for ex vivo T cell analysis has enabled precise monitoring of the differentiation of antigen-specific T cell populations. ICS and CCA are the most useful techniques for measurement of cytokine expression patterns (Figure 5.2), particularly when used in combination with improved multicolor flow cytometry technologies that allow simultaneous detection of several different cytokines on the single-cell level [51]. The effector function of cytotoxic T cell populations can also be analyzed in vitro [52] and in vivo [53] by CTL assays, which measure the ability to lyse epitope-presenting target cells. The cytotoxicity of CD8+ T cells is often so effective that CTL assays can detect very low frequencies of antigen-specific effector-memory T cells directly ex vivo (Figure 5.3). The sensitivity of such assays can be enhanced by cell enrichment (e. g., for CD8+ T cells [54]) or selective purification of the antigen-specific population with MHCstreptamer reagents [20].

5.5 T Cell Receptor Repertoire, Avidity Maturation, and Epitope Competition

5.5 T Cell Receptor Repertoire, Avidity Maturation, and Epitope Competition

The TCR repertoire of naive T cell populations specific for foreign, pathogen-derived epitopes is often highly diverse and consists of T cells with various binding affinities for their cognate MHC–epitope complexes [55–57]. The strength of the interaction between TCR and MHC molecules is an important factor in the regulation of immune responses. This is best characterized for the selection of T cells in the thymus, where positive selection occurs only within a distinct range of binding affinities to MHC–self-peptide complexes. However, TCR–MHC binding affinity in the periphery also plays an important role in the quality of antigen-specific T cell responses and the maintenance of T cell populations over time. It is generally believed that in–vivo activation and expansion of T cell subpopulations with relatively high binding affinities for MHC loaded with a pathogen-derived epitope is advantageous for rapid control of infection and the development of effective protective immunity [58]. As shown in both adoptive transfer experiments on animal models and preliminary clinical studies, high-affinity T cells can respond rapidly to low amounts of presented epitope, enabling early interruption of the progression of infection. Identification of the requirements for induction of high-affinity T cells is therefore important for vaccine design. Accordingly, many efforts have been made in recent years to develop assays to measure TCR affinities of polyclonal T cell populations. Techniques based on surface plasmon resonance, which have been the ‘gold standard’ of TCR MHC affinity measurements, require large-scale purification of each receptor–ligand pair and therefore cannot be used for analysis of complex polyclonal T cell populations. Various TCR affinity assays based on the binding and dissociation kinetics of MHC–epitope complexes [56, 59, 60] or changes in effector functions in response to decreasing concentrations of epitope (Figure 5.3 [56, 61]) have been developed; each can be used for direct ex vivo T cell analysis and applied to highly polyclonal T cell populations. Assays measuring dissociation kinetics of surface-bound MHC–peptide complexes are probably the best existing indicator of the relative binding strengths of MHC– peptide complexes on polyclonal T cell populations [60]. Values obtained from function-based avidity assays are influenced by a variety of additional factors, such as the quality of signal transduction required to trigger effector function, which may not be directly related to the structural binding complex [61]. The use of different readout systems can result in quite different experimental findings, even when analyzing the same T cell populations. It has therefore been proposed that the experimental source of T cell avidity data be indicated by the terms ‘structural’ and ‘functional’ avidity [61]. Despite the continuing debate, it is safe to say that studies based on these new methods have demonstrated that, although interaction of the TCR with epitope-presenting MHC complexes affects the overall binding strength, coreceptor binding (e. g., CD8 [62]), membrane composition (e. g., organization of TCR in membrane rafts [63]), and T cell activation status [64] are also important factors. Most people therefore prefer to use the term ‘avidity’ to describe the binding strength of a T cell. The in vivo dynamics of

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T cell avidity have been most impressively demonstrated by experiments using T cells derived from TCR-transgenic mouse lines, in which all cells have exactly the same T cell receptor; even under these truly monoclonal conditions, T cells with different avidities can be found [64]. These avidity differences seem to depend primarily on the in vivo activation status of the T cell. Primary T cell responses to pathogen-derived epitopes usually result in the expansion of populations characterized by diverse TCR repertoires and different structural and functional avidities. By using experimental mouse models, we and others recently demonstrated that repetitive antigen challenge further shapes the composition of antigen-specific T cell populations [56, 65]: during antigen challenge, cells with higher structural avidity for MHC–epitope complexes respond faster and, through selective expansion, increasingly become the predominant subpopulation. These changes can also be observed on the level of the TCR repertoire, which becomes more restricted and oligoclonal during avidity maturation [55]. Selective expansion and avidity maturation might be explanations for the requirement of booster vaccinations for effective protective immunity against many pathogens. Selective expansion of high-avidity T cell populations might also have certain disadvantages. For example, T cells with high avidity for one specific epitope can suppress in vivo development of T cell responses with specificity for other epitopes [65]. This so-called ‘superdomination’ of T cell responses, which may occur during vaccination with multiple pathogen-derived epitopes, may be due to T cell competition on the APC cell surface [32–34]. The consequences of epitope competition are believed to be more pronounced during antigen rechallenge, when responding T cells rapidly

5.6 Functional Heterogeneity of T Cell Memory

exert effector functions that can reduce the half-life of the APC. Because high-avidity T cells bind faster to MHC–epitope complexes, form contact zones (synapses) more rapidly, and respond to very low amounts of epitope, they interfere with the activation of lower-avidity T cells. Suboptimal activation of T cells can result in diminished survival [66], leading to the loss of specific T cell populations. Different epitopes may also be presented with distinct kinetics by the same APC; for example, viral and bacterial antigens are turned on or off at different stages during infection. Here, T cells specific for epitopes presented early during infection may have an advantage. Focusing of immune responses through superdomination might be especially problematic for vaccination against chronic viral infections, in which the pathogen often has time to employ mechanisms of escape from specific immune responses. For example, effective epitope-specific T cell populations responding to HIV infection have been circumvented by mutations within the viral genome that specifically eliminated the T cell epitope [67]; if the T cell response is focused on a single epitope, the consequences of such a mutation are dramatic. In other situations, the greater peptide sensitivity of high-avidity T cells may prove disadvantageous to the immune response, not by interfering with other T cell populations (through superdomination), but via negative effects on the high-avidity T cells themselves. High-avidity T cells are sensitive to high antigen concentrations, and data from experimental models suggest that, in the presence of too much antigen, deletion of high-avidity cells by overstimulation-induced cell death or exhaustion may result [68, 69]. Thus, it will be important to optimize antigen-rechallenge conditions that promote induction of high-avidity T cells. T cell avidity, epitope presentation kinetics, and epitope competition are central mechanisms controlling immunological phenomena, including immunodominance and T cell affinity maturation, and they must be taken into consideration during the design of T cell-based vaccines. In particular, although ex vivo measurement of T cell avidities is still new, the potential clinical value of avidity as a parameter for assessment of the quality and efficacy of vaccination strategies should encourage scientists to include avidity assays in their future studies.

5.6 Functional Heterogeneity of T Cell Memory

Most vaccines are designed to induce protective immunity in an individual who might require protection at an undefined time in the future (preventive vaccination). Design of preventive vaccines must focus on the generation of long-living memory cells. In contrast, for other clinical applications of vaccination, such as therapeutic vaccination of a patient with an ongoing infectious disease or post-exposure vaccination given immediately after a potential infection with the pathogen, it might be advantageous to support the generation of effector cells (Figure 5.4 [70]). Memory T cells are characterized by their ability to functionally respond more rapidly than naïve T cells to antigen reexposure, and they can – in contrast with effector cells – survive for a long period of time, sometimes an entire lifetime [71]. Most

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recent studies support the view that persistence of antigen depots is not required for long-term survival of memory T cells, although this is still a matter of debate. Continuous interactions with self–MHC complexes [72] and increased responsiveness to cytokines such as IL-7 and IL-15 [73], which trigger anti-apoptotic pathways (e. g., bcl2– bclXL up-regulation [74]), are believed to be primary factors in the longevity of memory T cells, but the exact underlying mechanisms are not well understood. The mechanisms of memory T cell generation also remain unclear. Are they generated during the priming period as a distinct cell lineage, developing in parallel with expanding effector T cells, or do they represent a subpopulation of effector T cells that receive (stochastically?) a signal during the contraction phase to survive as post-effector cells in the memory T cell pool? Although the ‘post-effector cell’ model was favored in the recent past [75, 76], more recent data have shown that T cells with a typical memory phenotype can be found early during the priming period, an observation that fits the ‘lineage model’. Antigen-specific memory T cells cannot be regarded as a homogeneous cell population because the pool of memory T cells contains several subpopulations with distinct differentiation patterns and migration characteristics (Figure 5.5 [77]). A central memory T cell subpopulation (TCM) resides preferentially in lymphoid organs, lacks immediate effector functions, and is characterized by vigorous homeostatic and antigen-driven proliferation; maintenance of TCM appears to be dependent on T cell growth factors such as IL-2, IL-15, and IL-7, which signal through common g-chain receptors. The peripheral effector memory T cell subpopulation (TEM), in contrast, is characterized by immediate effector function upon antigen reencounter and migrates preferentially to nonlymphoid organs, where cell maintenance depends on survival factors provided by the local micromilieu. In humans, the different memory T cell subsets can be discriminated by CCR7 expression in combination with staining for CD45RA [77]. In the mouse, L-selectin (CD62L) expression, which mediates T cell migration into lymphoid organs, has been used as a marker to distinguish

5.7 Vaccination Strategies and Their Efficacy for T Cell-based Vaccination

TCM from TEM [7, 78]. However, better markers are needed to allow the subpopulations to be clearly distinguished and to facilitate detailed analysis of their functional roles in protection and long-term maintenance of T cell memory. Our current working hypothesis is that TEM are the main players in conferring protection against invading pathogens; they are preferentially located in nonlymphoid, mucosa-associated tissues (e. g., lung and gut [79, 80]), the entry site for most pathogens, and they rapidly activate their effector mechanisms. When this first line of antigen-specific defense cannot control the local infection and antigen reaches lymphoid tissue, TCM become the source for expansion of antigen-specific effector T cells. TCM are characterized by slow baseline proliferation in the absence of antigen; perhaps some of their daughter cells permanently differentiate into TEM, refreshing the TEM pool in nonlymphoid tissues. According to this hypothesis, TCM is the memory subset crucial for maintenance of T cell memory. The relatively new concept of T cell memory diversification, with different subpopulations contributing distinct functions, has direct consequences for the future development of T cell-based vaccines. The heterogeneity of memory T cells may help explain why several vaccination procedures, in particular the use of particulate vaccines made from inactivated organisms or purified antigens, induce protection inefficiently despite clearly demonstrable generation of antigen-specific memory T cells [81]. It is important to understand the exact mechanisms required for in vivo generation of the various memory T cell subtypes, since they may become attractive targets for modulation of the efficiency or quality of vaccine-induced protective immunity. Recent studies have demonstrated that the composition and quality of the CD8+ memory T cell pool is shaped by instructive, T helper cell-dependent events early during the initial priming period [82–84]. These data suggest that the crucial time window for interventions designed to modulate in vivo memory T cell generation exists early after vaccination.

5.7 Vaccination Strategies and Their Efficacy for T Cell-based Vaccination

As mentioned previously, nearly all vaccines made from inactivated organisms or purified antigens generate poor T cell responses, inducing detectable T cell-mediated protection only when combined with strong adjuvants. The function of adjuvants and their specific immunogenicity-improving qualities, the different adjuvant compositions currently in use, and the recent development of improved adjuvants, especially those based on current knowledge of the Toll-like receptor (TLR) system, are discussed extensively in other chapters of this book. In the context of T cell-based vaccination, it is particularly important to understand the ability of adjuvants to activate APC, especially DC, and create a micromilieu at the site of T cell priming that promotes development of T cell responses. Induction of IL-12 secretion, which is required for strong TH1 and Tc1 responses, and local inhibition of regulatory immune cells (such as Treg), which might otherwise suppress the development of T cell responses [45], are two examples of adjuvant-mediated ef-

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CCR7 Fig. 5.5 Heterogeneity of antigen-specific memory T cell populations. (a) Human peripheral blood lymphocytes from a donor who suffered years ago from infection with influenza virus were analyzed by multiparameter flow cytometry. Dot plot shows double-staining for HLA-A*0201/MP 58–66 multimer reagents (y-axis) and CD8 surface expression (x-axis); frequencies of cell populations within the two regions are indicated. (b) CD8+, HLA-A*0201/MP58–66 tetramer-positive memory T cells can be further subdivided into CCR7+ central memory T cells and CCR7- effector memory T cells. CCR7 (xaxis) versus CD127 (y-axis) expression is shown; percentages of cells within the different quadrants are indicated. (c) Same staining as in (b), but cells are gated on CD8 +, HLA-A*0201/ MP58–66 tetramer-negative cells. Note the presence of an additional subpopulation of CD127/CCR7 double-negative cells, which probably represent a population of ‘ real ’ effector cells.

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5.7 Vaccination Strategies and Their Efficacy for T Cell-based Vaccination

fects that can have a major impact on the efficacy of T cell-based vaccination. However, even in combination with potent adjuvants, the T cell immunogenicity of most particulate vaccines remains poor, especially in comparison with results obtained from vaccination with live vectors such as attenuated vaccinia virus strains (e. g., modified vaccinia virus Ankara or MVA [85]) or attenuated bacteria (Salmonella, Yersinia, Listeria [29]). These differences probably reflect the optimal combination of antigen delivery and local induction of inflammatory immune responses provided by live vectors, conditions that have yet to be effectively simulated by strategies employing adjuvants. Direct conjugation of purified antigens with defined TLR ligands is a promising approach for future vaccine development [86, 87]; because TLR expression is celltype-specific, with preferential expression on various APC subpopulations, TLR ligands might be able to shuttle antigen to distinct APC subtypes and simultaneously induce activation of the antigen-exposed APC. Preliminary studies in experimental models have demonstrated that antigen–TLR-ligand conjugates can substantially increase the T cell immunogenicity of purified antigens [86, 87]. Although live vectors induce potent T cell responses, the simultaneous elicitation of strong vector-specific immune responses is a clear disadvantage of this system; as discussed above, a dominating anti-vector response can suppress T cell responses to other antigens (e. g., the recombinant antigen of interest), especially during antigen challenge. For this reason, boost vaccinations should be performed with different vector systems or, as often necessitated by the limited availability of live vector systems, other vaccination strategies must be included in prime–boost protocols [28]. A boost-vaccination protocol combining DNA vaccination and recombinant MVA challenge induces strong antigen-specific CD8+ memory T cell responses [88]. DNA vaccination, the use of naked plasmid DNA encoding antigenic proteins, is most commonly applied by intramuscular injection [89]. In recent studies, plasmid DNA has also been successfully transduced into eukaryotic cells by using live bacterial vectors (such as Listeria or Salmonella [90, 91]) as vehicles to target the vaccines to professional APC. Coexpression of immunomodulatory molecules (e. g., IL-2, IL-12, GM-CSF) and increasing the content of nonmethylated, immunostimulatory DNA motifs (CpG motifs), which activate innate immune cells through TLR9, are two strategies that have been used to increase the immunogenicity of plasmid DNA. The development of culture protocols for efficient in vitro generation of large quantities of DC, the most potent APC, has made DC immunization possible. Protocols in which DC are loaded with antigens and subsequently transferred into recipients have, depending on the source of DC and their degree of maturation, proven to be effective T cell vaccines in some experimental animal models as well as in humans [92]. These examples of recent vaccine developments, which are discussed in more detail in the chapters of part IV ‘Classical and Novel Vaccination Stretegies: A Comparison’ summarizing the current status of specific vaccination strategies, demonstrate that our increasing knowledge of the basic mechanisms of in vivo antigen-specific T cell responses have already started to influence the design of new vaccines.

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5.8 Concluding Remarks

The development of vaccines capable of inducing strong T cell-mediated immunity requires consideration of certain aspects of T cell responses, several of which are addressed in this chapter. Major advances in analysis of antigen-specific T cells ex vivo and a more detailed knowledge of the in vivo mechanisms that regulate and modulate specific T cell responses are building the basis for a more rational design of vaccines targeting this distinct arm of the immune system. MHC multimer techniques and function-based, ex vivo T cell assays allow us to monitor the success of vaccination in vivo. These techniques will have a major impact on the speed and quality of future vaccine development. Until recently, T cell research in vaccinology was focused mainly on the identification of pathogen-derived, immunodominant T cell antigens and epitopes and analysis of their potential contribution to protective immunity. The close interaction between the innate and adaptive immune systems is now being increasing addressed in vaccine research, and we are beginning to understand that the requirements for an effective vaccine will depend strongly on the pathogen, the kinetics of disease (acute or chronic), and the clinical goal of the vaccination (preventative, therapeutic, or post-exposure). Several types of specialized APC exist; in vivo induction of T cells with distinct effector functions requires targeting of antigen to the appropriate APC subpopulation. APC activation is a necessary prerequisite for up-regulation of coregulatory molecules and secretion of soluble immunomodulatory molecules, factors essential for the development of protective, antigen-specific T cell responses. With the identification of receptors such as the TLR, which can specifically stimulate subtypes of APC, we are now able to develop more defined adjuvants, some of which will be especially effective for induction of distinct types of T cell responses. Protocol design for therapeutic and post-exposure vaccinations will likely focus on the rapid development and expansion of effector T cell populations capable of immediately fighting infection. Induction of long-term memory, on the other hand, requires induction of sufficient frequencies of effector-memory T cells and/or maintenance of this population over time through the presence of central-memory T cells. Some epitope-specific T cell responses might negatively interfere with others, which could lead to the suppression of cell populations crucial for protection; potentially counterproductive mechanisms that may surface during immune responses to different pathogens or antigens need to be identified and subsequently considered during vaccine design. Modulation of the function of distinct regulatory T cell populations may even provide a promising target for increasing the immunogenicity of otherwise weak vaccines.

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References 78 E. J. Wherry,V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H. von Andrian and R. Ahmed, Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol, 2003, 4, 225–234. 79 R. L. Reinhardt, A. Khoruts, R. Merica, T. Zell and M. K. Jenkins, Visualizing the generation of memory CD4 T cells in the whole body. Nature, 2001, 410, 101–105. 80 D. Masopust,V. Vezys, A. L. Marzo and L. Lefrancois, Preferential localization of effector memory cells in nonlymphoid tissue. Science, 2001, 291, 2413–2417. 81 G. Lauvau, S. Vijh, P. Kong, T. Horng, K. Kerksiek, N. Serbina, R. A. Tuma and E. G. Pamer, Priming of memory but not effector CD8 T cells by a killed bacterial vaccine. Science, 2001, 294, 1735–1739. 82 E. M. Janssen, E. E. Lemmens, T. Wolfe, U. Christen, M. G. von Herrath and S. P. Schoenberger, CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature, 2003, 421, 852–856. 83 D. J. Shedlock and H. Shen, Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science, 2003, 300, 337–339. 84 J. C. Sun and M. J. Bevan, Defective CD8 T cell memory following acute infection without CD4 T cell help. Science, 2003, 300, 339–342. 85 B. Moss, M. W. Carroll, L. S. Wyatt, J. R. Bennink,V. M. Hirsch, S. Goldstein, W. R. Elkins, T. R. Fuerst, J. D. Lifson, M. Piatak, N. P. Restifo, W. Overwijk, R. Chamberlain, S. A. Rosenberg and G. Sutter, Host range restricted, non-replicating vaccinia virus vectors as vaccine candidates. Adv Exp Med Biol, 1996, 397, 7–13. 86 A. Heit, T. Maurer, H. Hochrein, S. Bauer, K. M. Huster, D. H. Busch and H. Wagner, Cutting edge: Toll-like receptor 9 expression is not required for CpG DNA-aided cross-presentation

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Part III Adjuvants

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6 Microbial Adjuvants Klaus Heeg, Stefan Zimmermann, and Alexander Dalpke

6.1 Introduction

The central goal of vaccination is the achievement of an efficient, protective, and long-lasting immune response of the adaptive immune system. Immunological memory and secondary responses are the major characteristic hallmarks of the adaptive response of T and B lymphocytes. This system evolved late in phylogeny and has reached its most sophisticated form in mammals. In contrast, innate immunity is phylogenetically old, does not include memory, and lacks enhanced secondary responses. Until recently, both systems were considered principally as two independent limbs of immunity. This view was changed when researchers recognized that innate immunity holds the key for initiation and primary activation of adaptive immune responses [1]. This new perception was fruitful in explaining the adjuvanticity of certain compounds and may lead to new strategies for developing effective vaccines. Innate immune cells control adaptive immune responses by two interrelated restriction points. First, they take up antigen, process it, and present it as peptide fragments to lymphocytes. Second, they recognize infectious danger, become activated, and are then able to provide a second signal to lymphocytes necessary for their effective activation (Figure 6.1). Secondary signals constitute costimulatory signals delivered by specialized ligand–receptor interactions (e. g., B7–CD28) and the secretion of regulatory cytokines like TNF, IL-1, IL-18, and IL-12. Dendritic cells (DC) are the most crucial innate immune cell type in this process. Antigen alone fails to initiate an adaptive immune response, but rather, induces immunological tolerance. During microbial infection the infectious agents provide both signals. It is well known that specific microbial proteins and even carbohydrates can be recognized as humoral antigens. However, it also became clear that in vaccination, antigen alone fails to induce an effective adaptive immune response. Instead, addition of ill-defined components to antigen was necessary to elicit the desired response. These compounds were termed adjuvants; however, their mode of action was elusive. During natural infection, microbial infectious agents obviously induce protective secondary responses and thus are capable of delivering a sufficient activation signal

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6 Microbial Adjuvants microbe

PAMP

antigen

Danger TLR

INNATE SYSTEM

⊕ SIGNAL 2

Innate immune cell (DC)

costimulation

antigen processing and presentation

SIGNAL 1 TCR

ADAPTIVE SYSTEM

T cell

Figure 6.1 Innate and adaptive immunity are interconnected during infection.

to innate immune cells. However, the nature of this natural adjuvant signal was not known until recently. The compounds triggering these signals were recently defined and characterized as conserved microbial molecular patterns. These conserved microbial structures are composed of various microbial cell wall components, specific microbial structural proteins, lipoproteins and lipopeptides, and microbial nucleic acids. The complete set of these structures has been termed pathogen-associated molecular pattern (PAMP), which can be sensed by the innate immune system. An array of receptors are expressed in the host, called PAMPrecognition receptors (PRR); they include the family of Toll-like receptors (TLR), scavenger receptors, and carbohydrate receptors in cell-bound and soluble forms. TLR seem to play a pivotal role in recognizing microbial infection (Table 6.1). PAMPs can be typified as evolutionarily conserved danger signals leading to activation of innate immunity in the host, which is crucial for subsequent activation of adaptive immune responses. Indeed, some previously defined adjuvants now have been characterized as microbial or microbial-derived components that activate innate immune cells in TLR-dependent and TLR-independent manners. Moreover, new synthetic TLR agonists have been identified that possess adjuvant activity and which can be used as well-defined pharmaceutical agents in new vaccine formulations (Table 6.2). New antigendelivery system such as emulsions, liposomes, microparticles, and ISCOMs might possess intrinsic adjuvant activity too that might be enhanced by addition of defined adjuvants. This chapter focuses on microbial and microbial-derived natural and synthetic danger signals that can be used as adjuvants in vaccination.

6.2 Microbial Danger Signals Tab. 6.1 Toll-like receptors (TLR) and their natural ligands. TLR

Microbial ligand

TLR1

Cooperation with TLR2, triacylated lipoproteins.

TLR2

Lipoproteins and lipopeptides (E. coli, M. tuberculosis, B. burgdorferi, T. pallidum, M. fermentans, S. epidermidis, H. influenzae, S. flexneri). Peptidoglycan, yeast cell walls, lipoarabinomannan, glycolipids (Treponema), GPI anchors (T. cruzi), lipoteichois acid (LTA), lipopolysaccharide (Leptospira, Porphyromonas).

TLR3

Viral RNA, poly (I:C).

TLR4

LPS, F-protein from RS-virus, fungal components (Aspergillus, Cryptococcus).

TLR5

Flagellin (L. monocytogenes, S. typhimurium).

TLR6 + TLR2

Peptidoglycan, zymosan, MALP-2 (Mycoplasma), lipoproteins.

TLR7

? (synthetic: imidazoquinolines).

TLR8

? (human: synthetic: imidazoquinolines).

TLR9

Bacterial DNA (synthetic: CpG–ODN).

TLR10

?

Tab. 6.2 Microbial and microbial-derived adjuvants. Activators of the innate immune system Toxins cholera toxin, heat-labile toxin (E. coli) Toll-like receptor-dependent bacterial compounds derivatives of lipid A (MPL) muramyl dipeptides (MDP) lipopeptides, MALP other components (flagellin) bacterial CG DNA Toll-like receptor-dependent synthetic compounds synthetic CpG DNA AGPs Other low molecular synthetic agonists imidazoquinolines

6.2 Microbial Danger Signals 6.2.1 Toxins (CT and LT)

During infection, microbes not only activate innate immune cells by their PAMPS but also are able to influence cellular responses by specifically released toxins. Some of these toxins act as adjuvants. The most powerful toxin adjuvanticity found so far

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is that of cholera toxin (CT) and heat-labile toxin from E coli (LT). Both agents act as strong mucosal adjuvants in experimental models. CT and LT are the underlying pathophysiological principles of cholera and traveler’s diarrhea. Therefore, the use of wild-type CT and LT in humans is precluded. CT and LT are composed of two subunits (A and B). Subunit B binds to cellular receptors, and subunit A crosses the cellular membrane and influences the activity of adenylate cyclase, causing its permanent activation and accumulation of cAMP. Genetically defined mutants of CT and LT have been obtained that lack toxicity yet retain their immunological activity. These mutants are currently being tested for applicability in humans. However, recent studies have shown that intranasally applied CT and LT might be taken up and found in the brain [2], which would preclude their use as local mucosal adjuvants. Although the powerful mucosal adjuvanticity of CT and LT has been demonstrated convincingly, their mode of action is still unknown. CT-aided responses are characterized by a predominant activation of the Th2 type of CD4 T cells. This is due to increased induction of IL-4, IL-5, and IL-10 while IL-12 receptor expression is suppressed. In contrast, LT seems to activate Th1 and Th2 cells concomitantly. Recently CT and LT were shown to activate human monocyte-derived dendritic cells (DC) in a cAMP-dependent way [3]. Thus, direct DC activation might be one underlying mechanism of CT- and LT-mediated adjuvanticity that could be orchestrated by direct release of regulatory cytokines from various innate immune cells. 6.2.2 Toll-like Receptor-dependent Microbial Adjuvants 6.2.2.1 Lipopolysaccharide and Lipid A Derivatives Lipopolysaccharide (LPS) is a constituent of the outer membrane of gram-negative bacteria. Its proinflammatory activity and its role as a sepsis-inducing factor in severe infections were recognized long ago. In simplified terms, LPS consists of a core polyacylated disaccharide (lipid A) and various attached carbohydrate chains. Lipid A was defined as the active part of LPS. The number and positions of acyl side chains were found to be important for the proinflammatory activity of LPS [4]. Although known for a long time, only recently has the receptor system mediating recognition of LPS been elucidated. TLR4 molecules in combination with CD14 and a soluble protein (MD-2) sense LPS and lead to cellular activation [5]. LPS preparations are highly toxic; however, heroic trials in the early 20th century suggested that LPS might possess immunomodulatory and immunostimulatory activity. Chemically defined derivatives of LPS were identified that display reduced toxicity yet retain the immunomodulatory properties of wild-type LPS. One of these components is monophosphoryl lipid A, which was further refined and which is now used as adjuvant (MPL) [6]. MPL has been evaluated in numerous studies and was tested in more than 10 000 individuals in various vaccine formulas: it was an effective mucosal adjuvant and activated humoral as well as cellular immune responses. Vaccination against infectious diseases (herpes, HBV, influenza) induced lasting, effective immune responses. Although shown to be effective, its exact mode of action has still to be defined. How-

6.2 Microbial Danger Signals

ever, since TLR4 gene-defective mice do not respond to MPL, involvement of TLR4 in the adjuvanticity effect of MPL is strongly suggested. MPL activates human DC to maturate, express costimulatory molecules (CD80, CD86, CD40, CD83), enhance antigen presentation (HLA-DR), and induce secretion of regulatory cytokines (IL-12). However, its intrinsic activity in vitro is lower than that of wild-type LPS. Moreover, MPL also might directly act on T cells via TLRs [7]. 6.2.2.2 Peptidoglycan and Lipoteichoic Acid Peptidoglycans (PG) are essential constituents of all bacterial cell walls. PGs are recognized by innate immune cells in context with TLR2 and TLR6. Although PG clearly represent a PAMP, their role as an adjuvants is limited. So far, isolated PG was shown to possess only limited immunostimulatory activity relative to other TLR ligands. Similar conclusions can be drawn for lipoteichoic acid (LTA). LTA clearly represents a PAMP; however, again its intrinsic activity is rather low. Recently, pure alanylated fractions of natural LTA with improved activity were successfully prepared [8]. However, compared to other TLR ligands, the activity was rather low, and in addition, LTA was a very poor inducer of IL-12. Thus, natural LTA does not seem to be a promising candidate for a new adjuvant. 6.2.2.3 Other Microbial Components (Lipopeptides, Flagellin) Bacterial lipopeptides exert potent immunostimulatory activity [9]. Because bacteria of the species Mycoplasma contain neither LPS nor PG, they have been used to study the effects of bacterial lipoproteins in detail. A mycoplasmal-derived lipopeptide, termed macrophage-activating lipopeptide-2 (MALP-2) was shown to stimulate DC activation and maturation via TLR2 and TLR6 [10]. MALP therefore was used as a mucosal adjuvant and proved to be applicable [11]. Flagellin is a protein of many gram-negative bacteria, which also may be secreted. Flagellin is a member of the PAMP family and is recognized by innate immune cells. A rather intriguing property of flagellin is its recognition in combination with TLR5 [12]. However, flagellin is a rather weak stimulus for innate immune cells. Although it stimulates up-regulation of costimulatory molecules of DC, its intrinsic ability to stimulate cytokine secretion is low. Nevertheless, flagellin has been used in experimental animal models and was effective as adjuvant for CD4+ T cells [13]. Due to its complicated isolation process, its practical use as an adjuvant will be limited. 6.2.2.4 Bacterial DNA It is well known that the base compositions of bacterial and vertebrate DNA differ. Vertebrate DNA exhibits so-called CG suppression, that is, a reduced overall frequency of the dinucleotide CG compared to the statistically expected value of 1/16. Moreover, CG islands in vertebrate DNA are DNA regions that are involved in gene regulatory processes and possess methylated cytosine residues. In contrast, regulative mechanisms relying on DNA methylation are lacking in bacterial genomes. As a consequence, bacterial DNA contains much more unmethylated (free) CG dinucleotides than vertebrate DNA does. Accordingly, the frequent presence of CG dinucleotides in DNA molecules distinguishes bacterial from vertebrate DNA and thus ful-

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fills the requirements of a PAMP. These concepts were not known when bacterial DNA was recognized as a natural adjuvant in the early 1980s [14]. Preparations of Mycobacteria exert anti-tumor effects when injected at the tumor-bearing site. Delineation of the active principle revealed that mycobacterial DNA was responsible for the immunological effects. Moreover, it was shown that DNA containing CG dinucleotides was most effective. These discoveries have led to multiple approaches that are now used in clinical immunology, including the use of Mycobacteria (and thus bacterial) DNA to treat patients with urinary bladder carcinomas [14]. Similar to many PAMPs, TLRs are responsible for recognition of bacterial DNA. Gene knockout experiments have revealed a central role of TLR9 in recognition of bacterial DNA [15]. TLR9 belongs to a subset of TLRs (TLR7, 8, 9) that share some unique features. Canonical TLRs are expressed on the cell surface by multiple innate immune cell types, including DC, macrophages, and granulocytes, and even epithelial cell types and lymphocytes. In contrast, TLR7–9 show a rather restricted cellular expression profile, confined to intracellular endosomal compartments. Accordingly, bacterial DNA has to be taken up from extracellular sources into endosomes or has to be released from phagocytosed microbes prior to triggering TLR9 receptors. The expression profiles of TLR9 differ in mice and humans. Although mice express TLR9 on macrophages, DC, and B cells, in humans macrophages and conventional DC fail to express TLR9. Only a minor subset of peripheral DC, the plasmacytoid DCs, show TLR9 expression and are thus fully responsive to bacterial DNA (see below). In-vitro isolated and purified bacterial DNA triggers strong innate immune responses, including cellular activation and release of proinflammatory cytokines, which eventually lead to lethal shock syndrome, as does injection of LPS. However, the role of bacterial DNA during infection is still elusive [16]. So far, only limited information on the fate of infections in TLR9 gene-deficient mice is available. However, purified bacterial DNA is a strong inducer of IL-12 and thus preferentially triggers Th1 immune responses. Moreover, bacterial DNA prevented Th2-dominated allergic immune reactivities [17]. These observations support the hygiene hypothesis of the prevalence of allergies in developed countries. Lack of childhood infections with bacteria in developed counties, due to excessive cleanliness is believed to cause a reduced Th1 profile and thus favor allergy development [18]. Accordingly, bacterial DNA is now considered to be a major factor during bacterial infection, which leads to a Th1 bias in subsequent immune responses, thus preventing induction of allergic reactivities. Immunization with naked plasmid DNA encoding antigen induces strong humoral and cellular immune responses. Interestingly, ongoing immune responses were skewed towards a Th1 type of reactivity [19]. It turned out that plasmid DNA sequences not encoding the antigen were responsible for the efficacy as well as the induced Th1 bias [20]. CG motifs in these regions were found to be important in this respect. Hence, CG motifs seem to contribute to the overall effectiveness of DNAbased vaccines. DNA plasmid vaccines can therefore be classified as a two-component vaccine. DNA encodes the vaccine antigen, and at the same time, plasmid DNA acts as an intrinsic adjuvant [21]. However, the matter might be more complicated than initially thought. First, DNA motifs in viral, bacterial, and mammalian DNA

6.2 Microbial Danger Signals

have been described that inhibit CG-mediated effects. Thus, certain DNA sequences might specifically suppress activation of innate immune cells and therefore prevent activation and immunization against antigens [22]. Second, in contrast to mice, in humans the expression of TLR9 seems to be tightly restricted to subpopulations of DC. Therefore, the contribution of CG sequences in DNA vaccines could be less effective in humans. Indeed, recent experience with DNA vaccines in human trials seems to support this notion. 6.2.3 Toll-like Receptor-dependent Synthetic Compounds 6.2.3.1 Synthetic CpG DNA A major breakthrough in research on immunostimulatory CG DNA was the finding that short synthetic single-stranded oligonucleotides mimic the effects of bacterial DNA [23]. Moreover, it now was possible to synthesize defined oligonucleotides (ODN) to elucidate the DNA sequence requirements necessary for optimal activation. It turned out that a minimal hexameric DNA motif (5´-R-R-C-G-Y-Y-3´) within an oligonucleotide was sufficient to induce activation of innate immune cells. These active ODN were termed CpG oligonucleotides (CpG ODN) (where p stands for the phosphate bond). Moreover, CpG ODN proved to be of superior usefulness compared to bacterial DNA, because CpG ODN could be produced with high purity [24]. CpG ODN could additionally be manufactured with synthetic backbone modifications different from the natural phosphodiester backbone (PO), which conveyed nuclease resistance and in turn allowed greater in-vivo stability. Usually phosphorothioate backbone modifications (PTO) are used. CpG ODN have to be taken up to meet their receptor, presumably within the endosome. CpG ODN induce activation, maturation, and induction of effector function in innate immune cells (like DC and macrophages), but also induce proliferation and Ig secretion in B lymphocytes. A special peculiarity of CpG ODN is their profound capacity to induce IL-12 production, which may explain their capacity to preferentially induce Th1-biased immune responses (Figure 6.2). The underlying mechanisms for these effects are not yet known. In mice, IL-12 and IL-18 mediated activation of NK cells induces high amounts of IFN-gamma. DC respond with upregulation of their machinery for antigen processing and antigen presentation. Macrophages activate their innate effector functions, such as phagocytosis, iNOS induction, and production of reactive oxygen species (ROS). Therefore, CpG ODN are not only excellent candidates as vaccine adjuvants but also potent immune activators that can augment the effector phase of anti-microbial immune responses (Figure 6.2) [25]. Although direct injection of CpG ODN into joints induces autoimmune arthritis, no such autoimmune phenomena have been observed so far after local injection in the course of an experimental vaccination [26]. Hence, CpG ODN could be used as an agent to prevent Th2-mediated immune reactions or even as drug to treat Th2-dominated diseases. Intensive recent work on DNA sequences and ODN modifications has shown that CpG ODN effects are much more complicated than initially anticipated. Although

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6 Microbial Adjuvants CpG DNA

Macrophage

DC

B cell

activation of innate effector functions: phagocytosis, iNOS, ROS

IL-12 IL-18

NK cell

induction of adaptive immune responses: antigen presentation costimulation cytokine milieu

antimicrobial immune response

Figure 6.2 CpG DNA and the initiation of innate and adaptive immune responses in mice.

backbone modifications are advantageous when stability and long-term activity are concerned, at the same time, in experimental murine models they show undesired effects, including long-term lymphadenopathy and DNA sequence-independent stimulation of B lymphocytes. In vivo, natural PO backbone CpG ODN were almost ineffective; however, addition of certain DNA sequences restored their activity [27, 28]. This indicates that the pharmacology of CpG ODN can still be improved. In addition, species-specific sequence differences became evident. CpG ODN sequences that were optimal for murine cells were almost ineffective on human cells, and vice versa. Species-specific sequence alterations in TLR9 were responsible for these effects [29]. As mentioned above, there exist further differences in TLR9 expresion between mouse and man. In human peripheral blood, in addition to B cells, only plasmacytoid DCs express TLR9 and are thus CpG reactive. However, some ODN effects can be triggered independently on TLR9, indicating that other receptor systems (e. g., scavenger receptors) are involved too. Extensive DNA sequence analyses have further revealed that different types of ODN exist for human cells (Figure 6.3). D-type ODN require an unmethylated CpG dinucleotide, a palindrome with CG in the center, and a phosphorothioate backbone in the palindrome-flanking regions. D-type ODN preferentially stimulate IFNgamma production by human NK cells. In contrast, K-type ODN consist of multiple

6.2 Microbial Danger Signals Mice: 5'-x-x-...-G-A-C-G-T-T-...-x-x-3' (PTO modification allowed)

murine TLR9

Human: "K"-type ODN 5'-x-x-..T-C-G-T-T(A)-..-T-C-G-T-T(A)-..x-x-3' (PTO modification allowed)

human TLR9

"D"-type ODN 5'-x-x-.. ..-x-x-3' A •• T T ••A C–G

? other receptors ?

(or other palindromic sequences with central CG) palindrome: no PTO modification allowed

Figure 6.3

CpG DNA sequence motifs.

CpG dinucleotides, with a thymidine immediately 5´, and a TpT or TpA at the 3´ of the CpG. K-type ODN effectively stimulate monocytes and B cells, inducing proliferation, cytokine secretion, and Ig secretion [30]. Preferentially, K-type ODN have been used for preclinical studies in primates and proved their applicability [31]. Moreover, CpG ODN seem to require fewer booster injections of classical antigen to achieve protective antibody responses, as compared to vaccinations using conventional adjuvants. Finally, direct coupling of antigens to CpG ODN combines cellular delivery of antigen to DCs with concomitant activation and increased capability of antigen presentation within the same immune cell. This allows the amount of CpG ODN used for vaccination to be reduced and might enhance the immunogenicity of CpG antigen preparations in vaccination trials [32]. Although one advantage of CpG ODN is their marked adjuvanticity for the induction of T-cell responses, as shown in murine models, in primates so far, no information on CTL induction is available. Nevertheless, the data so far qualify CpG ODN as a novel vaccine adjuvant that could be used in vaccines aimed to vaccinate B and T lymphocytes and, in addition, induce a Th1-prone status of the immune system [33]. 6.2.3.2 Other Synthetic TLR ligands Due to the increasing knowledge of TLR4 and its interaction with LPS or lipid A analogues, novel synthetic aminoalkylglucosaminide phosphates (AGP) have been developed. These acylated monosaccharides are structurally related to lipid A [6] and possess strong adjuvant activity. Since AGPs are chemically synthesized, changes in

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their chemical moieties can be tested for improvement of activity, stabilization, and degradation with less pyrogenicity. Preclinical studies suggest that AGPs might be as effective as adjuvant as MPL is [6]. The minimal essential structure of peptidoglycan is MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine). MDP has been synthesized and tested for adjuvant activity; it stimulated human DC and induced up-regulation of costimulatory molecules. However, HLA DR was not affected [34]. Therefore, in comparison to other TLR ligands, the results are not yet promising. However, new chemical modifications (adamantylacetyl derivative of peptidoglycan monomers) seem to be more encouraging [35]. Synthetic lipopeptides have been used as adjuvant and combined antigen/adjuvant compositions. They amalgamate adjuvanticity and peptide-based antigen delivery. Synthetic lipopeptides can deliver CTL epitopes into the class I presentation pathway and concomitantly activate APC. Thus, effective T cell activation is brought about. TLR2 is crucially involved in activation of DC and thus in the adjuvant activity of lipopeptides [10]. In addition, preclinical trials have been performed to study vaccinations against HIV, HBV, HCV, CMV, HSV, and malaria [36]. Moreover, synthetic lipopeptides also promise future use in mucosal vaccines. 6.2.3.3 Low Molecular Weight TLR Agonists

Imidazoquinolines (Imiquimod, R848) Low molecular weight compounds of the imidazoquinoline family are active in stimulating adaptive immunity. They induce prophylactic and therapeutic reactivities against HSV infection and enhance Th1-mediated immune responses. Accordingly, these agents are called immunomodifiers. When compared with CpG DNA, imidazoquinolines induced similar cytokine and reaction profiles, albeit slightly less effectively [37]. Importantly, imidazoquinolines also induce IFN-alpha and thus might play an important role in aiding anti-viral responses. It was a surprise when TLR7 was identified as the recognition structure of imidazoquinolines in mice and humans [38]. Accordingly, imidazoquinolines activate the innate immune system in a MyD88-dependent pathway and thus can be classified as a synthetic ligand of an undefined microbial PAMP acting on TLR7 and possibly also TLR8. It is so far unknown whether the low molecular weight imidazoquinolines act directly on TLR7 or have to be bound to so-far-undefined matrixes. Since cellular and subcellular expression profiles of TLR7 and TLR9 seem to broadly overlap, and since imidazoquinolines share some properties of CpG DNA (e. g., Th1-inducing capacity), these compounds might be a new alternative to a microbial-derived synthetic PAMP that can be used effectively as adjuvants [39]. Because these compounds have been used already as an immune-response modifier in viral infection, they might be included in vaccine formulas soon. Moreover, their marked Th1-inducing capacity might qualify imidazoquinolines as Th1-promoting agents useful in allergy vaccines. Moreover, chemical modifications of the imidazoquinolines, which can be achieved easily, could sharpen the reactivity profile and enhance the intrinsic activity. Nevertheless, the natural ligands for TLR7 and TLR8 are not yet defined. One could speculate that low molecular compounds of mi-

6.3 Conclusion

crobial metabolism are natural ligands. This in turn would further catalyze the development of new synthetic TLR ligands with high and selective adjuvanticity.

6.3 Conclusion

Delineation of microbial PAMP and their receptors on innate immune cells has led to tremendous progress in understanding the mechanisms of sensing infectious danger and initiating innate and adaptive immune responses. The TLR systems and the respective microbial ligands play fundamental roles in this process. This knowledge has allowed us to understand the means by which microbial PAMPs and their derivatives, which were defined empirically, act as vaccine adjuvants (Figure 6.4). Based on these findings, natural microbial PAMPs can be modified and further developed, or synthetic compounds can be improved to be utilized as new efficient adjuvants in vaccination.

Cholera Toxin (CT) Heat labile Toxin (LT) adenylate cyclase MDP MALP-2 lipopeptides

MPL

TLR 2/6

TLR 4

ACTIVATION

TLR 5

flagellin

TLR 9

CpG DNA imidazoquinolines

TLR 8

endosome

innate immune cell

Figure 6.4

Microbial adjuvants and TLR.

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8. Morath, S., Stadelmaier, A., Geyer, A., Schmidt, R.R., and Hartung, T., Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release. J Exp Med 2002, 195, 1635–1640. 9. Bessler, W.G., Heinevetter, L., Wiesmuller, K.H., Jung, G., Baier,W., Huber, M., Lorenz, A.R., Esche, U.V., Mittenbuhler, K., and Hoffmann, P., Bacterial cell wall components as immunomodulators. I. Lipopeptides as adjuvants for parenteral and oral immunization. Int J Immunopharmacol 1997, 19, 547–550. 10. Hertz, C.J., Kiertscher, S.M., Godowski, P.J., Bouis, D.A., Norgard, M.V., Roth, M.D., and Modlin, R.L., Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2. J Immunol 2001, 166, 2444–2450. 11. Rharbaoui, F., Drabner, B., Borsutzky, S.,Winckler, U., Morr, M., Ensoli, B., Muhlradt, P.F., and Guzman, C.A., The Mycoplasma-derived lipopeptide MALP-2 is a potent mucosal adjuvant. Eur J Immunol 2002, 32, 2857–2865. 12. Hayashi, F., Smith, K.D., Ozinsky, A., Hawn, T.R.,Yi, E.C., Goodlett, D.R., Eng, J.K., Akira, S., Underhill, D.M., and Aderem, A., The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001, 410, 1099–1103. 13. Krogsgaard, M., Wucherpfennig, K.W., Canella, B., Hansen, B.E., Svejgaard, A., Pyrdol, J., Ditzel, H., Raine, C., Engberg, J., and Fugger, L., Visualization of myelin basic protein (MBP) T cell epitopes in multiple sclerosis lesions using a monoclonal antibody specific for the human histocompatibility leukocyte antigen (HLA)DR2-MBP 85–99 complex. J Exp Med 2000, 191, 1395–1412. 14. Yamamoto, S.,Yamamoto, T., and Tokunaga, T., The discovery of immunostimulatory DNA sequence. Springer Semin Immunopathol 2000, 22, 11–19. 15. Hemmi, H., Takeuchi, O., Kawai, T.,

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Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S., A Toll-like receptor recognizes bacterial DNA. Nature 2000, 408, 740–745. Dalpke, A., Zimmermann, S., and Heeg, K., CpG–DNA in the prevention and treatment of infections. BioDrugs 2002, 16, 419–431. Van Uden, J. and Raz, E., Immunostimulatory DNA and applications to allergic disease. J Allergy Clin Immunol 1999, 104, 902–910. Parronchi, P., Brugnolo, F., Sampognaro, S., and Maggi, E., Genetic and environmental factors contributing to the onset of allergic disorders. Int Arch Allergy Immunol 2000, 121, 2–9. Raz, E., Tighe, H., Sato,Y., Corr, M., Dudler, J.A., Roman, M., Swain, S.L., Spiegelberg, H.L., and Carson, D.A., Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization. Proc Natl Acad Sci USA 1996, 93, 5141–5145. Sato,Y., Roman, M., Tighe, H., Lee, D., Corr, M., Nguyen, M.D., Silverman, G.J., Lotz, M., Carson, D.A., and Raz, E., Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 1996, 273, 352–354. McCluskie, M.J.,Weeratna, R.D., and Davis, H.L., The role of CpG in DNA vaccines. Springer Semin Immunopathol 2000, 22, 125–132. Krieg, A.M.,Wu, T., Weeratna, R., Efler, S.M., Love-Homan, L.,Yang, L., Yi, A.K., Short, D., and Davis, H.L., Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. Proc Natl Acad Sci USA 1998, 95, 12631–12636. Krieg, A.M.,Yi, A.K., Matson, S., Waldschmidt, T.J., Bishop, G.A., Teasdale, R., Koretzky, G.A., and Klinman, D.M., CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995, 374, 546–549. Krieg, A.M., Now I know my CpGs. Trends Microbiol 2001, 9, 249–252. Wagner, H., Toll meets bacterial CpG– DNA. Immunity 2001, 14, 499–502.

26. Deng, G.M., Nilsson, I.M., Verdrengh, M., Collins, L.V., and Tarkowski, A., Intra-articulary localized bacterial DNA containing CpG motifs induces arthritis. Nat Med 1999, 5, 702– 705. 27. Agrawal, S. and Kandimalla, E.R., Medicinal chemistry and therapeutic potential of CpG DNA. Trends Mol Med 2002, 8, 114–121. 28. Dalpke, A., Zimmermann, S., and Heeg, K., Immunopharmacology of CpG DNA. Biol Chem 2002, 383, 1491– 1500. 29. Bauer, S., Kirschning, C.J., Hacker, H., Redecke,V., Hausmann, S., Akira, S., Wagner, H., and Lipford, G.B., Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad SciUSA 2001, 98, 9237– 9242. 30. Gursel, M.,Verthelyi, D., Gursel, I., Ishii, K.J., and Klinman, D.M., Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotide. J Leukoc Biol 2002, 71, 813–820. 31. Verthelyi, D., Kenney, R.T., Seder, R.A., Gam, A.A., Friedag, B., and Klinman, D.M., CpG oligodeoxynucleotides as vaccine adjuvants in primates. J Immunol 2002, 168, 1659–1663. 32. Tighe, H., Takabayashi, K., Schwartz, D., Marsden, R., Beck, L., Corbeil, J., Richman, D.D., Eiden, J.J., Jr., Spiegelberg, H.L., and Raz, E., Conjugation of protein to immunostimulatory DNA results in a rapid, longlasting and potent induction of cellmediated and humoral immunity. Eur J Immunol 2000, 30, 1939–1947. 33. Singh, M. and O'Hagan, D.T., Recent advances in vaccine adjuvants. Pharm Res 2002, 19, 715–728. 34. Todate, A., Suda, T., Kuwata, H., Chida, K., and Nakamura, H., Muramyl dipeptide-Lys stimulates the function of human dendritic cells. J Leukoc Biol 2001, 70, 723–729. 35. Ljevakovic, D., Tomasic, J., Sporec,V., Spoljar, B.H., and Hanzl-Dujmovic, I., Synthesis of novel adamantylacetyl derivative of peptidoglycan mono-

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with CpG ODN. CellImmunol 2000, 204, 64–74. 38. Hemmi, H., Kaisho, T., Takeuchi, O., Sato, S., Sanjo, H., Hoshino, K., Horiuchi, T., Tomizawa, H., Takeda, K., and Akira, S., Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 2002, 3, 1–5. 39. Hengge, U.R., Benninghoff, B., Ruzicka, T., and Goos, M., Topical immunomodulators: progress towards treating inflammation, infection, and cancer. Lancet Infect Dis 2001, 1, 189–198.

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7 Host-derived Adjuvants Norbert Hilf, Markus Radsak, and Hansjörg Schild

7.1 Introduction

The immunogenicity of most antigens, such as proteins or peptides, is very limited when applied as pure substances. Therefore, these antigens are mixed with substances known as adjuvants (adiuvare (Latin): to help), which facilitate and amplify the induction of an immune response. The earliest discovered adjuvants are aluminum salts, which have been in use since Glenny and coworkers discovered in 1926 that alum-precipitated diphtheria toxoid is a more potent vaccine than the toxoid alone [1]. As early as 1937, Freund developed the most effective adjuvant known, an oil-in-water emulsion containing killed mycobacteria [2]. This mixture became famous as ‘complete Freund's adjuvant’ (CFA). The components of these and other classical adjuvants are chemicals or are derived from pathogenic microorganisms. However, several substances of mammalian origin possess adjuvant-like features and are therefore also candidates for use in vaccinations. Today, adjuvants are believed to contribute to the immune response in at least two distinct ways. First, they convert the antigen into a form that is more accessible to the immune system. For example, alum-precipitated antigen is more particulate and therefore more readily ingested by phagocytes. Moreover, the antigen is released with delayed kinetics. Second, adjuvants provide immunostimulatory signals that tell the adaptive immune system to take the provided antigen seriously. During a challenge with pathogenic microorganisms or viruses, this second signal is provided by danger signals that are either pathogen-derived structures or substances released by the host itself. This chapter focuses on the latter: endogenous substances with immunostimulatory capacities. Several cytokines, low molecular weight substances, and proteins are able to provide immunostimulatory signals. However, heat shock proteins (HSPs) possess in addition amazing ‘immunological’ properties that serve the adaptive arm of immunity. Therefore, they are the focus of this chapter.

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7.2 Heat Shock Proteins in Immunology 7.2.1 General Remarks

Elevated expression of HSPs is initiated in every cell in response to a variety of stress situations, such as high temperature, anoxia, heavy metals, or glucose starvation, which promote the denaturation of proteins. Because the structural integrity of proteins is crucial for all biological processes, the survival of cells is threatened under these conditions. HSPs bind to hydrophobic stretches of denatured proteins, thereby preventing their aggregation (chaperone function). Moreover, HSPs initiate the refolding of their bound substrate or proteolytic degradation if the unfolding was irreversible. These proteins thus confer elevated stress tolerance to the cells they are expressed in [3]. However, also under nonstressed conditions most HSPs are constitutively expressed in high copy numbers and fulfil their chaperone function – assistance in de novo protein folding, subunit assembly, and transport between cellular compartments [4]. HSPs are divided into six protein families that are named according to their molecular weight: small HSPs, HSP40, HSP60, HSP70, HSP90, and HSP100. The individual members of these families – although most are very similar in sequence – are located in different subcellular compartments and are differentially regulated. For example, the HSP70 family is represented in the ER by BIP, in the mitochondrion by Grp75, and in the cytosol by the constitutively expressed Hsc70 and the stress-inducible Hsp70. The most intensely studied HSP from the immunological point of view is the ER-resident Hsp90 homologue Gp96, but other proteins mainly from the HSP60, HSP70, and HSP90 families have also been described as possessing immunological functions (Table 7.1). Tab. 7.1 Chaperones with immunogenic functions. Chaperone family

Member with immune functions

Localization

Peptide APC binding 1) activation2)

HSP60 HSP70

Hsp60 Hsc70 (Hsp73) (constitutive form) Hsp70 (Hsp72) (stress-inducible form) Hsp110 (Hsp105) Grp170 (Orp150) Hsp90 a/b (Hsp86/Hsp84) Gp96 (Grp94, endoplasmin) calreticulin (calregulin, Erp60)

mitochondrial cytoplasmic

– ++

++ +

cytoplasmic

++

+

cytoplasmic ER lumen cytoplasmic ER lumen ER lumen

+ + ++ ++ +

– – + + –

HSP110 subfamily GRP170 subfamily HSP90 Calreticulin family

1) – not demonstrated or proposed; + indirect evidence by cross-presentation or immunization experiments; ++ additional direct proof by peptide elution or x-ray structure for HSP70 chaperones 2) – not demonstrated or proposed; + some evidence, but contradicting reports; ++ clear evidence, well accepted

7.2 Heat Shock Proteins in Immunology

7.2.2 Heat Shock Proteins Are Immunogenic

The immunogenic potential of HSPs was first demonstrated by Srivastava and coworkers (reviewed in [5]). Gp96 purified from tumor cells was able to confer protective immunity in mice against a subsequent challenge with the same tumor, but not with a distinct one. Moreover, treatment of mice with HSPs could be used for therapy against a pre-existing primary tumor and its metastases [6]. Again, only tumorderived, but not liver-derived, Gp96 was able to elicit a specific immune response against the tumor. But in these experiments the HSPs purified from the tumor were not mutated compared to HSPs in healthy tissue. Instead, immunity was induced by tumor-specific peptides bound to the HSPs. The requirement for peptide binding has been validated by findings that HSPs deprived of peptides do not induce immunity [7] and that loading of ‘empty’ HSPs with peptides results in reconstitution of HSP immunogenicity [8]. Although Gp96 is the best described HSP concerning its role in the induction of specific immune responses, other HSPs have since been shown to perform similar functions: the constitutive and inducible cytosolic members of the HSP70 family [7], Hsp90 [9], calreticulin [10, 11], Hsp110, and Grp170 [12]. All these proteins have the ability in common to bind intracellular peptides and form potent immunogenic HSP-peptide complexes that can be used as vaccines. HSP-mediated immunity is independent of the MHC haplotype of the cell from which the HSPs were isolated. Peptides complexed by HSPs purified from cells with one haplotype induce immunity in mice of a different MHC haplotype [13]. This phenomenon – the independence of priming from cellular MHC haplotype – has been called cross-priming [14] and was recently shown to be required if the antigen is not expressed by the professional APC itself [15]. In vivo, cross-priming would be essential for inducing immune responses against non-APCs that have been infected by viruses or other pathogens or have turned into tumor cells. Viral or tumor antigens would then be transferred to APCs, where they are cross-presented to elicit a potent T cell response. A model for the mechanism by which HSP-bound peptides are cross-presented by APCs to prime specific CD8+ T cells is depicted in Figure 7.1 and explained in the following sections. 7.2.3 Heat Shock Proteins Bind Peptides

The fact that several HSPs can act as peptide carriers is not really surprising, in terms of their primary chaperone function: they bind to partially unfolded proteins preventing their aggregation. This is achieved by interaction with hydrophobic stretches, which are normally buried and not exposed on the surface. The HSP70 family members bind with high affinity to small hydrophobic segments in extended conformation. Due to negatively charged surfaces at the ends of the hydrophobic binding tunnel, basic residues flanking a hydrophobic core of 4 to 5 residues are preferred. Since the affinity for the bound peptide segment seems not to be altered by distant parts of the substrate proteins [16], small peptides bind as strongly as pep-

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Fig. 7.1 The current model for the physiological role of HSPs in cross-priming. During the courseof, for example, a viral infection, pathogen-derived proteins are produced by the infected host cell. Due to the usual protein turnover, viral peptide fragments are generated by the proteasome and may be presented on MHC class I molecules. HSPs are associated with such peptides in vivo. If the virus causes necrotic death of the infected cells, the HSP–peptide complexes are released and can be taken up by APCs in a receptor-mediated fashion via CD91 and LOX-1. The APC can represent the HSP-ligated viral peptides on its MHC class I molecules, thereby enabling its recognition by naive CD8+ T cells. Moreover, released HSP activates APCs via TLR4 and TLR2, resulting in the up-regulation of costimulatory molecules and in the secretion of various proinflammatory cytokines and chemokines. This second signal enables the priming of the naive CD8+ T cells, triggering their development into effector cytotoxic T cells (CTLs). These cells are now able to kill virus-infected cells without further costimulatory signals (see colour plates page XXXV).

tides, in the context of partially denatured proteins. Substrate binding to HSP70s is regulated by adenosine nucleotides. Binding of ATP to the conserved N-terminal domain of the chaperone induces a conformational change that allows rapid substrate exchange. However, spontaneous hydrolysis of the cofactor to ADP traps the bound substrate, which can then be released only after ADP–ATP exchange [17]. The 90kDa heat shock proteins also possess a nucleotide binding pocket, but its role is less clear. ATP binds with low affinity to Hsp90 and induces the formation of a ‘molecular clamp’ [18]. The function of this conformational transition is still unclear, but

7.2 Heat Shock Proteins in Immunology

ATP binding to Hsp90 is crucial for the function of this chaperone in vivo [19]. The anti-tumor drug geldanamycin binds exclusively to the unusual nucleotide binding pockets of Hsp90 and Gp96 [20]. Several functional studies with nucleotide derivatives have been performed, and an influence of nucleotide binding on oligomerization and substrate affinity was observed for both HSPs [21–23]. Therefore, it was proposed that at least for Gp96, ADP and ATP work as negative regulators of substrate binding [23]. The peptide binding motif of Gp96 is even less well defined than that of Hsp70, but a preference for uncharged residues at positions 2 and 9 of the peptide has been reported [24]. HSPs can be loaded with peptides in vitro [8], but how do they acquire peptides inside the cell? Most peptides inside the cell are generated by the proteasomes. They are produced as intermediates of protein turnover and are further degraded by cytosolic and ER-resident peptidases [25–29]. There are hints that the concentration of free peptide in the cytosol is very low and that their half-life is in the range of seconds [30]. Therefore, only a very small fraction of the generated peptides escape via the TAP transporter into the ER and can be presented on MHC class I molecules. Nevertheless, there are hints that HSPs can associate with peptides in vivo. The cytosolic Hsp70 and Hsc70 are physically linked to the proteasome via BAG-1 and might therefore be able to bind to peptides directly after their generation and before their destruction by peptidases [31]. Similarly, Hsp90 binds directly to the proteasome, thereby inhibiting its proteolytic activity [32, 33]. Gp96 and Grp170 are peptide acceptors in the ER in vivo, although their peptide binding capacities are clearly exceeded by that of protein disulfide isomerase (PDI) [24, 34]. It has been proposed that the peptides that end up in the MHC binding groove never occur as free molecules, but are chaperoned by HSPs from their place of origin to the MHC class I molecule, in the cytosol as well as inside the ER. But so far, little functional evidence supports this intriguing ‘relay line hypothesis’ [35], although the drug deoxyspergualin, which specifically binds to Hsp70 and Hsp90, inhibits the presentation of generated antigenic peptides on MHC class I molecules, for example after virus infection [36]. In the same study, HSP chaperoned peptide elicited a stronger immune response than free peptide when introduced into the cytosol. Because of their broad binding specificity, HSPs are associated with a pool of peptides that represent the protein content of the cell they originate from. This pool also includes several possible MHC ligands. Purification of HSPs from tumor cells or infected cells therefore represents an important alternative for the acquisition of immunogenic material without the need to identify individual antigens. 7.2.4 Receptor-mediated Uptake of HSPs

The simple fact that HSPs bind peptides does not explain the amazing immunogenic potential of HSP–peptide complexes. As little as 9 mg of tumor-derived Gp96 injected subcutaneously into a mouse is sufficient to initiate a protective immune response against a subsequent tumor challenge [9]. Moreover, only a very small portion of the injected HSP carries immunogenic peptides. It was proposed very early that

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receptor-mediated uptake of the antigenic material by specialized APCs might contribute to this enormous sensitivity [35]. The first evidence to support this hypothesis was provided by EM binding studies using gold particle-labeled Hsc70 and Gp96 [37]. In this study, specific binding to monocytic and dendritic cell lines and subsequent endocytosis was shown. This observation was confirmed in confocal microscopy studies [38]. Evidence for binding to specific receptors was finally provided by flow cytometry studies, using fluorochrome-labeled Hsc70 [39] or Gp96 [40]. In these reports, saturation and competition by nonlabeled Gp96 was shown, demonstrating the specificity of the HSP–receptor interaction. It was also revealed that only APCs like macrophages, DCs, and B cells, but not T cells, bind Gp96 in a receptormediated fashion. Our current knowledge of HSP receptors is summarized in Table 7.2. CD91, also known as a2-macroglobulin receptor or low-density lipoprotein receptor-related protein, was identified as the first HSP receptor by cross-linking experiments with immobilized Gp96 [41], and it was suggested that CD91 is a common receptor for Gp96, Hsp90, Hsp70, and calreticulin [42]. The classical ligands for CD91 are proteases or proteases complexed by their cognate inhibitors (e. g., a2-macroglobulin) Tab. 7.2 Proposed HSP receptors and their functions. Receptor

Interacting HSPs (ligands)

Expressing cells

CD91

Gp96, Hsp90, Hsp70, calreticulin, (activated a2-macroglobulin)

DCs, macrophages, endocytosis, platelets signaling

CD36

Gp96, (thrombospondin, macrophages, Plasmodium falsiparum, platelets oxidized LDL)

scavenger receptor

unknown

LOX-1

Hsp70, (oxidized LDL)

DCs

scavenger receptor

important for cross-presentation of ligand associated peptides

CD40

mycobacterial Hsp70, (CD40L)

APCs

APC activation CC-chemokine release

TLR4, MD-2, Hsp70, Hsp60, Gp96, CD14 (LPS)

APCs, monocytes, neutrophils, regulatory T cells

cell activation

upregulation of costimulation, cytokine and chemokine secretion, NO synthesis

TLR2

APCs, monocytes, neutrophils

cell activation

upregulation of costimulation, cytokine and chemokine secretion, NO synthesis

Hsp70, Hsp60, Gp96, (lipoproteins, zymosan)

General function

Immunological function important for cross-presentation of ligand associated peptides

7.2 Heat Shock Proteins in Immunology

[43]. These ligands are then endocytosed and degraded, thereby regulating the extracellular proteolytic activity. Interestingly, a2-macroglobulin itself can associate with peptides, and the resulting complexes can prime CD8+ T cell responses in vivo [44]. However, open questions remain regarding the role of CD91 as an HSP receptor, because CD91+ Chinese hamster ovary (CHO) cells do not bind GP96 [45]. Also, scavenger receptors have been proposed to be at least partly responsible for HSP binding to APCs: macrophages from CD36-deficient mice show a 50 % reduced Gp96 binding compared to wild-type cells, and expression of this scavenger receptor in an otherwise nonexpressing cell line conferred on the cells the ability to bind Gp96 [46]. Concerning Hsp70 binding on dendritic cells, the scavenger receptor lectin-like oxidized lipoprotein receptor-1 (LOX-1) (and not CD36) was shown to be the main receptor that mediates cross-presentation of Hsp70-bound peptides [47]. Finally, CD40 has been identified as an HSP receptor, which is expressed on APCs and was originally described as transmitting activating signals from CD4+ T helper cells mediated by CD40 ligand. Direct interaction of mycobacterial and human Hsp70 with CD40 has been demonstrated, and this binding is enhanced in the presence of Hsp70 peptide substrates [48, 49]. However, the interplay of all these receptors and their relevance for the interaction with HSPs still have to be worked out in more detail. On the other hand, it is now well established that the receptor-mediated targeting of chaperone–peptide complexes to APCs is an important feature that strongly enhances their immunogenicity. 7.2.5 Cross-presentation Pathways for HSP–Peptide Complexes

The receptor-mediated uptake of HSP–peptide complexes is a prerequisite for efficient re-presentation of the associated peptide, but it is not the only requirement: antigens presented by MHC class I molecules usually derive from cytosolic proteins [50]. However, the so-called cross-presentation of exogenous antigens on MHC class I molecules has been shown for antigens supplied in several different forms; among these are HSP–peptide complexes [51]. The pathways taken by the chaperone-associated peptides inside the cell are still not fully understood and remain controversial. It is well accepted, however, that receptor-mediated endocytosis of HSPs is crucial for the re-presentation of associated peptides [39;40]. Some reports indicate that uptake via CD91 is required for the re-presentation of HSP-associated peptides [41, 42] and can be inhibited by the CD91 ligand a2-macroglobulin and antibodies against CD91. But CD91-independent re-presentation has also been reported [45]. In these experiments Gp96 did not colocalize with CD91 after endocytosis and re-presentation was not inhibited by excess of a2-macroglobulin. During the next step, endocytosed Gp96, in contrast to HSP taken up by pinocytosis, travels to vesicles enriched in MHC class I and II molecules but does not reach late endosomes [40, 52]. Two distinct pathways for the cross-presentation have been described (Figure 7.2): on the one hand, a cytosolic route depending on proteasomal activity and TAP [53]; on the other hand, an endosomal pathway in which the endocytosed antigen meets MHC molecules in endocytic vesicles. As a variation of the lat-

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Fig. 7.2 The two pathways of cross-presentation. Two pathways for the presentation of HSP-ligated peptides inside the APC have been proposed after their receptor-mediated uptake. (A) The endosomal pathway includes the transfer of HSP–peptide complexes into special endosomal compartments, where they colocalize with MHC class I and II. There the peptides are possibly transferred onto recycled empty MHC molecules from the cell surface. (B) The cytosolic pathway requires the transfer of peptide or HSP–peptide complex into the cytosol. This process remains enigmatic. However, once the peptides have reached the cytosol, they are processed via the usual class I presentation pathway. The proteasome is required for peptide trimming, and the TAP transporter shuttles the generated peptides into the ER, where they are presented on MHC class I molecules.

ter pathway, the peptides generated by processing in endocytic vesicles might be ‘regurgitated’ and loaded onto empty MHC molecules at the cell surface [54]. Concerning Gp96–peptide complexes, hints for both pathways have been described. Re-presentation of the peptide from Gp96 complexed with the Kb-restricted CTL epitope SIINFEKL from ovalbumin probably occurs via the endosomal pathway, because it does not require de novo synthesis of MHC class I molecules [52]. The re-presentation of an elongated epitope derived from vesicular stomatitis virus requires proteasomal and TAP function, favoring the cytosolic pathway [42]. For Hsp70, a dependency of the preferred pathway on the nature of the associated peptides has been demonstrated [39]: C-terminally extended epitopes have to take the cytosolic route and require proteasomal processing; N-terminally extended peptides can be trimmed via

7.2 Heat Shock Proteins in Immunology

the endosomal pathway. However, the mechanism of antigen delivery to the cytosol is still enigmatic, and the rules that define whether an antigen is processed via either or both of these routes still have to be determined in more detail. 7.2.6 Danger Signals – The Importance of the Second Signal

The surprising path of HSP-associated peptides into the binding groove of MHC molecules of APCs is not sufficient for the induction of a specific immune response. In 1975 Lafferty and Cunningham performed experiments that suggested the requirement of a second signal for priming of naive T cells [55]. This signal, named ‘costimulation’, is received by T cells from stimulator cells (today known as antigenpresenting cells, APCs). Costimulation was later assigned to molecules on the APC site including CD80, CD86, and CD137, which specifically interact with receptors on the T cell site (e. g., CD28). This interaction leads to interleukin-2 (IL-2) responsiveness and IL-2 expression in T cells and therefore allows their proliferation and maturation in an autocrine and paracrine fashion. The absence of costimulation during TCR engagement leads to tolerance instead of priming. But the expression of costimulatory signals on APCs is activation-dependent, and APC activation is a critical key point in the induction of a specific immune response. In the original self–nonself (SNS) model proposed by Burnet and supported by experiments of Medawar, the induction of an immune response depends only on cells of adaptive immunity (B and T lymphocytes), which are taught the definitions of ‘self ’ early in their development. Janeway refined the SNS model to account for the importance of costimulation and proposed a distinct sense of self–nonself discrimination [56]. He proposed that APCs are poor T cell stimulators (due to lack of costimulation) until they are activated by pathogen-associated molecular patterns (PAMPs) recognized via a few germline-encoded pattern-recognition receptors. According to this, the decision made by APCs is between ‘infectious-nonself ’ and ‘noninfectious-self ’. In 1994, the model was further refined by Matzinger, who introduced the ‘danger model’ (reviewed in [57]): she proposed that the immune system does not care about self and nonself or infectious and harmless, but is activated upon receiving danger signals, either exogenous PAMPs or endogenous substances released from suffering cells, such as those exposed to pathogens, toxins, or mechanical damage. 7.2.7 Heat Shock Proteins as Danger Signals

Bacterial lipopolysaccharide (LPS) was perhaps the first stimulus with proven APCstimulating capacities, but discoveries of many other pathogen-derived substances followed [58]. Moreover, the content of cells undergoing necrosis (but not apoptosis) can activate macrophages and DCs [59]. This is in line with Matzinger’s model, because harmful (necrotic) cell death should lead to the release of danger signals. Surprisingly, the first component of this immunostimulatory cocktail to be identified was a heat shock protein. Chlamydial [60], as well as human Hsp60 [61], was demon-

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strated to activate macrophages, vascular endothelium, and smooth muscle cells; and this activation is mediated in a CD14-dependent manner [62] via Toll-like receptor (TLR) 4 in combination with the secreted cofactor MD-2 or via TLR2 [63, 64]. It is unlikely that Hsp60 is involved in cross-presentation, as shown for the HSPs from the HSP90 and HSP70 families, because no peptide-binding capacities have been found so far. HSP90 and HSP70, both have peptide-binding capacities and are able to activate APCs: Gp96 [65, 66], Hsp90 [65], and Hsp70 [67] activate dendritic cells, and their signal is mediated via TLR4/MD-2 and TLR2 [68, 69]. Interestingly, Hsp60 and Gp96 have to be endocytosed for transduction of activating signals, possibly because they meet the TLRs in endocytic vesicles [64, 69]. The maturation signals provided by HSPs are mediated inside the cell via the usual downstream TLR signaling pathways [70]: the adaptor protein myeloid differentiation factor 88 (MyD88) links the activated receptor to the serine kinase IL-1 receptor-associated protein kinase (IRAK), which is subsequently phosphorylated. Phosphorylated IRAK dissociates from the receptor complex and associates with TNF receptor-activated factor 6 (TRAF6). Via further unknown mechanisms, this leads to activation of the transcription factors c-Jun N-terminal kinase (Jnk), p38 mitogen-activated protein kinase (MAPK), and NF-kB. Finally, stimulation of APCs with HSPs leads to secretion of proinflammatory cytokines, such as TNF- a and IL-6, which strongly promote TH1 responses, to the production of the inflammatory mediator nitric oxide by macrophages [63, 71], and to up-regulation of costimulatory molecules that are required for priming naive T cells. Therefore, these antigen-independent immunostimulatory capacities accomplish the function of HSPs as cross-priming carrier perfectly, making them a powerful tool for the induction of specific immune responses. The activating stimuli provided by HSPs are still controversial. It is a major concern that these observations might be due to minor endotoxin contaminations in the protein preparations. Two groups report that recombinant human Hsp70 with very low endotoxin content showed no APC-activating capacities [72, 73]. In one report, human Gp96 failed to induce activation of human monocytes and DCs under serum-free conditions [74]. But there is growing evidence that, for Gp96 at least, the protein itself is responsible for APC maturation. First, Gp96-induced, but not endotoxin-induced, DC maturation is heat-sensitive and polymyxin-insensitive (Braedel et al., unpublished observations). Second, cells transfected with membrane-bound Gp96 [75] (and Hilf et al., unpublished observations) and cells that secrete Gp96 [76] activate APCs in the absence of bacterial contamintions. Third, the threshold for DC activation is independent of the observed endotoxin content of the protein preparations and reaches saturation at about 50 mg mL –1 Gp96 (Hilf et al., unpublished observations). Interestingly, this amount equals the concentration that favors complete dimerization of the cytosolic equivalent Hsp90 [77]. Therefore, the activating capacities of Gp96 might depend on the protein dimer. Of course, the fact that endotoxins and HSPs use the same receptors to mediate their maturation signal tempts speculation about endotoxin contaminations, and it is still possible that Gp96 potentiates the effect of very low LPS concentrations by binding and delivering it via receptormediated uptake to cells. But on the other hand, several different exogenous PAMPs from very different pathogens have been shown to bind to, for example TLR2. There-

7.2 Heat Shock Proteins in Immunology

fore, and in line with Matzinger's danger hypothesis [57], it is not surprising that several and – for HSPs – also endogenous danger signals bind to a single TLR. The message given by LPS and released HSPs is the same: something is wrong, and adaptive and innate immunity have to be alert. The APC activation mediated by HSPs might even be sufficient to lead to the expansion of T cells of a certain specificity in the absence of antigenic peptide [78]. Recently, a publication from Nicchitta’s laboratory attributed the protective immunity conferred by Gp96 mainly to the general immunostimulatory features of the HSP [76]. In these experiments, immunization with transfected cells that secrete soluble Gp96 induced some tumor protection (slower tumor growth) irrespective of the cell type chosen as source of the protein. Moreover, Gp96 retarded tumor growth even in the absence of its C-terminal domain, which possesses a peptide binding site [79]. These results obviously contradict the early observations of Srivastava and of others that tumor protection is restricted to the tumor the Gp96 had been purified from. However, with the tumor system chosen by Nicchitta and coworkers, no specific immune response could be induced, even with tumor lysate – the tumor line was simply not immunogenic. In the experiments published by Srivastava, the tumor lysate did induce tumor protection and the success of Gp96 vaccination was limited to the tumor from which the protein was purified [80]. But even in this work a slight tumor-independent retardation of tumor growth was observed, which is likely to be mediated by the general immunostimulatory capacities of Gp96. Therefore, it is reasonable to ascribe the immunogenicity of HSPs to both their cross-presentation function, serving specifically the adaptive immune system, and their APC activating capacities, which generally support immune responses. 7.2.8 Heat Shock Proteins as Endogenous Adjuvants

It is a matter of speculation whether the in vivo concentrations of Gp96 and other endogenous heat shock proteins after necrotic cell death are high enough to elicit APC activation. However, measurable concentrations of Gp96 have been detected in the supernatant of cells infected with a lytic virus [81] and in the wound fluid of patients with severe tissue damage (S. Herter, unpublished observations). Gp96 is the most abundant protein in the ER. Release of the ER contents after necrosis might therefore lead to a very high local concentration of this HSP. Moreover, the intradermal injection of as little as 1 mg of Gp96 led to massive infiltration of CD11c+ DCs into the draining lymph nodes from the point of injection. These DCs were of the mature phenotype and were highly efficient in stimulating T cells [82]. Taking into account that all endogenous danger signals released after necrotic cell death may act synergistically it is very likely that sufficient concentrations for APC activation can occur in vivo. If cell death leads to the release of danger signals at APC-activating concentrations, this signal has to be controlled in situations in which sustained inflammation and potential autoimmune responses have to be prevented, such as in wound healing or systemic shock after release of inflammatory substances in the blood. The ef-

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fects of Gp96 in the blood might be neutralized by excessive amounts of the CD91 ligand, a2 macroglobulin [41], and by the huge number of platelets that have been shown to bind this HSP [83]. The control of Gp96 action by platelets might be even more effective in the prevention of chronic inflammation after injury. In this scenario, activated platelets express 10-fold higher amounts of Gp96 receptors and are able to interfere with Gp96-induced DC activation. But APCs and platelets are not the only bone-marrow-derived HSP-targeted cells. Recently, it was shown that granulocytes and monocytes express Gp96 receptors [84]. In particular, neutrophils comprise the major fraction of blood leukocytes and are an important part of the innate immune response. The binding of Gp96 to these cell types triggers IL-8 release and enhances phagocytic activity of neutrophils. Interestingly, the cross-priming induced by Gp96–peptide complexes requires a positive feedback loop between NK cells and DCs to allow expansion of specific CD8+ T cells, which also requires perforin and IFN- g [85]. Although the mechanism of this interaction is not clear, these data demonstrate that HSP-induced immunity results from a complex interplay of several components of the immune system. The direction in which an immune response is guided by an adjuvant is another important point that has to be considered. For HSPs, this question has not yet been definitely answered. It is reported that Gp96 expressed on the surface of LPS-activated B cells functions as a TH2-promoting molecule [86]. In contrast to this, recent data from our laboratory indicate that Gp96-activated DCs, in contrast to LPS-activated DCs, favor the expansion of antigen-specific CD8+ T cells over CD4+ T cell expansion in vivo and in vitro [87]. 7.2.9 Clinical Use of Heat Shock Proteins

The combined properties of HSPs to prime specific immune responses by the transfer of antigenic peptides with concurrent activation of innate immune cells is the rationale behind utilization of HSPs in clinical settings. These approaches include the induction of therapeutic immune responses by vaccination in patients with malignant and also infectious diseases. HSP-based immunization therapy using a personalized tumor vaccine is currently under clinical evaluation. This vaccine is produced from individual tumor tissue and is called heat shock protein peptide complex 96 (HSPPC-96; Oncophage). HSPPC96 consists of purified Gp96 from patient cancer tissue complexed with peptides specific for a patient's tumor and has been tested in several clinical trials, including on patients with melanoma, colorectal cancer, gastric cancer, kidney cancer, and several other cancers. The clinical results of using HSPPC-96 in various cancer patients so far demonstrate that this kind of treatment is well tolerated and can induce favorable immune responses against tumor or virus [88]. On the other hand, the results also show that HSP-based vaccination might not be effective in all patients, but only in a subpopulation. Several studies, including a phase III trial with HSPPC-96, are still under way, but at this point no research group has reported of any severe adverse events,

7.3 Cytokines as Adjuvants

again indicating the feasibility and safety of this approach. Further studies are needed to determine the efficacy of such treatments and to define patient populations who will benefit most from HSP-based vaccination treatment. Over all, these studies are encouraging and indicate that it is possible as well as feasible to improve an antigen-specific immune response by using adjuvants that mediate distinct signals to innate immune cells, for example, via TLRs. In particular, HSPs enhance and influence the immune response in several ways that are more than a simple, unspecific immunostimulatory function.

7.3 Cytokines as Adjuvants

The HSPs discussed so far as endogenous adjuvants are unique in that these proteins can deliver antigens to antigen-presenting cells and concurrently provide the inflammatory signals needed in addition to mount an adaptive immune response. As already mentioned, these inflammatory signals include the up-regulation of costimulatory molecules, as well as the release of proinflammatory cytokines by local cells, for example, resident DCs. Therefore, another way to induce specific immune responses by vaccination is to provide the antigen and add proinflammatory substances, to mimic or induce an inflammatory environment and recruit immune cells to present the respective antigen. A variety of such inflammatory mediators of endogenous origin are known, available as recombinant proteins, and used as adjuvants in vaccination protocols. Several reports have shown that subcutaneous or intradermal administration of granulocyte macrophage colony-stimulating factor (GM-CSF) together with an antigen is able to induce CTL responses in cancer patients [89–91]. Also IL-1a and IL-1b or fragments of IL-1b have proven efficacy, for example, in mucosal immunizations [92, 93]. Further studies have evaluated the adjuvant application of T cell-stimulating cytokines such as IL-2 or IL-12 for cancer immunotherapy. IL-2 has already demonstrated beneficial effects as single agent without additional antigen administration against renal cancer and other tumor entities [94]. Although studies using IL-12 as a single agent were disappointing, since it lacked efficacy in the course of repeated administration at the maximal tolerated dosages [95], IL-12 at low doses, as well as IL-2, have demonstrated powerful properties as adjuvants in both animal models and humans [96, 97]. Although tremendous efforts have been put into research on utilizing cytokines as adjuvants, and the basic proof-of-concept has been made, it is still unclear what cytokines will give the best results in vaccination [96]. More recent studies suggest that the maximum dose may not be the one that will give the best response [98]. In addition, evidence is accumulating that a combination of cytokines might be more effective than a single cytokine. For example, GM-CSF in combination with IL-12 or CD40L is synergistic in CTL induction [99, 100]. Also, the triple combination of GMCSF, IL-12, and TNF-a was most effective in some mouse models [101].

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7.4 Concluding Remarks

Recent discoveries clearly underline the importance of the influence of the innate immune system on the development of adaptive immune responses. Cytokines produced and surface molecules expressed during the activation of innate immune cells determine whether a TH1- or TH2-biased immune response is initiated. Learning more about this interaction will allow us to modulate immune responses in vaccination protocols. HSPs might be of special interest in this regard, since they have the unique feature of combining antigenicity and adjuvanticity, which determine immunogenicity, within the same molecule. Their use in clinical trials will determine if these features can be exploited in a therapeutic setting.

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8 Microparticles as vaccine adjuvants and delivery systems Derek T. O’Hagan and Manmohan Singh

8.1 Introduction

Traditional vaccines have mainly consisted of live attenuated pathogens, whole inactivated organisms, or inactivated bacterial toxins. These approaches have mainly been successful due to the induction of antibody responses, which neutralize viruses or toxins, inhibit the binding of microorganisms to cells, or promote their uptake by phagocytes. However, to develop vaccines against more ‘difficult’ pathogens, which often establish chronic infections, for example HIV, HCV, TB, and malaria, the induction of more potent cellular immune responses may be required, including the induction of Th1 cytokines and cytotoxic T lymphocyte (CTL) responses. Unfortunately, nonliving vaccines have generally proven ineffective at inducing potent Th1 responses or CTL. Although live vaccines can induce CTL, live attenuated vaccines may cause disease in immunosuppressed individuals, and some pathogens are difficult or impossible to grow in culture (e. g., HCV) or present significant safety concerns (e. g., HIV). As a result of the limitations of traditional approaches, several new approaches to vaccine development have emerged in recent decades, including (1) recombinant protein subunits, (2) synthetic peptides, (3) protein–polysaccharide conjugates, and (4) plasmid DNA. Although these new approaches offer advantages, a general problem is that they are often poorly immunogenic. Traditional vaccines often contained contaminating components that can elicit additional T cell help or function as adjuvants, for example, bacterial cell components, including LPS. However, these components have been largely eliminated from new-generation vaccines, to make them safer and less reactogenic. Therefore, they are likely to need more potent adjuvants to allow them to be effective vaccines. A notable exception is protein– polysaccharide conjugate vaccines, which contain the same bacterial toxoids that were included in traditional vaccines. The toxoids function by providing T cell help for antibody induction against the conjugated polysaccharides. Therefore, conjugate vaccines have so far proven effective with only ‘traditional’ adjuvants, based on insoluble aluminum salts. Immunological adjuvants were originally described by Ramon [1] as ‘substances used in combination with a specific antigen that produced a more robust immune

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response than the antigen alone’. This broad definition encompasses a very wide range of materials [2]. However, despite extensive evaluation of a large number of candidates over many years, the only adjuvants included in products approved by the US Food and Drug Administration are aluminum-based mineral salts, which are generically called alum. Although alum has an excellent safety record, comparative studies show that it is a weak adjuvant for antibody induction to recombinant protein vaccines and induces Th2, rather than Th1 cellular response [3]. In addition, alum is not effective for the induction of mucosal immunity and can induce IgE antibody responses, which have been associated with allergic reactions in some people [3, 4]. Although alum has been used as an adjuvant for many years, its mechanism of action remains poorly defined. It was originally thought to provide a ‘depot’ effect, resulting in persistence of antigen at the injection site. However, more recent studies suggest that alum does not establish a depot [5], but appears to up-regulate costimulatory signals on human monocytes and promotes the release of IL-4 [6], a Th2 cytokine. Importantly, alum may contribute to a reduction in toxicity for some vaccines, due to the adsorption of contaminating LPS [7]. A key issue in adjuvant development is toxicity, since safety concerns have restricted the development of adjuvants since alum was first introduced more than 70 years ago [8]. Many experimental adjuvants have advanced to clinical trials and some have demonstrated high potency, but most have proven too toxic for routine clinical use. For standard prophylactic immunization of healthy individuals, only adjuvants that induce minimal adverse effects will prove acceptable. Additional practical issues that are important for adjuvant development include biodegradability, stability, ease of manufacture, cost, and applicability to a wide range of vaccines.

8.2 The Role of Adjuvants in Vaccine Development

Adjuvants can be used to improve the immune response to vaccine antigens in several different ways, including (1) increasing the immunogenicity of weak antigens, (2) enhancing the speed and duration of the immune response, (3) modulating antibody avidity, specificity, isotype, or subclass distribution, (4) stimulating CTL, (5) promoting the induction of mucosal immunity, (6) enhancing immune responses in immunologically immature or senescent individuals, (7) decreasing the dose of antigen in the vaccine to reduce costs, and (8) helping to overcome antigen competition in combination vaccines. Unfortunately, the mechanisms of action of most adjuvants are complex and often remain poorly understood. Nevertheless, significant advances have been made in this area recently, following the identification of key receptors on cells of innate immunity which are activated by a variety of known adjuvants. However, if one accepts the geographical concept of immune reactivity, in which antigens that do not reach the local lymph nodes do not induce responses [9], it becomes easier to propose mechanistic interpretations for adjuvants which work predominantly through a ‘delivery’ mechanism, including microparticles. If antigens that do not reach lymph nodes

8.2 The Role of Adjuvants in Vaccine Development

do not induce responses, then any adjuvant or delivery system that enhances delivery of antigen into the cells that traffic to the lymph node may enhance the response. A subset of dendritic cells (DC) are thought to be the key cells for antigen presentation; these cells circulate in peripheral tissues and act as ‘sentinels’, being responsible for the uptake of antigens and their transfer to lymph nodes. Circulating immature DCs are efficient for antigen uptake, which then mature and present antigen to T cells in lymph nodes. Hence, antigen uptake into DC, the trafficking of DC to lymph nodes, and the triggering of DC maturation are thought to be key components in the generation of potent immune responses. Although DC are thought to be the most effective antigen-presenting cells (APC), macrophages are also effective APC. Although adjuvants are notoriously difficult to classify, and many examples resist easy definitions, they can be divided into two broad groups based on their principal modes of action, according to whether or not they have direct immunostimulatory effects on APC or function as ‘delivery systems’ to promote antigen uptake into APC. Adjuvants that have direct effects on APC are often derived from pathogens and are referred to as displaying pathogen-associated molecular patterns (PAMP) [10, 11]. PAMP adjuvants are perceived as ‘danger signals’ and induce the release of proinflammatory cytokines from innate immune cells, which triggers and controls the adaptive immune response [12, 13]. Traditional vaccines, including bacterial toxoids and inactivated viruses, often contain most of the components of pathogens and induce potent immune responses. In contrast, new-generation vaccines, including recombinant proteins, are highly purified, lack many of the features of the original pathogen, and do not evoke strong responses. Therefore, the role of adjuvants in recombinant vaccines is to ensure that the vaccine sufficiently resembles an infection to initiate a potent immune response [10, 12]. In addition, adjuvants can control the type of acquired immune response induced [14]. Although this chapter focuses primarily on microparticles as vaccine-delivery systems, microparticles have also been used as delivery systems for adjuvants. Therefore, we also briefly introduce some of the immunostimulatory adjuvants that have been used in conjunction with microparticles. In addition, we briefly discuss alternative particulate-delivery systems, including emulsions, liposomes, and ISCOMs, which have also been used as delivery systems for antigens and adjuvants. Formulating adjuvants into delivery systems, including microparticles, serves two principal purposes – the adjuvants can be targeted to APC to enhance their effects, and their potential for toxicity can be minimized by limiting their systemic distribution. In relation to the minimization of toxicity, controlled release of adjuvants has significant potential, since the peak concentration of adjuvant can be dramatically reduced.

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8.3 Immunostimulatory Adjuvants 8.3.1 MPL

MPL (monophosphoryl lipid A) is derived from the LPS of the gram-negative bacteria Salmonella minnesota and therefore represents an archetypal PAMP. Like LPS, MPL is thought to interact with Toll-like receptor 4 (TLR4) on APC, resulting in the release of proinflammatory cytokines. In several preclinical studies, MPL was shown to induce the synthesis and release of IL-2 and IFN-g, which promote the generation of Th1 responses [15, 16]. Clinically, MPL has often been used as a component of complex formulations, including liposomes and emulsions, and has also been used in combination with alum and QS21 [17]. Overall, MPL has been extensively evaluated in the clinic (>12,000 subjects immunized) for cancer (melanoma and breast) and infectious disease vaccines (genital herpes, HBV, malaria and HPV), and for allergies, with an acceptable profile of adverse effects. MPL is marketed in Europe for use in combination with allergy vaccines [18]. 8.3.2 CpG

In the past few years, a whole new class of adjuvants have been identified, following the demonstration that bacterial DNA, but not vertebrate DNA, had direct immunostimulatory effects on immune cells in vitro, inducing B cells to proliferate and produce immunoglobulins [19, 20]. The immunostimulatory effect is due to the presence of unmethylated cytosines in the CpG [21], which are under-represented and methylated cytosines in vertebrate DNA. Unmethylated CpG, in conjunction with selective flanking sequences, is believed to be recognized by the innate immune system to allow discrimination of pathogen-derived DNA from self DNA [22]. Active CpG exert their effects on many cell types, including a subset of DC, called plasmacytoid DC, which mature and secrete cytokines in response to CpG activation. Cellular responses to CpG DNA depend on the presence of TLR9 [23]. In addition, CpG are taken up by nonspecific endocytosis, and endosomal maturation is necessary for cell activation and the release of proinflammatory cytokines [24]. The potent Th1 adjuvant effect of CpG appears to be maximized by their conjugation to protein antigens [25]. CpG appears to have potential for the modulation of preexisting immune responses, which may be useful in various clinical settings, including treatment of allergies [26]. 8.3.3 QS21

A third group of immunostimulatory adjuvants is the triterpenoid glycosides, or saponins, derived from Quil, which is extracted from the bark of a Chilean tree,

8.4 Particulate Vaccine Delivery Systems

Quillaja saponaria. Saponins have been widely used as adjuvants for many years and have been included in several veterinary vaccines. QS21, which is a highly purified fraction from Quil A, is a potent adjuvant for Th1 cytokines (IFN-g) and antibodies of the IgG2 a isotype, which indicates a Th1 response in mice [27]. Saponins intercalate into cell membranes, through interaction with structurally similar cholesterol, forming holes or pores [28]. However, it is currently unknown if the adjuvant effect of saponins is related to pore formation in cells. Several clinical trials have been performed, using QS21 as an adjuvant, initially in cancer vaccines (melanoma, breast and prostate), and subsequently in vaccines for infectious diseases, including HIV-1, influenza, herpes simplex, malaria, and hepatitis B [29]. More than 3500 people have been immunized with QS21. Doses of 200 mg or higher of QS21 have been associated with significant local reactions [29], but lower doses appear to be better tolerated. 8.3.4 Cytokines

Most cytokines have the ability to modify and redirect the immune response. The cytokines that have been evaluated most extensively as adjuvants include IL-1, IL-2, IFN-g, IL-12, and GM-CSF [30]. However, all of these molecules exhibit dose-related toxicity. In addition, since they are proteins, they have stability problems, have a short in vivo half-life, and are relatively expensive to produce. Therefore, it is unlikely that cytokines will prove broadly acceptable for use as adjuvants in vaccines designed to protect against infectious diseases. Nevertheless, considerable progress has been made in the use of cytokines for the immunotherapy of cancer [31]. Microparticles have been used as delivery systems for cytokines, including GM-CSF [32] and IL-12 [33], mostly for use in oncology.

8.4 Particulate Vaccine Delivery Systems

Particulates (e. g., emulsions, microparticles, ISCOMs, liposomes, virosomes, and virus-like particles) have comparable dimensions to the pathogens that the immune system evolved to combat and are efficiently taken up by APC. Therefore, these agents have been used as vaccine-delivery systems. Immunostimulatory adjuvants may also be included in the delivery systems to enhance the level of response or to focus the response toward a desired pathway. In addition, formulating potent immunostimulatory adjuvants into delivery systems may limit adverse events, through restricting the systemic circulation of the adjuvant.

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8.4.1 Lipid-based Particles as Adjuvants

A potent oil-in-water (o/w) adjuvant, the Syntex adjuvant formulation (SAF) [34], was developed by using a biodegradable oil (squalane) in the 1980s, as a potential replacement for Freund’s adjuvants. Freund's adjuvants are potent, but toxic, water-inmineral oil adjuvants, which may also contain killed mycobacteria [35]. However, SAF contained a bacterial cell wall-based synthetic adjuvant, threonyl muramyl dipeptide (MDP), and a nonionic surfactant, poloxamer L121, and proved too toxic for widespread use in humans [8]. Therefore, a squalene o/w emulsion was devloped (MF59), without the presence of additional immunostimulatory adjuvants, which proved to be a potent adjuvant with an acceptable safety profile [36]. MF59 enhanced the immunogenicity of influenza vaccine [37–39] and is a more potent adjuvant than alum for hepatitis B vaccine (HBV) in baboons [40] and in humans [41]. Subsequently, the safety and immunogenicity of MF59-adjuvanted influenza vaccine (FLUAD) was confirmed in elderly subjects in clinical trials [42, 43], and these data allowed this product to be approved for licensure in Europe in 1997. In addition, MF59 was shown to be safe and well tolerated in newborn infants in a HIV vaccine trial [44]. MF59 has also been used as an effective booster vaccine, following a priming immunization with live virus [45] or DNA [46] vaccines. In summary, MF59 is a safe and well tolerated vaccine adjuvant in humans and is effective for the induction of potent antibody responses. In many subsequent studies, similar emulsions have been used as delivery systems for immunostimulatory adjuvants, including MPL and QS21. Liposomes are phospholipid vesicles and have been evaluated both as adjuvants and as delivery systems for antigens and adjuvants [47, 48]. Liposomes have been commonly used in complex formulations, often including MPL, which makes it difficult to determine the contribution of the liposome to the overall adjuvant effect. Nevertheless, several liposomal vaccines based on viral membrane proteins (virosomes) without additional immunostimulators have been extensively evaluated in the clinic and are approved as products in Europe for hepatitis A and influenza [49]. The immunostimulatory fractions from Quillaja saponaria (Quil A) have been incorporated into lipid particles comprising cholesterol, phospholipids, and cell membrane antigens, which are called immune-stimulating complexes or ISCOMs [50]. In a study in macaques, an influenza ISCOM vaccine was more immunogenic than a classical subunit vaccine and induced enhanced protective efficacy [51]. A similar formulation has been evaluated in human clinical trials and was claimed to induce CTL responses [52]. The principal advantage of the preparation of ISCOMs is that it allows a reduction in the dose of the hemolytic Quil A adjuvant and targets the formulation directly to APCs. In addition, within the ISCOM, the Quil A is bound to cholesterol and is not free to interact with cell membranes. Therefore, the hemolytic activity of the saponins is significantly reduced [50, 53]. However, a potential problem with ISCOMs is that inclusion of antigens in the adjuvant is often difficult and may require extensive antigen modification [54].

8.4 Particulate Vaccine Delivery Systems

8.4.2 The Adjuvant Effect of Synthetic Particles

The adjuvant effect achieved as a consequence of linking antigens to synthetic particles has been known for many years and was previously reviewed by O’Hagan [55]. However, the particles used in the early studies were nondegradable and were therefore not appropriate for development as vaccine adjuvants for human use. In addition, antigens were often chemically conjugated to the nondegradable particles, adding an extra level of complexity and making commercial development less likely [56]. Particulate delivery systems present multiple copies of antigens to the immune system and are thought to promote trapping and retention of antigens in local lymph nodes. In addition, antigen uptake by APC is enhanced by association of antigen with particles. Biodegradable and biocompatible polyesters, the polylactide-coglycolides (PLG), are the primary candidates for the development of microparticles as adjuvants, since they have been used in humans for many years as resorbable suture material and as controlled-release drug-delivery systems [57, 58]. The adjuvant effect achieved through the encapsulation of antigens into PLG microparticles was first demonstrated by several groups in the early 1990s [59–62]. 8.4.3 Uptake of Microparticles into APC

It is assumed that the uptake of microparticles into APC is important for the particles’ ability to function as vaccine adjuvants, but the uptake of microparticles (< 5 mm) by phagocytic cells has been demonstrated on many occasions. An early paper [63] described the uptake of microparticles (1–3 mm) by macrophages, but showed that 12-mm microparticles were not taken up. It was subsequently shown that maximal uptake of microparticles occurred with particles of < 2 mm [64, 65]. In addition, the surface charge and hydrophobicity of the microparticles modified their uptake [65]. It has also been reported that macrophages that carry microparticles to lymph nodes can mature into DC [66]. In addition, uptake of PLG microparticles directly into DC has been demonstrated both in vitro [67] and in vivo [68]. 8.4.4 Microparticles as Adjuvants for Antibody Induction

The adjuvant effect achieved through the encapsulation of antigens into PLG microparticles was first demonstrated by several groups in the early 1990s [59–62]. Both O’Hagan et al. (1991) and Eldridge et al. (1991) showed that microparticles with entrapped antigens, ovalbumin (OVA) and staphylococcal B enterotoxoid, respectively, had immunogenicity comparable to that of Freund’s adjuvant. Particle size is an important parameter affecting the immunogenicity of microparticles, since smaller particles (< 10 µm) were significantly more immunogenic than larger ones [59, 62]. The effect of particle size on immunogenicity is likely to be a consequence of enhanced uptake of smaller particles into APC. The adjuvant effect of microparticles can be

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8 Microparticles as vaccine adjuvants and delivery systems Fig. 8.1 Serum IgG antibody responses following intramuscular immunization of mice with recombinant p55gag protein adsorbed to PLG microparticles (PLG/ p55) or p55 gag alone (25 mg), at 0 and 4 weeks. Geometric mean titer (GMT) ± s.e. is shown for each group (n = 10).

further enhanced by their coadministration with additional adjuvants [61, 69]. Early studies in mice suggested that microparticles induced antibodies of predominantly the IgG2 a isotype, indicating a Th1 response [70]. However, the potential of microparticles as adjuvants has been limited by many reports describing the denaturation of antigens during and following microencapsulation [71]. Therefore, we developed a novel approach, in which the antigen is adsorbed onto the surface of PLG microparticles that have been modified to promote adsorption [72]. This approach allowed the induction of significantly enhanced antibody titers in mice with an adsorbed recombinant p55 gag antigen from HIV-1 (Figure 8.1). Similar anionic microparticles have also been used to induce potent antibody responses against a recombinant antigen from Neisseria meningitides type B, and the potency of the microparticles did not depend on the choice of anionic surfactant used to make them (Table 8.1). A somewhat related approach was also described, in which a novel charged polymer was used to prepare nanoparticles, which were able to adsorb tetanus toxoid for mucosal delivery of antigens [73].

Tab. 8.1 Immunogenicity of a model meningitis B vaccine on PLG microparticles. Formulation PLG/DSS/MB1 + CpG PLG/SDS/MB1 + CpG PLG/PVA + MB1 + CpG Alum/MB1 + CpG

Two weeks after 3rd immunization Geometric mean titer MB1 Bactericidal titer 67 921 29 911 1065 12 236

4096 4096 < 16 1024

Serum antibody and bactericidal titers in groups of mice immunized with PLG microparticles prepared with two alternative anionic detergents, dioctyl sodium sulfosuccinate (DSS) and sodium dodecyl sulfate (SDS). The anionic PLG microparticles performed as a delivery system for an adsorbed recombinant antigen from Neisseria meningitides type (MB1) and were compared with alum formulations ± CpG adjuvant and also with the antigen in Freund's complete adjuvant (CFA). Geometric mean titers and BCA for each group (n = 10) are shown.

8.4 Particulate Vaccine Delivery Systems

8.4.5 The Induction of Cell-mediated Immunity with Microparticles

Several early studies showed that microparticles exerted an adjuvant effect for the induction of CTL responses in rodents [74–77]. Moreover, microparticles also induced a delayed-type hypersensitivity (DTH) response and potent T cell proliferative responses [75]. In a recent study, we evaluated the ability of microparticles with adsorbed p55 gag to induce CTL responses in rhesus macaques. Previously, we showed that this formulation induced CTL responses in rodents (Figure 8.2) [72] and was more potent than microparticles with entrapped antigen (unpublished data). Although the anionic PLG microparticles were not effective for CTL induction in nonhuman primates, they did induce potent antibody and T cell proliferative responses. Although we did not evaluate microparticles with entrapped antigens, we do not believe that they would have been any more potent than the formulations with adsorbed antigen. Hence, microparticles appear to be capable of inducing CTL responses in rodents with protein antigens, but are ineffective in nonhuman primates. In contrast to the adjuvanted protein approaches, naked DNA was a potent inducer of CTL responses in macaques. 8.4.6 Microparticles as Delivery Systems for DNA Vaccines

DNA vaccines have been repeated shown to offer significant potential for the induction of potent CTL responses [78]. Nevertheless, the potency of DNA vaccines in humans has so far been disappointing, particularly in relation to their ability to induce antibody responses [79, 80]. This has prompted investigators to work on adjuvants and delivery systems for DNA vaccines and also to use DNA in a prime–boost setting with alternative modalities [81–83].

Fig. 8.2 Cytotoxic T lymphocyte (CTL) responses from splenocytes (measured as specific lysis of peptide-pulsed target cells at different effector/target cell ratios) in groups of mice ( n = 5) immunized intramuscularly with soluble recombinant p55 gag protein at 10 mg, 25 mg, or 50 mg, or with 10 mg p55 adsorbed onto PLG microparticles (PLG/gag). As a positive control, a group of mice were infected intraperitoneally with recombinant vaccinia virus vector expressing the p55 gag and polymerase proteins.

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An early study suggested that microparticles with entrapped DNA may have potential as an improved DNA vaccine, although there was no direct comparison with naked DNA [84, 85]. Nevertheless, it soon became clear that the approach of encapsulating DNA into microparticles had several limitations, similar to those previously described for protein antigens, involving damage during microencapsulation and low encapsulation efficiency [86–88]. To avoid these problems, we developed a novel cationic PLG microparticle formulation, which adsorbed DNA onto the surface [85]. Importantly, the cationic microparticles induced enhanced immune responses in comparison to naked DNA (Figure 8.3), and this enhancement was apparent in all species evaluated, including nonhuman primates (Figure 8.4). In addition, the cationic microparticles efficiently adsorbed DNA and could deliver several plasmids simultaneously at high loading levels [89, 90]. The microparticles appear to be effective predominantly as a consequence of efficient delivery of the adsorbed DNA into DC [91]. A similar approach was recently described, in which DNA was adsorbed onto the surface of novel cationic emulsions [92].

Fig. 8.3 Serum IgG antibody responses following intramuscular immunization in mice at 0 and 4 weeks with PLG/DNA (encoding p55 gag) administered in doses of 1 and 10 mg, in comparison to naked DNA at the same dose levels. Geometric mean titer (GMT) + s.e. is shown for each group (n = 10).

Fig. 8.4 Serum IgG antibody responses in two groups of rhesus macaques after intramuscular immunization at 0 and 4 weeks with PLG/DNA (encoding p55 gag) in 500 mg dosesin comparison to naked DNA at the same dose level. Geometric mean titer (GMT) + s.e. is shown for each group (n = 5).

8.4 Particulate Vaccine Delivery Systems

8.4.7 Microparticles as Delivery Systems for Adjuvants

The potency of microparticles with adsorbed antigens has been significantly improved by their coadministration with adsorbed adjuvants [93]. Simultaneous delivery of antigens and adjuvants on microparticles ensures that both agents are delivered into the same APC population. We used cationic PLG microparticles to adsorb the polyanionic adjuvant CpG, to induce enhanced serum antibody responses against HIV-1 gp120 protein that was also adsorbed to microparticles [93]. Tabata and Ikada were the first to entrap an adjuvant, muramyl dipeptide (MDP), in microspheres [94], and Puri and Sinko [95] showed that MDP entrapped in microspheres induced enhanced immune responses. However, Tabata and Ikada [64] previously showed that the pyrogenicity of MDP was reduced by microencapsulation, establishing that microparticles can improve potency and also reduce reactogenicity for adjuvants. PLG microparticles have also been used for delivery of QS21 adjuvant in combination with gp120 antigen [96]. However, although studies with an implantable osmotic pump showed that higher titers were obtained with a discontinuous rather than a continuous release profile for the adjuvant [96], the studies also showed that, although the adjuvant was critical for induction of high initial titers, it was not required for the secondary response. Therefore, the easiest approach was to suspend the microparticles containing the antigen in a solution containing the adjuvant, so that it was immediately available to enhance titers. It remains to be determined whether it is more attractive to encapsulate adjuvants, to adsorb them onto microparticles, or to simply to coadminister them, which may vary for different adjuvants. Encapsulation or adsorption offers the opportunity to minimize the peak local concentrations of adjuvant, which might reduce adverse effects, and to ensure that the adjuvant is taken up efficiently into APCs. Although both approaches offer the potential for controlled release of the adjuvant, the duration of release is expected to be longer for an entrapped agent. Nevertheless, it may be more desirable to have the adjuvant available early in the response, for optimal enhancement. Hence, simple coadministration might work for some adjuvants, but this might depend on their mechanism of action. If they need to be internalized in APC to interact with receptors, then association with the microparticle might be preferred. Adsorption might be preferred to ensure maximal availability of adjuvant after uptake into APC. Microparticles can also be used in conjunction with traditional adjuvants, including alum [97, 98]. In addition, microparticles can also be combined with emulsionbased adjuvants to improve their potency [99, 100]. In a recent study, we highlighted the potential of microparticles as a cancer vaccine [101]. 8.4.8 Microparticles as Single-dose Vaccines

In the early to mid 1990s, several studies were undertaken to evaluate the potential of controlled-release microparticles for the development of single-dose vaccines,

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which would result in improved vaccine compliance, particularly in the developing world. The majority of this work focused on tetanus toxoid (TT), although additional work was also performed with diphtheria toxoid (DT), gp120, hepatitis B surface antigen, and human chorionic gonadotrophin [102]. Commercially, PLG microparticles have been developed as controlled-release formulations for a variety of drugs, including a recombinant protein (human growth hormone). Although microparticle formulations with entrapped antigens could induce potent, long-lasting immune responses in rodents, it was clear that microencapsulated toxoids had significant instability problems [103, 104]. Various approaches have been undertaken to attempt to stabilize toxoids in microparticles, but with limited success [105, 106]. Although the successful development of a product with an entrapped recombinant protein shows that that instability problems can be overcome for PLG microparticles, the solution applied to hGH involved both a novel process and also protein-specific modifications [71]. Unfortunately, experience suggests that each protein for microencapsulation will present its own difficulties, requiring specific solutions, and there is unlikely to be a universal formulation approach that can be applied to all vaccine antigens. Recently, a multivalent approach was described, with four childhood vaccines entrapped in PLG microparticles [107]. Nevertheless, although vaccines entrapped in PLG microparticles have induced long-term responses in small-animal models, early studies showed that alum formulations could also induce similar responses [74, 108]. Hence, the slow decay kinetics of antibody responses in rodents makes them less than ideal for evaluating the potential of controlled-release formulations in which ‘pulses’ of antigen release are designed to provide booster responses. Overall, there is little evidence for formulations providing in vivo boosting in rodents, but it is difficult to see how a response that is maintained at a high level can be boosted. An additional problem relates to the optimal release profile that might be preferred to induce long-term responses in larger animals, including humans. The maintenance of high titers for extended periods in small animals makes them less than ideal for evaluating the effects of different release formulations. Although there have been many claims that a ‘pulse’ of antigen release is preferable to continuous release, the earliest studies showing the benefits of controlled-release technology for vaccines showed that a continuous release profile induced high titers [109]. Moreover, many subsequent studies have shown that both continuous- and discontinuous-release formulations can induce potent immune responses in small-animal models. When we developed a PLG microparticle formulation that was designed specifically for continuous release, we were surprised to find that it induced immune responses comparable to those of PLG microparticles previously designed for discontinuous release [110]. Overall, which release profile is desirable to induce the most potent immune responses in larger animals, including humans, remains unclear. Moreover, it seems unlikely that such questions can be adequately addressed in small-animal models. It is disappointing to note that, after more than 10 years of work, we cannot find any studies in which PLG microparticles with entrapped vaccines have been evaluated in nonhuman primates or humans, although there have been suggestions for some time that clinical trials are imminent [111].

8.5 Alternative Routes of Immunization

8.4.9 Alternative Particulate Delivery Systems

Alternative polymers, including polyanhydrides, poly(ortho)esters, hyaluronic acid, chitosan, and starch, have been evaluated as microparticles for antigen delivery [102], as have polymers that self-assemble into particulates (poloxamers) [112] and soluble polymers (polyphosphazenes) [113]. However, the potency, safety, and tolerability of these approaches remain to be further evaluated. Although advantages are often claimed for these approaches, they are often not clear and are rarely demonstrated in comparative studies. The commercial use of PLG in several marketed products continues to make it an attractive approach, despite some well-documented limitations [102]. Recombinant proteins that naturally self-assemble into particulates are also an attractive approach for enhancing delivery of antigens to APC. The first recombinant protein vaccine, based on hepatitis B surface antigen (HBsAg), was expressed in yeast as a particulate protein [114]. Recombinant HBsAg is potently immunogenic and can be used to prime CTL responses [115]. HBsAg and other virus-like particles (VLPs) can also be used as adjuvants for coexpressed proteins [116]. For example, recombinant VLPs expressed from the yeast Saccharomyces cerevisiae prime CTL responses in mice after a single immunization [117]. In addition,VLPs induce CTL activity in macaques against coexpressed SIV p27 [118]. In addition, clinical trials of VLPs showed them to be safe and immunogenic in humans [119].

8.5 Alternative Routes of Immunization

Although most vaccines have traditionally been administered by intramuscular or subcutaneous injection, mucosal administration of vaccines offers several important advantages, including easier administration, reduced adverse effects, and the potential for frequent boosting. In addition, local immunization induces mucosal immunity at the sites where most pathogens initially establish infection. In general, systemic immunization fails to induce mucosal IgA antibody responses. Oral immunization would be particularly advantageous in isolated communities, where access to health care professionals is difficult. Moreover, mucosal immunization would avoid the potential problem of infection due to the reuse of needles. Several orally administered vaccines are commercially available, which are based on live-attenuated organisms, including vaccines against polio virus,Vibrio cholerae, and Salmonella typhi. In addition, a wide range of approaches is currently being evaluated for mucosal delivery of vaccines [120], including many approaches involving nonliving adjuvants and delivery systems, including microparticles [121, 122]. The most attractive route for mucosal immunization is oral, due to the ease and acceptability of administration through this route. However, due to the acidity of the stomach, the broad range of digestive enzymes in the intestine, and a protective coating of mucus that limits access to the mucosal epithelium, oral immunization with

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nonliving antigens has proven extremely difficult. However, novel delivery systems and adjuvants may be used to significantly enhance immune responses following oral immunization. 8.5.1 Mucosal Immunization with Microparticles

In mice, oral immunization with PLG microparticles induces potent mucosal and systemic immunity to entrapped antigens [55, 123–125]. In addition, mucosal immunization in rodents with microparticles induces protective immunity against challenge with Bordetella pertussis [126–129], Chlamydia trachomatis [130], Salmonella typhimurium [131], Streptococcus pneumoniae [132], and ricin toxin [133]. In primates, mucosal immunization with microparticles has induced protective immunity against challenge with SIV [134] and staphylococcal enterotoxin B [135]. Comparative studies in small-animal models have shown that microparticles are one of the most potent adjuvants available for mucosal delivery of vaccines [136]. In addition, microparticles have shown promise for the mucosal delivery of DNA [137, 138]. The ability of microparticles to perform as effective adjuvants following mucosal administration is largely a consequence of their uptake into specialized mucosal-associated lymphoid tissue (MALT) [139], including MALT in the nasal cavity of rodents [140]. The potential of microparticles and other polymeric delivery systems for mucosal delivery of vaccines was recently reviewed [121, 122], as was the use of alternative delivery systems [141]. Mucosal delivery of vaccines has been undertaken with microparticles prepared from a range of polymers, including PLG, starch, chitosan, sodium alginate, and hydrogels. However, encouraging data is largely restricted to small-animal models, and studies in larger animals, including studies in humans, have largely been disappointing [142, 143]. Hence, although microparticle formulations have significant potential for the mucosal delivery of vaccines, accumulated experimental evidence suggests that simple encapsulation of vaccines into microparticles is unlikely to result in the successful development of oral vaccines and that improvements in the current technology are clearly needed [144]. Nevertheless, microparticles may contribute to the successful development of mucosal vaccines by functioning as delivery systems for both antigens and adjuvants. 8.5.2 Microparticles as Delivery Systems for Mucosal Adjuvants

The most potent mucosal adjuvants currently available are the bacterial toxins from Vibrio cholerae and Escherichia coli, cholera toxin (CT) and heat-labile enterotoxin (LT). However, since CT and LT are respectively the causes of cholera and travelers’ diarrhea, they are considered too toxic for mucosal administration in humans. Therefore, they have been genetically manipulated to reduce toxicity [145–147]. Single amino acid substitutions in the enzymatic A subunit of LT allowed the development of mutant toxins that retained potent adjuvant activity but showed negligible or dramatically reduced toxicity [148–150]. LT mutants have been administered by the oral

8.5 Alternative Routes of Immunization

route to induce protective immunity in mice against H. pylori challenge [151]. In addition, LT mutants have been shown to be potent oral adjuvants for influenza vaccine [152] and model antigens [153]. Nevertheless, due to the significant challenges associated with the development of oral vaccines, which should not be under-appreciated, LT mutants have been evaluated for alternative routes of immunization, including nasal, intravaginal, and intrarectal. Of these, intranasal immunization offers great promise, because of the potent responses induced by this route, the easy access, and the simple administration devices that already exist. On many occasions, the ability of LT mutants to induce potent immune responses after intranasal immunization has been demonstrated [154]. Moreover, in recent studies, LT mutants have induced protective immunity against challenge with B. pertussis [155], S. pneumoniae [156], and herpes simplex virus [157]. In addition, intranasal immunization with LT mutants induces potent CTL responses [158, 159]. We recently showed that the potency of LT mutants can be enhanced by their formulation into a novel bioadhesive microsphere delivery system (Figure 8.4) [93]. A similar observation was made in a second study, confirming the ability of bioadhesive microspheres to enhance the potency of LT mutants [160]. Importantly, we showed recently that the potency of LT mutants for intranasal immunization with a second vaccine was not affected by the presence of preexisting immunity to the adjuvant [161].

Tab. 8.2 Evaluation of an influenza vaccine on bioadhesive HYAFF microparticles. Formulation

Route

HA alone (25 mg)

Intramuscular

HA (25 mg) + LTK63 (100 mg)

Intranasal

HA (25 mg) + LTK63 (100 mg) + HYAFF (25 mg)

Intranasal

Hemagglutination inhibition (HI) titers 640 160 160 80 1280 640 320 40 2560 1280 640 320

Serum hemagglutination inhibition titers at week 6, after immunization at weeks 0 and 4, in 3 groups of 4 micropigs with either 25 mg hemagglutinin (HA) alone by the intramuscular (IM) route, intranasally (IN) with the mucosal adjuvant LTK63 (HA+LTK63), or IN with the mucosal adjuvant LTK63 delivered with bioadhesive microspheres (HYAFF) prepared from esterified hyaluronic acid (HA+LTK63+HYAFF). The table shows responses of individual animals.

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8.6 Adjuvant for Therapeutic Vaccines

It seems increasingly likely that novel adjuvants may prove sufficiently potent to allow the development of therapeutic vaccines and that microparticles may have a role to play here by improving the potency of the adjuvants. Rather than preventing infection, therapeutic vaccines would be designed to eliminate or ameliorate existing diseases, including (1) chronic infectious diseases, for example those caused by HSV, HIV, HCV, HBV, HPV, or H. pylori; (2) tumors, for example melanoma, breast, or colon cancer; and (3) allergic or autoimmune disorders, for example multiple sclerosis, Type I diabetes, and rheumatoid arthritis. Generally speaking, the level of toxicity acceptable for an adjuvant to be used in a therapeutic situation is likely to be higher than for a prophylactic vaccine designed to be used in healthy individuals, particularly if the vaccine is designed to treat cancer or the life-threatening consequences of an infectious disease. Recently PLG microparticles with encapsulated DNA have been evaluated in humans as a cancer vaccine and have shown some evidence of clinical benefit [162]. In addition, cationic microparticles with adsorbed DNA have also shown potency as a cancer vaccine in a preclinical study [101].

8.7 Future Developments in Vaccine Adjuvants

Several recent problems highlight the urgent need for the development of new and improved vaccines. These problems include (1) the lack of success of traditional vaccine approaches against ‘difficult’ organisms, for example HIV and HCV; (2) the emergence of new diseases, for example Ebola, West Nile, and Hanta viruses; (3) the reemergence of ‘old’ infections, for example TB; (4) the continuing spread of antibiotic-resistant bacteria; and (5) the potential use of microorganisms for bioterrorism. The induction of CTL responses may be necessary for some vaccines, including HIV, but accumulated information shows that induction of potent CTL is difficult with protein-based vaccines. Therefore, DNA is an attractive approach, but needs to be delivered more effectively to improve its potency in humans. Microparticles may have a significant role to play in improving the potency of DNA vaccines (Figures 8.3 and 8.4). Targeted delivery of antigens and adjuvants to specific APC populations via microparticles may reduce the adverse effects of adjuvants or help to achieve a specific desired response. Targeting may be achieved at several different levels, including tissue-specific delivery to local lymph nodes, cell-specific targeting to APC, or targeting to subcellular compartments within APC, for example the proteasome to promote CTL or the nucleus for DNA vaccines. However, targeting may also be achieved through the use of ligands designed to specifically interact with receptors on APC, including TLR, which evolved to recognize various components of bacteria and viruses. An alternative target is the mannose receptor, which has been used to target

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Acknowledgments

We thank our colleagues at Chiron Corporation for their contributions to the ideas contained in this review, particularly Rino Rappuoli. We also thank all the members of the Vaccine Adjuvants and Delivery Group at Chiron. Thanks are also due to Nelle Cronen for her help in preparing the manuscript.

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139. O’Hagan DT. The intestinal uptake of particles and the implications for drug and antigen delivery. J Anat 1996, 189(3), 477–482. 140. Eyles JE, Spiers ID, Williamson ED, Alpar HO. Tissue distribution of radioactivity following intranasal administration of radioactive microspheres. J Pharm Pharmacol 2001, 53, 601–607. 141. Michalek SM, O'Hagan DT, GouldFogerite S, Rimmelzwaan GF, Osterhaus ADME. Antigen delivery systems: nonliving microparticles, liposomes, cochleates, and ISCOMs. In: Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstrock J, McGhee JR, eds. Mucosal Immunology. San Diego: Academic Press, 1999, 759–778. 142. Tacket CO, Reid RH, Boedeker EC, et al. Enteral immunization and challenge of volunteers given enterotoxigenic E. coli CFA/II encapsulated in biodegradable microspheres. Vaccine 1994, 12, 1270–1274. 143. Lambert JS, Keefer M, Mulligan MJ, et al. A Phase I safety and immunogenicity trial of UBI microparticulate monovalent HIV-1 MN oral peptide immunogen with parenteral boost in HIV1 seronegative human subjects. Vaccine 2001, 19, 3033–3042. 144. Brayden DJ. Oral vaccination in man using antigens in particles: current status. Eur J Pharm Sci 2001, 14, 183–189. 145. Dickinson BL, Clements JD. Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity. Infect Immun 1995, 63, 1617–1623. 146. Douce G, Turcotte C, Cropley I, et al. Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants. Proc Natl Acad Sci USA 1995, 92, 1644–1648. 147. Douce G, Fontana M, Pizza M, Rappuoli R, Dougan G. Intranasal immunogenicity and adjuvanticity of site-directed mutant derivatives of cholera toxin. Infect Immun 1997, 65, 2821– 2828. 148. Di Tommaso A, Saletti G, Pizza M, et al. Induction of antigen-specific antibodies in vaginal secretions by using a

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by cholera toxin A1 subunit. J Immunol 1997, 158, 3936–3946. 170. Agren LC, Ekman L, Lowenadler B, Nedrud JG, Lycke NY. Adjuvanticity of the cholera toxin A1-based gene fusion protein, CTA1-DD, is critically dependent on the ADP-ribosyltransferase and Ig-binding activity. J Immunol 1999, 162, 2432–2440. 171. Agren L, Sverremark E, Ekman L, et al. The ADP-ribosylating CTA1-DD adjuvant enhances T cell-dependent and independent responses by direct action on B cells involving anti-apoptotic Bcl-2- and germinal center-promoting effects. J Immunol 2000, 164, 6276– 6286. 172. Goletz TJ, Klimpel KR, Arora N, Leppla SH, Keith JM, Berzofsky JA. Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein. Proc Natl Acad Sci USA 1997, 94, 12059–12064.

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9 Liposomes and ISCOMs Gideon Kersten, Debbie Drane, Martin Pearse, Wim Jiskoot, and Alan Coulter

9.1 Introduction

A dilemma currently facing vaccinologists is that the often rule is: the more defined and purified an antigen, the lower its immunogenicity. Starting with a pathogen, the removal of nucleic acids, endotoxin, cell wall components, host cell components, and other ‘irrelevant’ components in general yields a well-defined, pharmaceutically acceptable and safe vaccine. Unfortunately, such a vaccine rarely elicits strong, longlasting immune responses. After the process of stripping, purification, and polishing, the antigen needs to be reformulated and presented in a multimeric, particulate form together with an adjuvant with a high safety profile. The development of antigen carriers that mimic the outside of pathogens is a logical, and in principle, straightforward approach. Therefore, it is not surprising that within 10 y after the first description of liposomes in 1965 [1] and of ‘virus like particles’ after saponin treatment of membrane viruses in 1973 [2] their potential as antigen carriers was investigated [3, 4]. Since then, more than a 1000 papers about lipidic vesicles related to vaccinology and immunology have appeared in the literature (reviewed in [5–8]). Although immune stimulating complexes, (ISCOMs) have a profoundly different structure than liposomes (an important structural component is the Quillaja saponin adjuvant) (Figure 9.1D), they are included in this chapter because they belong to the large family of amphiphile-based vesicular antigen carriers (Table 9.1). The most important developments in the preparation and characterization of these carriers, their mechanism of action, their in vivo effects, and their status with respect to use in registered vaccines is discussed in this chapter. The archetypal, classical liposome is a vesicle composed of phospholipids (Figure 9.1A). Liposomes are highly versatile with regard to composition, size, physicochemical, and immunological characteristics. This allows a flexible design with the aim of maximizing functionality. Instead of liposomes as a single entity, one can define a whole group of liposome-like structures, many of which are being tested as antigen carriers. Most of these structures are discussed in this chapter, although

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9 Liposomes and ISCOMs Fig. 9.1 Diagrams and electron micrographs of liposomes (A), virosomes (B), cochleates (C), and ISCOMs (D). Liposome s shown are small unilamellar vesicles (about 100 nm) prepared by detergent removal. The virosome size is about 150 nm. The virosome diagram shows hemagglutinin (trimers) and neuraminidase. Cochleates are fused bilayers kept rolled up by intercalated calcium ions. ISCOMs are profoundl y different from lipid vesicles. Their main constituents are cholesterol and saponin from Quillaja saponaria. Size is 40 nm. Micrographs A and C are freeze-fracture images. B and D are negative staining. Sources: (A, diagram) W. Jiskoot, Universit y of Utrecht, (A, micrograph) A. Verkleij, University of Utrecht, (B, diagram) T. Wyler, University of Berne. Reprinted from Vaccine 21, Zurbriggen, 921, 2003 Elsevier Science,with permission, (B, micrograph) Waeltie and Gluck, Int. J. Cancer 77, 728, 1998 Wiley-Liss. Used by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc, (C, micrograph) L. Zarif et al., Drug Delivery 2, 2002, with permission, (D, diagram) Reprinted from [5] 1995 Elsevier Science with permission, (D, micrograph) courtesy of K. Teppema, M. Burger, RIVM, Bilthoven.

some may disagree on the classification ‘liposome-like’ for some vehicles, e. g., niosomes, cochleates, or outer membrane vesicles. Sometimes a different name for a certain vesicle type is merely semantic or guided by commercial motives, and sometimes the structures are really different from classical liposomes. Unlike ISCOMs, most liposomal structures contain an internal space separated from the environment (except for cochleates, Figure 9.1C). This allows the incorporation of hydrophilic antigens (or adjuvants), apart from bilayer association of more amphiphilic antigens, although it is difficult to achieve high loading yields. ISCOMs, on the other hand, are spherical structures, typically 40 nm in diameter. Unlike liposomes, ISCOMs cannot be loaded with water-soluble antigens because the vesicle contains pores [9].

9.1 Introduction Tab. 9.1 Colloidal antigen delivery systems discussed in this chapter. Name

Composition

Structure and characteristics

Liposome

phospholipids, cholesterol

bilayer vesicles, 50 nm to 10 mm characteristics highly versatile

Transfersome

phospholipids, cholate

ultradeformable bilayer vesicles optimised for transdermal delivery

Niosome

nonionic surfactants, cholesterol

nonphospholipid liposomes high chemical stability, modest physical stability

Virosome

viral phospholipids, viral membrane proteins (HA)

bilayer vesicles, ~ 150 nm intrinsic targeting properties

Proteosome/OMV

membrane proteins, phospholipids, LPS

bilayer(?) bacterial vesicles, 60–100 nm high protein : lipid ratio

Archaeosome

Archaeobacterial lipids

bilayer vesicles high chemical stability

Cochleate

phospholipids, cholesterol, calcium

nonvesicular; rolled up bilayer sheets rigid and stable, no internal aqueous space

ISCOM

Quillaja saponin, cholesterol, phospholipid

spherical aggregates of ringlike micelles, ~ 40 nm, no isolated internal volume; no bilayer(?) very high intrinsic adjuvant activity

OMV: outer membrane vesicle; HA: hemagglutinin; LPS: lipopolysaccharide; See text for details.

Another difference between liposomes and ISCOMs is that ISCOMs have potent intrinsic adjuvant properties, whereas liposomes generally have modest intrinsic adjuvant activity. Phospholipids, or other bilayer-forming molecules, are the key components of liposomes. The main building blocks of ISCOMs are saponins from Quillaia saponaria, cholesterol, and phospholipid. Phospholipid is necessary, probably as a fluidizer of the rigid cholesterol–saponin complexes, but bilayer structures are thought to be absent. Initially, ISCOMs were designed to carry membrane proteins, however today, ISCOM vaccines can be prepared with almost all types of antigens (see section 9.3.1.3). Furthermore, in some circumstances, physical association of the antigen with the ISCOM is not necessary to induce potent immune responses. Because of the noted differences in the structure and properties of liposomes and ISCOMs, they are discussed separately below.

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9.2 Liposomes and Related Structures 9.2.1 Composition, Characteristics, and Preparation Methods of Liposomes 9.2.1.1 Composition and Characteristics of Liposomes As mentioned in the Introduction, classical liposomes, i. e., vesicles composed of phospholipids and often cholesterol, are just one type of a relatively large group of colloidal particles composed of lipidic or lipid-like constituents suitable for antigen delivery. In this section the different vesicle types are described and their potential as vaccine component is discussed. Liposomes The physical properties of liposomes can vary widely as a function of composition and method of preparation. Phospholipids are the primary bilayer components. Cholesterol is often added to increase bilayer rigidity, resulting in increased membrane stability. Application of phospholipids with high phase-transition temperatures has the same effect. Surface charge and pH sensitivity (see section 9.2.2) are also modifiable. The size of liposomes ranges from 50 nm to several micrometers. They are uni- or multilamellar, depending on the method of preparation. Efficient incorporation of antigens may require extensive optimization. Liposomes can be freeze-dried, that is to say, in the drying phase liposomes partly disintegrate but reform after reconstitution. For membrane-associated antigens this is not necessarily a problem, but when hydrophilic antigens are present in the aqueous interior, freeze-drying is not possible without losing part of the load. In general, classical liposomes are good, but not very good, enhancers of the immune response. Incorporation of amphiphilic adjuvants circumvents this potential problem. Adjuvants of this type are monophosphoryl lipid A (MPL) and other LPS derivatives and dioctadecyl ammonium bromide (DDA). Interesting results in humans have been obtained with lipid A-adjuvanted liposomes (see also section 9.2.3). Liposomes can be included in oil-in-water emulsions, making it possible to employ the advantages of both delivery systems: induction of strong humoral responses by the emulsion and induction of CTL responses against liposome-encapsulated soluble antigen [9]. Liposomes can be optimized for certain tasks. For oral application, polymerization of the lipids protects the interior against low pH, proteolytic enzymes, and bile salts [10]. By using lipids conjugated to polyethylene glycol (PEG), liposomes acquire ‘stealth’ properties and are sterically stabilized. For vaccination purposes this generally is less desirable but, combined with specific targeting to antigen-presenting cells (APC), PEGylation may be beneficial [11] (see also section 9.2.2.2). Transfersomes A disadvantage of parenteral immunization is the use of needles. Reuse of needles in developing countries causes many hepatitis B and HIV infections. Second, fear of

9.2 Liposomes and Related Structures

needles and perceived pain in part of the population have encouraged vaccine developers to look for alternatives. One of these is dermal delivery of antigen by active or passive skin penetration. The rationale is that the number of dendritic cells in the viable epidermis, just under the top layer (the stratum corneum), is very high. On the other hand, the barrier function of the stratum corneum (consisting of keratin and multiple bilayers of ceramides) is formidable, and it is difficult to get macromolecules through the skin. Transfersomes are ultradeformable liposomes with enhanced skin-penetration properties. They are composed of phosphatidylcholine (PC) and cholate (9 : 2 molar ratio). It is claimed that the presence of cholate makes the vesicle ultradeformable. This enables them to squeeze through pores in the stratum corneum with a diameter down to 10 % of the transfersome size. As a result, 200- to 300-nm vesicles can pass the stratum corneum. It is thought that a hydration gradient from the dry surface to the wet viable tissue drives the transfersomes through the stratum corneum. Studies with water-soluble as well as membrane-associated model antigens have shown that dermal application of transfersomes induces responses equal to parenteral immunization [12]. There is some debate about the validity of claims that transfersomes are very effective (trans)dermal delivery systems. Experiments with other vesicular structures with potential skin-penetrating properties, such as fluid liposomes (i. e., consisting of lowmelting-point phospholipids), niosomes (see below), or ‘ethosomes’ (liposomes with high levels of ethanol), do not come close to the claimed delivery potential of transfersomes (see [13] for review). Niosomes Vesicles can be prepared without any phospholipids. As a substitute, nonionic surfactants are used. Niosomes (reviewed in [14]) are also called nonionic surfactant vesicles (NISV) [15] or Novasomes [16]. They have some advantages over classical liposomes. Synthetic nonionic surfactants are chemically more stable than phospholipids. Phospholipids are especially prone to oxidation (of unsaturated fatty acids). Surfactants are much cheaper than phospholipids. Finally, some surfactants have an intrinsic adjuvant activity, improving the immunogenicity of niosome-encapsulated antigens. Bovine serum albumin (BSA) in niosomes was shown to be as immunogenic as BSA with complete Freund’s adjuvant [11, 13]. Niosomes can be tailored to meet specific needs. Coincorporation of bile salts like deoxycholate into niosomes results in vesicles (bilosomes) that enhance immune responses after oral immunization [17]. The vesicles show some promise as transdermal delivery vehicles and may be suitable antigen carriers for this immunization route. Virosomes Liposomes prepared from viral envelopes supplemented with endogenous phospholipids are called virosomes (Figure 9.1B). They resemble small unilamellar vesicles. The presence of viral membrane proteins make virosomes more efficient in binding to APC and/or cytosolic delivery (see section 9.2.2). Virosomes prepared from influenza virus envelopes, also called IRIVs (immunopotentiating reconstituted influenza virosomes) are in advanced stages of development (see also section 9.2.3 and

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9 Liposomes and ISCOMs Tab. 9.2 Examples of clinical trials with liposome-like structures and products on the market. Vehicle

Antigen

Stage

Read out

Ref.

Lipid A-containing liposomes, Al(OH)3

malaria antigen (recombinant protein)

phase 1/2

seroconversion

95, 47

Meningococcal proteosomes

Shigella flexneri LPS

phase 1/2 (intranasal)

seroconversion

96

MDP-containing virosomes

influenza HA/NA

phase 1

functional seroconversion (HA inhibition)

97

Influenza virosomes

HepA

registered (Epaxal)

seroconversion

98–101

Influenza virosomes

influenza HA/NA

registered (Infexal V)

functional seroconversion (HA inhibition), protection

102–104

HLT containing influenza virosomes

influenza HA/NA

phase 1/2

functional seroconversion (HA inhibition)

105,106

Outer membrane vesicles

Neisseria meningitidis pore protein A

phase 1/2, phase 3

functional seroconversion (bactericidal activity), protection

49–52, 107–109

Al(OH)3 : alminum hydroxide; LPS: lipopolysaccharide; MPD: muramyl dipeptide; HA/NA: hemagglutinin/neuraminidase; HepA: hepatitis A; HLT: heat-labile toxin (E. coli).

Table 9.2). Coincorporation of membrane proteins generally induces enhanced (cellular) immune responses against these antigens. Virosomes not only show promise as parenteral delivery vehicles but also are able to boost the immune response after intranasal application (Table 9.2). Proteosomes and Outer Membrane Vesicles Proteosomes and outer membrane vesicles are of bacterial origin and, as a result, contain large amounts of bacterial components, including membrane proteins. They are prepared by solubilization of bacterial outer membranes, followed by purification steps such as ammonium sulfate precipitation and dialysis against detergent-containing buffer [18]. Electron micrographs reveal liposomal vesicles with a size of about 100 nm, but the protein:lipid ratio is higher than can be achieved with purified protein incorporated into liposomes. Proteins and peptides are noncovalently complexed to the proteosomes, making them highly immunogenic (see [19] for review). Several groups are or have been developing vesicle-based vaccines against Meningococci. These are outer membranes from Neisseria meningitidis type B containing pore proteins as antigens. Immunization with these vesicles leads to the induction of functional antibodies (see section 9.2.3 and Table 9.2).

9.2 Liposomes and Related Structures

Archaeosomes Archaeobacteria are extremophiles, often living in hostile habitats. They are resistant to low pH, high temperature, or high salt concentrations. Membranes of Archaeobacteria contain lipids that are chemically distinct from those of eukaryotic and prokaryotic species. The saturated, branched C-20, -25, and -40 phytanyl chains can form liposomes with unique properties with respect to physical and chemical stability and uptake by APCs [20]. They are less sensitive to oxidative stress, high temperature, alkaline pH, phospholipases, and bile salts. Immune responses comparable to immunization with complete Freund’s adjuvant and superior to immunization with conventional liposomes have been reported after immunization with archaeosomes [21, 22]. Cochleates Cochleates are elongated supramolecular assemblies and differ from other members of the liposome group in that they are not spherical (see [23] for review). They are bilayer sheets consisting of phosphatidyl ethanolamine (PE), negatively charged phosphatidyl serine (PS), and cholesterol. Calcium ions are added and intercalate with the bilayers. This results in a rolled-up bilayer sheet without internal volume. Despite the latter fact, cochleates can contain many types of molecules. Hydrophobic and amphiphilic molecules are taken up by the bilayer, whereas negatively and positively charged compounds interact with the calcium ions or with the phosphatidyl serine. respectively, by electrostatic interactions. The size and aggregation behavior of cochleates is influenced by the method of preparation (see section 9.2.1.2). The benefits of cochleate-based vaccines (compared to true liposomal formulations) are not yet clear and may be antigen dependent (see [24] for review). Influenza antigens in cochleates are as immunogenic (humoral response) as influenza antigens in liposomes. Cochleates may be efficient vehicles for DNA vaccines, however. Experiments with HIV-1 gp 160-encoding plasmid DNA resulted in superior induction of antigen-specific cytotoxicity in mice. For the oral route, cochleate vaccines may be beneficial because the delivery vehicle is very stable, due to the tight bilayer– bilayer interaction. It remains unclear whether the preparation of cochleates is really necessary to provide antigen protection via the oral route, since calcium levels in saliva and the stomach are high enough to fuse phosphatidyl serine liposomes into cochleates [24]. 9.2.1.2 Preparation Methods of Liposomes The preparation of plain liposomes on a small scale is not very difficult. This does not mean that high incorporation efficiency of the antigen, incorporation of the antigen with minimal activity loss, or scaling up of production is without problems. For the preparation of liposomes on a laboratory scale, many procedures have been published. The mechanisms behind three regularly used basic procedures are: (1) hydration of lipids by energy input, (2) mixing of a nonpolar phase containing the lipids with the aqueous phase, followed by removal of the organic solvent (also called reverse-phase evaporation when organic solvents are used that are not miscible with water), and (3) solubilization of the lipids by a detergent and subsequent removal of the detergent:

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(1) Lipid can be simply dispersed in water by vigorous shaking in the presence of glass beads, by bubbling a stream of nitrogen gas through the mixture, or by highshear homogenization and/or ultrasonic treatment. Liposomes prepared this way are generally large multilamellar vesicles (MLV), although rigorous ultrasonic treatment or extrusion may generate smaller, unilamellar vesicles (SUV). MLVs are suitable for the incorporation of water-soluble molecules in the liposome interior. However, high concentrations of lipids have to be used to achieve high incorporation efficiencies for this class of compounds. (2) The reverse-phase evaporation method provides large unilamellar liposomes with a higher encapsulation efficiency per mole of lipid for water-soluble, hydrophilic molecules. The lipids are solubilized in an organic solvent (chloroform), water is added, and the solvent is evaporated under vigorous stirring. (3) A method to obtain unilamellar vesicles without applying a high energy input or using an organic solvent, both of which may be destructive of (protein) antigens, requires the use of detergents [25]. The lipids are solubilized with a detergent. The detergent should have a high critical micelle concentration (CMC), so that it is easily removed by dialysis. Octylglucoside, with a CMC of 25 mM in water, is often used. During dialysis the liposomes are formed. A model for the conversion from mixed micelles to spherical structures via bilayer disks was described by Lasic [26]. Dilution is a fast way of lowering the detergent concentration. With this method SUVs can be produced with a narrow size distribution [27]. The average size of these vesicles can range from 80 to 200 nm, depending on lipid composition, protein content, and dilution rate. After the liposome-forming dilution step, the detergent is removed by dialysis. The detergent-removal method has proven to be useful for the incorporation of membrane proteins in the bilayer. The preparation of cochleates differs somewhat from that of liposomes. They can be prepared by the slow addition of a solution of calcium chloride to PE–PS liposomes. This results in liposome aggregation and subsequent formation of stacked sheets with macroscopic dimensions. Direct dialysis of mixed detergent–phospholipid micelles against calcium chloride solution generates intermediate-sized cochleates, probably without the formation of a liposome intermediate. Small cochleates are formed when calcium chloride intercalates slowly with small liposomes, which can be achieved by dialysis of preformed liposomes against a solution of calcium chloride. Relatively few immunogenicity studies have been performed with cochleates, and the importance of particle size on in vivo performance is not clear. Large-scale manufacturing processes for liposomes are available, although often these are optimized for the incorporation of relatively small drugs that can withstand the presence of organic solvents or high mechanical stress [28]. 9.2.2 Mechanisms of Action of Liposomes

Classical liposomes consisting of neutral phospholipids are relatively ineffective at enhancing the immunogenicity of antigens. The phospholipids do not act as adjuvants, and it is probably merely the multimeric presentation form that causes higher re-

9.2 Liposomes and Related Structures

sponses as compared to antigen alone. Among the exceptions are the archaeosomes. In vivo, these liposomes enhance immune responses by recruitment and activation of APC [29]. The adjuvant effect of liposomes without additional adjuvant can be optimized by manipulating the composition. The beneficial effect of lipid-based carriers may occur during one or more of the three phases between administration and antigen presentation by APC: transport from the site of administration to the APC, binding to the APC, or uptake by the APC and processing via the appropriate intracellular route. Based on this, the known mechanisms of action of liposomal systems are (1) protection and stabilization of the antigen, including the mimicry (of pathogen surface) function, (2) enhanced binding to and uptake by APC by passive or active targeting, and (3) enhanced or controlled antigen processing after uptake. 9.2.2.1 Protection, Stabilization, and Mimicry It is tempting to speculate that, for membrane antigens, the bilayer provides an environment that promotes native conformation and stabilization. Some experimental evidence suggests that this is true. Purified, detergent-solubilized Meningococcal pore protein A does induce high antibody levels but these are not functional, i. e., in the presence of complement the antibodies do not exhibit bactericidal activity. Worse, when they are mixed with bactericidal antibodies, they inhibit the bactericidal action [30]. Integration of the pore protein into liposomes does not lead to an increase in the antigen-specific antibody titers but the functional, i. e., bactericidal, titers are as high as after immunization with ‘natural’ outer membrane vesicles. Similar findings were reported for recombinant PorA [31]. Because protein conformation is important for the induction of bactericidal responses, this suggests that the liposomal formulation restores, to a certain extent at least, the conformation of the antigen. The relevance of this ability of course depends on the importance of the conformation for the induction of a functional immune response. There are indications that, in general, liposomes are no better in their capacity to refold proteins than mixed micelles consisting of lipids and detergents [32]. Mixed micelles are, however, less suitable as antigen carriers, because their structure is much more dynamic and concentrationdependent, and therefore pharmaceutically less acceptable. Dilution of mixed micelles leads to the formation of liposomes or aggregates. Some liposome-like formulations, e. g., cochleates, are likely to exert their immune-stimulating effect by efficient protection of the antigen against pH shifts, proteolytic enzymes, and, perhaps most important, dilution. The protective, containment-providing properties of liposomes are of utmost importance when demanding immunization routes, such as the oral route, are used. Inclusion of soluble antigen in the aqueous interior or membrane association by electrostatic interaction provides protection, but the classical liposomes themselves are sensitive to bile salts and therefore less suitable for immunization via the oral route. The stability of liposomes after parenteral immunization is not known, but rapid disintegration at the injection site may not be beneficial for the immune response. At least this is suggested by the observation that many physically stable formulations, such as cochleates, polymerized liposomes, and archeaosomes, are effective antigen carriers, some even via the oral route [10, 20, 24].

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9.2.2.2 Targeting Liposomal structure can be designed in such a way that binding to APC is facilitated. The most straightforward method is to include cationic lipids. Most eukaryotic cells are negatively charged. As a consequence, positively charged liposomes bind to APC (and other cells). There is evidence that this also leads to better uptake and in vivo CTL and humoral responses. Anionic and neutral liposomes are less potent in this respect (reviewed in [33]). Arigita et al. [34] showed that, in vitro, cationic liposomes containing meningococcal PorA interact much more efficiently with APC than neutral liposomes. In vivo, this resulted in more responders but not in higher bactericidal antibody titers. This type of targeting is of course not very specific, since most cells are negatively charged. The results that have been obtained with cationic liposomes show that an improved response is achievable, but it is not always clear whether this is due to enhanced binding to APC or a better loading of the liposomes or to other (unknown) mechanisms. Cationic liposomes, for example, are an excellent vehicle for genetic vaccination [35]. DNA entrapment is a simple procedure, resulting in more immunogenic (humoral as well as cellular responses) vaccines compared to naked DNA, regardless the immunization route. It is speculated that the response is more efficient because of higher direct uptake of liposomes by APC instead of transfection of myocytes, the main mechanism after intramuscular immunization with naked plasmid DNA. Specific targeting to APC can be achieved by incorporation of ligands or antibodies for receptors that are more or less specific for APC. These include the mannose receptor [34, 36, 37], the Fc receptor [38], surface IgG, or MHC molecules like HLA-DR [33]. Sterically stabilized liposomes with anti-HLA-DR Fab fragments attached to PEG chains resulted in enhanced accumulation in the lymphoid organs [11]. Although the latter approach was developed for anti-viral drug delivery, it may have applications for antigen delivery as well. Targeting with mannosylated entities can lead to improved binding in vitro [34, 37], as well as improved humoral (number of responders) [34] and CTL responses in vivo [39, 40]. The length of the spacer is critical for cellular uptake, at least for macrophages [41]. A last type of targeting that should be mentioned here is the use of influenza virosomes, containing HA as a ligand. HA binds to sialic acid residues on the cell surface, allowing receptor-mediated endocytosis by APC. For oral delivery, targeting strategies to the M-cells of intestinal Peyer’s patches have been developed [42]. The ligand used was lectin incorporated in polymerized liposomes. Double to triple increases in liposome uptake were observed, depending on the type of lectin. 9.2.2.3 Enhanced or Controlled Processing Cytoplasmic delivery of antigen is necessary for the induction of class 1 restricted antigen presentation. Fusogenic liposomes are efficient inducers of CTL responses. Three approaches are being investigated to make liposomes fusogenic. (1) The above mentioned influenza virosomes are taken up by receptor-mediated endocytosis. Endosomal acidity causes conformational changes in the HA, which render the virosome fusogenic, delivering its contents to the cytoplasm. (2) Sendai virus virosomes

9.2 Liposomes and Related Structures

containing at least F-protein seem to deliver their contents to the cytoplasm via an endocytosis-independent pathway, i. e., plasma membrane fusion [43, 44]. (3) The use of liposomes containing pH-sensitive lipids. Bilayers containing unsaturated phospholipids and dioleoylphosphatidyl ethanolamine (DOPE) are not very stable. This is because DOPE has a cone shape, reducing the ‘fit’ in bilayers and promoting nonbilayer structures such as reversed micelles. Actual pH-induced destabilization is achieved by incorporation of acidic amphiphiles such as palmitoylhomocysteine or cholesterol hemisuccinate. Protonation, for instance in the endosomal compartment, results in fusion with the endosomal membrane. These liposomes can generate CTL responses in vitro and in vivo [45]. Despite the ability of pH-sensitive liposomes to deliver their contents efficiently to the cytoplasm in vitro, in vivo the superiority of these liposomes largely disappears. It may be that in vivo APC are present at different stages of maturation, meaning that pH-sensitivity contributes only marginally to the eventual immune response [46]. Liposomes that are not fusogenic are also able to deliver antigen to the MHC class 1 route. An example are the ‘Walter Reed liposomes’ containing monophosphoryl lipid A (see [45] for review). 9.2.3 Liposome Performance and Products It is difficult to rank antigen-delivery systems according to their potential. Comparative studies are seldom performed, and with good reason. Comparative studies are large and expensive and do not provide general answers. Probably, there are no general answers. The antigen–adjuvant–carrier combination is what determines immunogenicity. A carrier that works well with antigen A may give moderate results with antigen B. Then there is the problem of reproducible preparation of these vaccines. Also, apparently insignificant differences in the methods of preparation, the quality of the raw materials, the purity of the antigens, the immunization scheme, the dose, and the animal species and strain can make the difference between failure or success. Finally, a success-determining factor is the willingness of research institutes and industry to move to human trials as soon as preclinical data permit. Numerous studies have been published demonstrating the ability of liposomal formulations to protect experimental animals (see [47] for review), but further product development and human trials are extraordinarily expensive. Nevertheless, a few general conclusions about safety and potency of liposomal structures can be drawn. To start with, liposomes without additional adjuvant are able to improve the humoral and cellular immune response against incorporated antigens. Addition of adjuvants, targeting molecules, or certain lipids may lead to more potent products without compromising safety. Second, liposomes are very safe. We are not aware of human trials reporting serious side effects. Several liposomal vaccine formulations have been tested in the field (Table 9.2). What has to be taken into consideration, however, is that the results of trials with a negative outcome are often not published. Berna in Switzerland has two virosome-based products: Infexal V, a flu-virosome and Epaxal, a hepatitis A virosome vaccine (www.bernabiotech.com, March 2003). The so-called Walter Reed liposomes have been tested in several human studies. Reports of these studies show high immunogenicity and minimal adverse effects. It is thought that the Walter

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Reed formulations display these beneficial effects because of the presence of lipid A or MPL as well as Al(OH)3 as additional adjuvants. Al(OH)3 has the additional advantage that it neutralizes the potential residual endotoxin activity of the LPS derivatives [48]. A group of vesicular vaccines that is approaching market are meningococcal vesicle vaccines containing pore protein A. Several human trials have been performed by Norwegian, Cuban, and Dutch groups, demonstrating that these vaccines are safe and induce bactericidal antibody responses in humans [49–51]. A complicating issue is that the protective immune response is serosubtype-specific. For an acceptable coverage of such a vaccine, several serosubtypes have to be included in the vaccine. Hexavalent vaccines have been tested in human trials with good results. The vaccine was well tolerated by children and seroconversion rates were high [52].

9.3 ISCOMs 9.3.1 Composition, Characteristics, and Preparation Methods of ISCOMs 9.3.1.1 Composition Two basic types of ISCOM structures have been described. The first, termed the classical ISCOM vaccine [53], is formed by the combination of saponin, cholesterol, phospholipid, and amphipathic protein. The second, termed the ISCOMATRIX adjuvant, is essentially the same structure but without the protein [54]. Although these are the basic structures, there are several different types of formulations:

. . .

Classical ISCOM vaccines in which an amphipathic antigen is incorporated into the structure during formation. ISCOM vaccines in which the antigen is mixed with and associates with preformed ISCOMATRIX adjuvant. ISCOMATRIX vaccines in which the antigen is mixed with but does not associate with the ISCOMATRIX adjuvant.

The typical cage-like structures of ISCOM formulations (Figure 9.1D) are composed of saponin/cholesterol/phospholipid micelles that are held together by hydrophobic forces [55]. The components of the ISCOMATRIX adjuvant are saponin, cholesterol, and phospholipid. Saponins interact strongly with cholesterol and biological membranes containing cholesterol [8, 56]. The saponin used in ISCOM formulations is isolated from the bark of the Quillaja saponaria tree, is usually referred to as Quillaia saponin, and contains adjuvant activity [57]. Although the basic structure of the Quillaia saponin is known (Figure 9.2), many structural variations exist, making them a highly complex group of molecules [58, 59]. Early ISCOM vaccines used Quil A saponin. This is a commercially available, partly refined preparation that is still a very heterogeneous mixture of Quillaia saponins and is suitable for veterinary use only.

9.3 ISCOMs

Fig. 9.2 Important structural features of the Quillaia saponin. The core is the hydrophobic quillaic acid residue. Carbohydrates are attached to C3 and C28.

Further studies of the crude Quillaia saponin identified three groups of components, QH-A, QH-B, and QH-C, of particular interest for ISCOM formulation [60]. Criteria were low toxicity, strong adjuvant activity, and ability to form ISCOM structure. More recent characterization of these components has led to the development of ISCOPREP saponin by CSL Ltd. ISCOPREP saponin is a purified saponin fraction prepared by a proprietary process that is robust and highly reproducible. ISCOPREP saponin is well defined and has been extensively characterized. Product-development programs now focus on ISCOPREP saponin, which has been used in several human studies and is an acceptable component of human vaccines. Cholesterol is a second key component of the ISCOM formulation and is available commercially from many suppliers. The source of the cholesterol is not critical, and both natural and synthetic types are available. The importance of lipid in ISCOM formulations has been shown [61], and several lipids have been used. Synthetic dipalmitoylphosphatidyl choline (DPPC) has been used in the formulations tested to date in humans. 9.3.1.2 Characteristics of ISCOMs ISCOM formulations have been defined as an antigen-delivery system with built-in adjuvant providing an optimal immune response [53]. The induction of particular immune responses by ISCOM formulations is a key characteristic of the adjuvant system

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and is discussed in more detail in section 9.3.2. The ISCOMATRIX adjuvant has been extensively characterized and forms the basis of this discussion on characteristics. The ISCOMATRIX adjuvant morphology has been studied by a variety of approaches. Transmission electron microscopy (TEM) showed the ISCOMATRIX adjuvant to be composed of subunit rings in typically cage-like spherical structures of approximately 40 nm [54, 62]. Recent work using cryopreservation electron microscopy, atomic force microscopy, as well as a number of particle-sizing techniques confirms the TEM observations (unpublished). The hemolytic activity of saponin due to its action on biological membranes is well recognized. The hemolytic activity of the purified Quillaia saponin, QS21, has been linked with significant local reactions observed in humans with this adjuvant [63]. One of the characteristics of the ISCOMATRIX adjuvant is the substantial reduction (>100 fold) in the hemolytic activity of the saponin once it is incorporated into the structure [60]. Little to no hemolytic activity is detected with the ISCOMATRIX adjuvant. Other important characteristics of the ISCOM adjuvant system include the simplicity and scalability of the manufacturing process for ISCOMATRIX adjuvant and the stability of both the ISCOMATRIX adjuvant and ISCOM vaccines. Stability studies are continuing, but to date, both the physicochemical and biological properties of the ISCOMATRIX adjuvant have remained stable for over 4 years when stored at 2–8 °C at pH 7.2. ISCOMATRIX adjuvant also remained stable at 37 °C for up to 1 y and can be frozen, freeze-dried, and even spray-dried with no deleterious effects [64]. 9.3.1.3 Preparation of ISCOMs ISCOM preparation methods are based on detergent-removal techniques. Saponin, cholesterol, and phospholipid (with or without amphipathic antigen) are mixed in the presence of detergent. The ISCOM structure forms spontaneously as the detergent is removed [53]. Classical ISCOM vaccines were originally prepared by the centrifugation method [53] that was developed with viral membrane proteins as antigens. A dialysis procedure was also developed, which enabled the use of a broader range of proteins and is now most commonly used [65, 66]. More recently, ultrafiltration techniques have been used, enabling the process to be performed on an industrial scale. Traditionally, amphipathic antigens have been incorporated into ISCOM vaccines via hydrophobic regions found naturally in the antigen. This restriction limited ISCOM vaccines to use with predominantly membrane-associated antigens, and so a number of techniques have been developed to increase the range of antigens suitable for use in ISCOM vaccines. Exposure of hydrophobic regions, by methods such as low pH treatment, and chemical conjugation of lipids to proteins enabled some nonmembrane antigens to be incorporated into classical ISCOM vaccines during formation [67]. More recently, methods to produce recombinant fusion proteins with a hydrophobic tag have been developed; these potentially enable an even broader range of antigens to be incorporated into the ISCOM structure [68]. Several strategies have been developed to form ISCOM vaccines by association of antigens with the preformed ISCOMATRIX adjuvant. These include chemically cou-

9.3 ISCOMs

Fig. 9.3 Flow chart showing the ISCOMATRIX adjuvant manufacturing process. Mega10 is a nonionic detergent: N-(D-gluco-2,3,4,5,6-pentahydroxy hexyl)-N-methyldecanamide.

pling antigens to activated lipids in the ISCOMATRIX structure and, more recently, techniques in which simple admixing of the antigen and the ISCOMATRIX adjuvant results in association. Electrostatic interactions, which take advantage of the negative charge of the ISCOMATRIX adjuvant, allow association of antigens containing positively charged regions. Electrostatically driven association can be improved by increasing the negative charge of the ISCOMATRIX adjuvant or, alternatively, the positive charge of the antigen [69]. Other novel methods of achieving association include the chelating ISCOMATRIX adjuvant, in which a metal-chelating group is incorporated into the structure, which can then bind antigens containing a hexahistidine tag (Malliaros, J. et al., unpublished). The strategies described above should enable the development of procedures for formulating virtually any antigen as an ISCOM vaccine. Due to the ease of manufacture, vaccine development programs now focus on the use of preformed ISCOMATRIX adjuvant to formulate either ISCOM or ISCOMATRIX vaccines. Procedures for manufacture of the ISCOMATRIX adjuvant have been developed at industrial scale (Figure 9.3). This process is GMP-compliant and has been optimized to produce a robust procedure that is well controlled and highly reproducible. Batch sizes of hundreds of liters (approximately 10 million vaccine doses) can be manufactured by this process. 9.3.2 Immunology and Mode of Action of ISCOM Vaccines 9.3.2.1 Immune Responses to ISCOM Vaccines Parenteral Immunization of Mice It is now nearly 20 years since ISCOM vaccines were first described [53], and over that period ISCOM vaccines have demonstrated protective immune responses in

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many species [70]. In the mouse, ISCOM vaccines induce strong antibody and cellular immune responses. Please see the review of Sjölander et al [70] for a detailed summary of mouse immune responses to ISCOM vaccines. ISCOM vaccines are effective at low antigen and adjuvant doses. They also induce long-lived antibody and cellular responses [59, 67, 71]. ISCOM vaccines can induce CTL against many antigens, both naturally occurring immunogens and also recombinant proteins [72–75]. Parenteral Immunization of Nonhuman Primates Sjölander et al. [70] also summarized the nonhuman primate responses to ISCOM vaccines; a variety of antigens including influenza, HIV, and SIV were used. In the monkey models, ISCOM vaccines induced T-helper and CTL responses as well as antibody responses. Experiments in Rhesus macaques with ISCOM vaccines containing the hepatitis C core protein were recently shown to prime strongly for CD4+ and CD8+ T-cell responses [75]. The immune response to E1E2 was also improved when the core ISCOM vaccine was mixed with E1E2 protein. More recently, experiments in guinea pigs, sheep, and baboons have shown the benefits of ISCOM vaccines in terms of their dose-sparing capability. We have shown in guinea pigs that the HIV antigen MNrgp120, combined with ISCOMATRIX adjuvant, gave strong immune responses with 10–100 fold less antigen than was required with aluminum hydroxide adjuvant (Boyle, J. et al., unpublished). In sheep [76] and nonhuman primates (baboons) given influenza vaccine antigens, serum HAI titers were equal or better with an ISCOMATRIX vaccine that contained 1/10 the antigen dose compared to unadjuvanted vaccine (Figure 9.4).

Fig. 9.4 Baboons were vaccinated twice with inactivated vaccine containing three virus strains. The serum responses to the B strain are shown. Similar responses were obtained for the HINI and H3N2 strains. The time between doses was 3 weeks; animals were bled 3 and 4 weeks after the first dose.

9.3 ISCOMs

Mucosal Immunization The use of ISCOM vaccines for nasal vaccination was recently reviewed by Hu et al. [77]. Most of this work has been performed in mouse models where, almost always, good protective immune responses have been obtained after intranasal vaccination. In mice, influenza ISCOM vaccines induce CTL as well as strong mucosal and systemic immune responses when delivered intranasally [78]. These responses were equivalent to or better than those obtained with the strong mucosal adjuvant LT (E. coli heat-labile enterotoxin). Responses at distant mucosal sites after intranasal delivery with ISCOM vaccines have also been observed [76, 78]. Responses in other species have been obtained after intranasal delivery, although they were not as good as in the mouse. This may be due to inefficient nasal delivery of vaccine to immune effector sites in the species concerned. Effective Immunization with ISCOM Vaccines in the Presence of Preexisting Antibody Vaccination strategies that stimulate protective responses even in the presence of preexisting immune responses to a particular immunogen would be valuable against a range of human and veterinary diseases. Many diseases are capable of eliciting antibody responses that are unable to eliminate the disease, e. g., various cancers, hepatitis, HIV, and many others. A vaccine adjuvant capable of stimulating immune responses in the presence of these antibodies is critical if therapeutic intervention is to be considered (see also section 9.3.3.2). ISCOM vaccines have shown potential at overcoming the vaccine-neutralizing effects of passively acquired antibody in neonatal mice, horses, and macaques [79–81]. In nonhuman primates and horses, active immunity was induced in the presence of maternal antibody against measles virus and equine herpes 2 virus, respectively, with ISCOM vaccines, but not with conventional killed vaccines. Furthermore, ISCOM vaccines can induce Th1 immune responses in neonates [79]. Conventional vaccines, on the other hand, elicit mainly Th2 responses in this age group. 9.3.2.2 Mode of Action of ISCOM Vaccines As described above, ISCOM vaccines are potent inducers of antibody as well as CD4+ and CD8+ T cell responses. By necessity, such a broad immune response as this is dependent on the induction of multiple innate and adaptive immune mediators and cellular processes and on the interplay between these elements. The details of this complex process are yet to be fully elucidated. However, with the available information regarding the immune responses induced by ISCOM vaccines, together with a greater understanding of immunological processes such as antigen processing, a clearer understanding is emerging. When mice are immunized with ISCOM vaccines, many of the known cytokines are up-regulated as a result. Up-regulation of proinflammatory IL-1 was the first cytokine response observed to be related to the adjuvant activity of ISCOM vaccines [82]. Since then, many cytokines have been shown to be stimulated by the adjuvant action of ISCOM vaccines; these include IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IFNg [70, 71]. This broad range of cytokines is consistent with the mixed Th1/Th2 responses observed with ISCOM vaccines.

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The depot effect, by which antigen is trapped at the site of administration in order to attract antigen-presenting cells (APCs), is considered to be an important function of adjuvants [83]. However, unlike aluminum- and oil-based adjuvants, ISCOM vaccines are likely to be cleared rapidly from the site of injection to the draining lymph nodes, although there is some evidence of dose site effects such as cellular infiltration. Recent studies using a sheep afferent lymph node cannulation model have shown that ISCOMATRIX adjuvant alone, in the absence of antigen, results in effects at the level of the draining lymph node such as an increase in lymphocyte recruitment and the expression of proinflammatory cytokines (IL-1, IL-6, IL-8, and IFN-g) [84]. Furthermore, ovalbumin ISCOM vaccine injected intraperitoneally in mice recruited a range of inflammatory cells (macrophages, dendritic cells, and lymphocytes), resulting in the production of a variety of immune mediators including IL-1, IL-6, IL-12, and IFNg [85]. The production of inflammatory mediators by immune effector cells is now recognized as being critical for the induction of primary immune responses. Cellular recruitment and activation are greatly reduced in IL-12 knockout mice given ISCOM vaccines, suggesting that IL-12 is important for ISCOM adjuvant activity. Together, these observations suggest that ISCOM vaccines activate innate immune responses, with IL-12 playing a key role. IL-12 is a macrophage-produced cytokine, which regulates the expression of IFN-g and is required for the differentiation of CTL. Clearly, the saponin component is critical for the immunomodulatory properties of ISCOM vaccines. Saponin per se has adjuvant potential; however, optimal CTL induction is achieved when it is formulated as an ISCOM vaccine (Lenarczyk, A., et al., unpublished). The most likely explanation for this is that ISCOM vaccines, because of their particulate nature, are targeted to and more efficiently taken up by cells of the immune system such as APCs (see also section 9.2.2). Consistent with this, ISCOM vaccines are efficiently taken up in vitro by dendritic cells. Furthermore, the vaccine antigens are processed and presented to CD8+ T cells (Maraskovsky et al., unpublished). Soltysik et al. [86] have shown that the full adjuvant activity of Quillaia saponin-based adjuvants depends on the aldehyde group and the aliphatic sidechain of the saponin, suggesting that cellular uptake may be mediated by the interaction of these moieties with the cell membrane. To date, there is little evidence to suggest that ISCOM formulations bind to specific receptors, although this should not be discounted and, unlike other activators of innate immune responses such as CpGs, LPS, and DNA, they do not appear to activate Toll-like receptors (TLRs) (unpublished). It therefore remains an open question as to how ISCOM vaccines cause cellular activation and up-regulate cytokine expression. After cellular uptake, ISCOM structures were found to be approximately equally distributed between the cytosol and intracellular compartments [72], which in part explains their capacity to access both the endosomal and proteosomal compartments for MHC class I and II antigen-processing, respectively. Access to the class I pathway is clearly critical to the ability of ISCOM vaccines to induce strong CTL responses. Although not completely understood, it is thought that the membrane-disrupting properties of the saponin component of the ISCOM structure might aid endosomal escape, thereby releasing antigen to the cytosol. ISCOM vaccines also induce up-regulation of MHC class II expression [72].

9.3 ISCOMs

9.3.3 Performance and Products 9.3.3.1 Protection Afforded by ISCOM Vaccines in Animal Models As described in the preceding section, ISCOM vaccines are potent inducers of both humoral and cellular immunity. Consistent with this, ISCOM vaccines elicit protective immunity in a variety of animal species against a range of pathogens (summarized in Table 9.3 and reviewed by Sjolander et al. [70]). Of particular relevance to human vaccine development, ISCOM vaccines formulated with HIV-1 gp120, HIV-1 multicomponent antigens (gp120, p24,V2 and V3 peptides), SIV antigens [87, 88], and EBV gp340 [89] have been shown to protect monkeys against viral challenge with these pathogens. Another key feature of ISCOM vaccines is the longevity of response. Polakos et al. [75] have shown that Rhesus macaque monkeys receiving an HCV core ISCOM vaccine produced strong HCV core-specific CTL responses, which were detected up to 12 months post-vaccination (the last time point tested). In contrast, CTL responses had waned to baseline levels in animals vaccinated with an HCV core retroviralbased vaccine. Similarly, protection against clinical disease, and in some cases infection, 15 months after vaccination was observed in horses receiving an equine influenza ISCOM vaccine [90]. Two ISCOM vaccines are currently registered for veterinary applications. The first is an equine influenza ISCOM vaccine – to date, more than 1 million doses have been administered with no adverse effects reported. The second registered ISCOM vaccine, also for horses and referred to as EquityTM, consists of a peptide analogue of gonadotropin-releasing factor mixed with ISCOMATRIX adjuvant. Equity induces a state of immune anestrus, thus controlling estrusrelated behavior in mares and fillies. 9.3.3.2 Human Clinical Trials with ISCOMs To date, a range of ISCOMATRIX and ISCOM vaccines have been tested in humans. These include vaccines containing influenza viral proteins, the purified recombinant Tab. 9.3 ISCOM vaccines: protective immune responses in animals. Species

Infectious agent

Rhesus macaques Cottontop tamarins Horse Guinea pig Cat Rabbit

SIV EBV (gp340) equine influenza virus RSV FIV HIV, influenza, Eimeira falciformis, Plasmodium falciparum BVDV, Toxoplasma gondii pseudorabies virus Adenovirus, Boophilus microplus avian influenza virus, Salmonella

Sheep Pig Cow Turkey

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9 Liposomes and ISCOMs Tab. 9.4 Human data with ISCOM vaccines. Antigen

Immune response (% responders) 1) 2) CD4 CTL Antibody

Influenza NY-ESO-1 HPV E6E7

> 80 100 100

1) 2) 3) NT

NT 55 92

1

25–80 30 3 42 3

Comment

Rapid antibody responses [92, 93] To be published Evidence of viral load reduction in cervical cells

Determined using DTH or IFN-g expression Determined using 51Cr –release assay Determined using ELISPOT or tetramer assays Not tested

antigen HPV E6E7 fusion protein, and the purified recombinant antigen NY-ESO-1 (human cancer testis antigen, which is expressed in a variety of tumors [91]) (Table 9.4). In a randomized double-blind controlled clinical study, both ISCOM- and ISCOMATRIX-based influenza vaccines induced higher antibody responses within the first week of vaccination compared to conventional influenza vaccines. This accelerated development of antibody responses could be advantageous in influenza epidemics or any other setting where a rapid response may be of particular advantage in reducing morbidity or mortality. Furthermore, both the ISCOM- and ISCOMATRIX-based influenza vaccines induced virus-specific CTL responses in 25 %– 80 % of recipients after a single vaccination, compared to 5 % of those receiving a standard influenza vaccine [92, 93]. In mice, influenza-specific CTLs can contribute to protective immunity, even against antigenic-drift variants of influenza A viruses [94]. Whether this also occurs in humans remains to be determined. Double-blind placebo-controlled phase I studies of safety and immunogenicity were also recently completed for two cancer/neoplasia vaccines. In the first, women with cervical dysplasia (CIN 1–3) due to human papilloma virus (HPV) were vaccinated with varying doses of E. coli-expressed recombinant HPV16 E6E7 fusion protein (20, 60 and 200 mg) combined with 120 mg of ISCOMATRIX adjuvant. The vaccine was well tolerated, and vaccinated subjects mounted strong HPV16 E6E7-specific humoral and cellular immune responses (Frazer, I. et al., unpublished). E6- and E7-specific antibody (100 %), CD4 (92 %), and CTL responses (42 %) were detected in vaccine-treated subjects. Furthermore, there was evidence of reduced viral load in vaccinated subjects. A similar phase I study was also recently completed with an NY-ESO-1 ISCOM vaccine in patients with NY-ESO-1 positive cancers and minimal residual disease (Cebon et al., unpublished). The NY-ESO-1 ISCOM vaccine was well tolerated and induced strong humoral and cellular vaccine-specific immunity. Antibody responses were observed in 100 % of the vaccine recipients, and CD4+ and CD8+ T cell responses in 69 % and 38 %, respectively. Importantly, CD8+ T cell responses were observed in individuals with and without preexisting antibodies to NY-ESO-1, suggesting that the ISCOM vaccine was effective even in the presence of preexisting antibo-

9.4 Perspectives

dies to the vaccine components. This observation is consistent with the results of studies performed with mice (Lenarczyk, A. et al., unpublished). Currently, a popular approach to CTL induction is to prime with a DNA vaccine and boost with a live viral vector. ISCOM vaccines obviate the need for separate prime and boost vaccines, because no neutralizing antibody response is induced to the ISCOMATRIX adjuvant per se. As a result, ISCOM vaccines can be used for repeat dosing. In addition to the obvious manufacturing, registration, and cost advantages of using the same vaccine for all doses, ISCOM vaccines have additional safety benefits over DNA and live vector approaches, such as lack of integration potential. Together, the results of these trials indicate that ISCOM vaccines are potent inducers of both humoral and cellular immunity in humans and therefore suitable for a wide range of applications that require the induction of an integrated immune response, e. g., cancer and chronic infectious diseases. The issue of reactogenicity with saponin-based adjuvants has been raised as a potential impediment to their widespread use in human and veterinary vaccines. This suggestion was largely based on mouse studies involving the administration of large adjuvant doses. However, in all other species tested, including chickens, cats, monkeys, dogs, sheep, cattle, and horses, little reactogenicity has been observed with parenterally administered ISCOM vaccines [70]. GLP toxicity studies have been performed on ISCOPREP saponin, ISCOMATRIX adjuvant, and both ISCOM and ISCOMATRIX vaccines. In all instances, the formulations were well tolerated, with little evidence of reactogenicity after repeated dosing. To date, ISCOM vaccines have been administered to approximately 1000 people with no serious vaccine-related adverse events. The most common adverse event is pain or ache at the injection site, which is mild-to-moderate and transient (2–3 days), resolving without the need for medical intervention.

9.4 Perspectives

Liposomal vaccines have been around for about 30 years. Classical liposomes as antigen carriers have been studied in depth. Simultaneously, a number of liposome variants have been developed, some with evident immune-stimulating properties and an attractive safety profile. Some are now available as registered products or in advanced stages of clinical testing. Combined with the use of safe adjuvants, increased knowledge of immunology, and improved, scalable manufacturing methods, more products should reach the market within 10 years. From the many studies with ISCOM vaccines in animal models and in human clinical trials, we can conclude that ISCOM vaccines are safe and immunogenic. The key features of ISCOM vaccines for humoral responses are the magnitude, speed, and longevity of responses and the capacity for dose sparing of antigen. ISCOM vaccines are ideal for applications that require a rapid immune response, such as pandemic influenza, or in situations where antigen is limited due to the high cost of manufacture. The key features of ISCOM vaccines for cellular immune responses

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are their ability to induce long-lived T helper and CTL responses without the need for different formulations for prime and boost. These features of ISCOM vaccines support their use in immunotherapeutic vaccines directed at chronic infectious agents and diseases such as hepatitis B, hepatitis C, HIV, malaria, HSV-1 and 2, and H. pylori and also for use in cancer immunotherapy.

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18. G.H. Lowell, L.F. Smith, R.C. Seid, W.D. Zollinger, J. Exp. Med. 1988, 167, 658–663. 19. G.H. Lowell in: New Generation Vaccines. Second edition, M.M. Levine, G.C. Woodrow, J.B. Kaper, G.S. Cobon, eds., 1997, 193–206, Marcel Dekker, New York. 20. G.B. Patel, G.D. Sprott, Crit. Rev. Biotechnol. 1999, 19, 317–357. 21. J.W. Conlan, L. Krishnan, G.E. Willick, G.B. Patel, G.D. Sprott,Vaccine 2001, 19, 3509–3517. 22. L. Krishnan, C.J. Dicaire, G.B. Patel, G.D. Sprott, Infect. Immun. 2000, 68, 54–63. 23. L. Zarif, J. Control. Rel. 2002, 81, 7–23. 24. S. Gould-Fogerite, M.T. Kheiri, F. Zhang, et al., Adv. Drug Del. Rev. 1998, 32, 273–287. 25. J. Brunner, P. Skrabal, H. Hauser, Biochim. Biophys. Acta 1976, 455, 322– 331. 26. D.D. Lasic, Biochim. Biophys. Acta 1982, 692, 501–502. 27. W. Jiskoot,T. Teerlink, E.C. Beuvery, D.J. Crommelin, Pharm. Weekbl. Sci. 1986, 8, 259–265. 28. D.D. Lasic,Trends Biotechnol. 1998, 16, 307–321. 29. L. Krishnan, S. Sad, G.B. Patel, G.D. Sprott. J. Immunol. 2001, 166, 1885– 1893. 30. C. Arigita, G.F.A. Kersten,T. Hazendonk,W.E. Hennink, D.J.A. Crommelin,W. Jiskoot,Vaccine 2003, 21, 950– 960. 31. I. Idanpaan-Heikkila, S. Muttilainen, E. Wahlstrom, et al.,Vaccine 1995, 13, 1501–1508. 32. G. Zardeneta, P.M. Horowitz, Anal. Biochem. 1994, 223, 1–6.

References 33. G. Chikh, M.P. Schutze-Redelmeier, Biosci. Rep. 2002, 22, 339–353. 34. C. Arigita, Towards an improved Neisseria meningitidis type B vaccine: vesicular PorA formulations, 2003,Thesis, University of Utrecht. 35. G. Gregoriadis, A. Bacon,W. Caparros-Wanderley, B. Mccormack,Vaccine 2002, 20 Suppl. 5, B1-B9. 36. N. Garcon, G. Gregoriadis, M.Taylor, J. Summerfield. Immunology 1988, 64, 743–745. 37. M.J. Copland, M.A. Baird,T. Rades, J.L. McKenzie, B. Becker, F. Reck, P.C. Tyler, N.M. Davies,Vaccine 2003, 21, 883–890. 38. P. Machy, K. Serre, L. Leserman, Eur. J. Immunol. 2000, 30, 848–857. 39. M. Fukasawa,Y. Shimizu, K. Shikata, M. Nakata, R. Sakakibara, N.Yamamoto, M. Hatanaka,T. Mizuochi, FEBS Lett. 1998, 441, 353–356. 40. S. Toda, N. Ishii, E. Okada, K.I. Kusakabe, H. Arai, K. Hamajima, I. Gorai, K. Nishioka, K. Okuda, Immunology 1997, 92, 111–117. 41. A. Engel, S.K. Chatterjee, A. al Arifi, D. Riemann, J. Langner, P. Nuhn, Pharm. Res. 2003, 20, 51–57. 42. H. Chen,V. Torchilin, R. Langer, Pharm. Res. 1996, 13, 1378–1383. 43. J. Kunisawa, S. Nakagawa,T. Mayumi, Adv. Drug Deliv. Rev. 2001, 52, 177–186. 44. L. Bungener, K. Serre, L. Bijl, L. Leserman, J. Wilschut,T. Daemen, P. Machy,Vaccine 2002, 20, 2287–2295. 45. M. Rao, C.R. Alving, Adv. Drug Deliv. Rev. 2000, 41, 171–188. 46. L. Bungener, A. Huckriede, J. Wilschut,T. Daemen, Biosci. Rep. 2002, 22, 323–338. 47. C.R. Alving in: New Generation Vaccines. Second edition, M.L. Levine, G.C. Woodrow, J.B. Kaper, G.S. Cobon, eds., 1997, 207–13. Marcel Dekker, New York. 48. C.R. Alving,V. Koulchin, G.M. Glenn, M. Rao, Immunol. Rev. 1995, 145, 5–31. 49. G. Bjune, E.A. Hoiby, J.K. Gronnesby et al., Lancet 1991, 338, 1093–1096. 50. L.G. Milagres, M.C.A. Gorla, C.T. Sacchi, M.M. Rodrigues, Infect. Immun. 1998, 66, 4755–4761. 51. E.D. de Kleijn, R. de Groot, A.B. Lafe-

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9 Liposomes and ISCOMs 71. A. Sjölander, J.C. Cox, I.G. Barr, J. Leukocyte Biol. 1998, 64, 713–723. 72. M.C. Villacres, S. Behboudi,T. Nikkila, K. Lovgren-Bengtsson, B. Morein, Cell. Immunol. 1998, 185, 30–38. 73. A.D. Wilson, K. Lö vgren-Bengtsson, M. Villacres-Ericsson, B. Morein, A.J. Morgan,Vaccine 1999, 17, 1282– 1290. 74. J.T.M. Voeten, G.F. Rimmelzwaan, N.J. Nieuwkoop, K. Lovgren-Bengtsson, A.D.M.E. Osterhaus,Vaccine 2001, 19, 514–522. 75. N.K. Polakos, D. Drane, J. Cox et al., J. Immunol. 2001, 166, 3589–3598. 76. A. Coulter, R. Harris, R. Davis et al., Vaccine 2003, 21, 946–949. 77. K.-F. Hu, K. Lovgren-Bengtsson, B. Morein. Adv. Drug Del. Rev. 2001, 51, 149–159. 78. S. Sjolander, D. Drane, R. Davis, L. Beezum, M. Pearse, J. Cox,Vaccine 2001, 19, 4072–4080. 79. B. Morein, K.L. Bengtsson, Immunol. Cell Biol. 1998, 76, 295–299. 80. A. Osterhaus, G. van Amerongen, R. van Binnendijk,Vaccine 1998, 16, 1479–1481. 81. B. Morein, K. Lövgren-Bengtsson, J. Cox in: Concepts in Vaccine Development, S.H.E. Kaufmann, ed., 1996, 243– 263,Walter de Gruyter, Berlin, New York. 82. S. Behboudi, B. Morein, M.Villacres-Eriksson, Scand. J. Immunol. 1999, 50, 371–377. 83. J.C. Cox, A.R. Coulter,Vaccine 1997, 15, 248–256. 84. R.G. Windon, P.J. Chaplin, L. Beezum et al.,Vaccine 2001, 19, 572–578. 85. R.E. Smith, A.M. Donachie, D. Grdic, N. Lycke, A.M. Mowat, J. Immunol. 1999, 162, 5536–5546. 86. S. Soltysik, J.-Y. Wu, J. Recchia et al., Vaccine 1995, 13, 1403–1410. 87. A. Osterhaus, P. de Vries, B. Morein, L. Akerblom, J. Heeney, AIDS Res. Hum. Retroviruses 1992, 8, 1507–1510. 88. P. de Vries, J.L. Heeney, J. Boes et al., Vaccine 1994, 12, 1443–1452. 89. A.J. Morgan, S. Finerty, K. Lovgren, F.T. Scullion, B. Morein, J. Gen. Virol. 1988, 69, 2093–2096. 90. J.A. Mumford, D. Jessett, U. Dunleavy et al.,Vaccine 1994, 12, 857–863.

91. T.-T. Chen, M.J. Scanlan, U. Sahin et al., Proc. Natl. Acad. Sci. USA 1997, 94, 1914–1918. 92. F.A. Ennis, J. Cruz, J. Jameson, M. Klein, D. Burt, J. Thipphawong, Virology 1999, 259, 256–261. 93. G.F. Rimmelzwaan, N. Nieuwkoop, A. Brandenburg et al.,Vaccine 2001, 19, 1180–1187. 94. S. Sambhara, A. Kurichh, R. Miranda et al., Cell. Immunol. 2001, 211, 143–153. 95. L.F. Fries, D.M. Gordon, R.L. Richards et al., Proc. Natl. Acad. Sci. USA 1992, 89, 358–362. 96. L.F. Fries, A.D. Montemarano, C.P. Mallett, D.N. Taylor,T.L. Hale, G.H. Lowell, Infect. Immun. 2001, 69, 4545– 4553. 97. M. Kaji,Y. Kaji, M. Kaji, K. Ohkuma,T. Honda,T. Oka, M. Sakoh, S. Nakamura, K. Kurachi, M. Sentoku, Vaccine 1992, 10, 663–667. 98. M. Just, R. Berger, H. Drechsler, S. Brantschen, R. Gluck,Vaccine 1992, 10, 737–739. 99. Y. Poovorawan, A. Theamboonlers, S. Chumdermpadetsuk, R. Gluck, S.J. Cryz,Vaccine 1995, 13, 891–893. 100. L. Loutan, P. Bovier, B. Althaus, R. Gluck, Lancet 1994, 343, 322–324. 101. R. Gluck, R. Mischler, S. Brantschen, M. Just, B. Althaus, S.J. Cryz, J. Clin. Invest. 1992, 90, 2491– 2495. 102. R. Gluck, R. Mischler, B. Finkel, J.U. Que, B. Scarpa, S.J. Cryz, Lancet 1994, 344, 160–163. 103. C. Herzog, I.C. Metcalfe, U.B. Schaad,Vaccine 2002, 20 Suppl 5, B24-B28. 104. P. Marchisio, R. Cavagna, B. Maspes et al., Clin. Infect. Dis. 2002, 35, 168–174. 105. R. Gluck, R Mischler, P. Durrer et al., J. Infect. Dis. 2000, 181, 1129–1132. 106. U. Gluck, J.O. Gebbers, R. Gluck, J. Virol. 1999, 73, 7780–7786. 107. E. de Kleijn, L. van Eijndhoven, C. Vermont et al.,Vaccine 2001, 20, 352–258. 108. E.D. de Kleijn, R. de Groot, A.B. Lafeber et al.,Vaccine 2000, 19, 1141–1148. 109. E.D. de Kleijn, R. de Groot, A.B. Lafeber et al., J. Infect. Dis. 2001, 184, 98– 102.

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10 Virosomal Technology and Mucosal Adjuvants Jean-François Viret, Christian Moser, Faiza Rharbaoui, Ian C. Metcalfe, and Carlos A. Guzmán

10.1 Overview

Immune responses are remarkably versatile adaptive processes, in which an organism activates specifically reactive molecules and cells in response to contacts with dangerous entities, including pathogenic microorganisms. The global immune system is composed of two major lines of defense, innate immunity and adaptive or acquired immunity, both of which rely on complex inter-regulated networks of humoral and cellular components. Innate immunity may be considered an emergency fight mechanism and plays a pivotal role at the time of first contact with a pathogenic agent, not only by providing a rapid response but also in the fine-tuning of the ensuing and more specific adaptive response (Figure 10.1). In this context, the actual challenge of vaccinology is to develop new strategies able to prime the immune system in such a way that an optimal balance of all key components of the immune responses is achieved, resulting in effective long-term protection. The ability of a vaccine to induce protective immune responses requires optimal presentation of pathogen-specific epitopes, as well as qualitatively and quantitatively appropriate immunopotentiating properties. Advances in vaccinology and immunology have provided an increasing array of candidate antigens suitable for vaccination against a variety of diseases. However, these novel antigens (e. g., synthetic peptides, purified subunits) generally require additional adjuvants to optimize their immunostimulating potential. This is especially true for the stimulation of efficient cellular immune responses. Although often antibodies alone may be sufficient for protection, costimulation of a cellular immune response is essential to induce sustained immunity (i. e., memory) and is generally considered indispensable for therapeutic approaches. Adjuvants can be subdivided into two major groups, vehicles and immunomodulators, depending on their primary mechanism of action [1]. Further subdivision allows the categorization of three types of adjuvant according to the underlying effect on the immune response [2]. Namely, (1) the protection of vulnerable antigens from rapid extracellular degradation or elimination by formation of a depot of antigen at

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1

1

4

APC Epithelium

10

5

2 8

B-Cell

6

11

PMN, Mo, NK 3

CD8+

CD4+

7

9

Fig. 10.1 Interactions between microorganisms and the immune system. (A) The innate immune system. For many microorganisms, the first contact with a potential host is via epithelia, mainly at mucosal surfaces. This tight barrier forms the first line of defense against pathogens and often is sufficient to block productive infections by means of fast, unspecific antibacterial and antiviral activities, i. e., the innate immune system. Many cell types, in particular, epithelial and immune cells, feature a sophisticated sensing system (1) capable of recognizing specific pathogen-associated molecular patterns (PAMPs)via Toll-like receptors (TLRs) and similar molecules expressed on the cell surface (e.g., for bacterial cell wall components) or located inside the cell (e.g., for double-stranded RNA). These in turn feed into the intracellular signaling network, leading to a PAMP-specif ic response. An increasing number of closely related sensor molecules have been identified in recent years in a wide variety of organisms, such as plants, insects, and mammals, indicating that this defense mechanism has been conserved throughout evolution. The PAMP-induced changes in the concentrations and phosphorylation patterns of intracellular signaling molecules and transcription factors lead to activation of proinflammatory and antimicrobial genes, as well as metabolic pathways, depending on the specific type of PAMP and the activated sensing molecules (2). The activated state of epithelial cells in reaction to contact with PAMPs induces release of locally active chemokines, cytokines, and molecules directly active against pathogens, such as defensins. The release of these warning signals acts as a chemoattracta nt for effector cells of the innate immune system, such as polymorphonuclear cells

(PMNs), macrophages, and natural killer cells (NK cells) (3). Furthermore, the surrounding epithelial cells are also primed for reaction to the microbial threat. The sum of the local and semispecific defense mechanisms, the so-called innate immunity, is often sufficient to eliminate the pathogen, even before an adaptive immune response is fully induced. This information is reviewed in references [131–136]. (B) The adaptive immune system.Professional antigen-presenting cells (APC) and precursors are resident in most tissues and are attracted in larger numbers by chemokines secreted by activated or infected cells. APC take up pathogens and their degradation products via phagocytosis (4) and process these antigens for presentation (5). Two distinct pathways for presenting antigens are referred to as the endogenous and the exogenous pathway, which result in the presentation of antigen-derived peptides in the context of MHC I (6) and MHC II (8) molecules, respectively. The default pathway for extracellular antigen taken up by APC is degradation in endosomes followed by loading onto MHC II and transport to the cell membrane (8). MHC II is expressed only by professional APC such as dendritic cells (DC) and macrophages. Presentation of peptides on MHC II molecules is required, together with costimulatory signals, for activation of antigen-specific CD4+ T-lymphocytes, most of them being the T-helper type. Activated helper T cells secrete interleukins and other cytokines,which further stimulate proliferation and maturation of B and T cells already activated by antigen recognition (9). MHC-I is expressed on all nucleated cell types. Peptides presented in the context of MHC I usually originate from the intracellular synthesis of foreign antigens, e.g., of viral or bacterial origin, and are

10.1 Overview

the site of application, which enables prolonged exposure to the antigen, thereby leading to immunopotentiation; (2) the increased uptake of antigen into antigen-presenting cells (APC), particularly dendritic cells (DC), macrophages, and B lymphocytes; and (3) the induction of synthesis and secretion of immune response enhancing factors, such as cytokines. Certain adjuvants also enable specific antigen targeting to APC and its channeling to specific subcellular locations, allowing efficient presentation in the context of both MHC I and II molecules. Adjuvant-associated nonspecific immunostimulatory effects unrelated to the respective antigen may also be evoked, e. g., binding to receptors or interfering with signaling pathways involved in innate and adaptive immunity. Adjuvants may also be instrumental in inducing long-term immunological memory [3–6]. During the past two decades, a variety of technologies have been investigated to improve on the widely used but suboptimal aluminum-based adjuvants. Aluminum adjuvants tend to strengthen the antigen-specific antibody response but lack the ability to stimulate CTL responses and may present undesirable side effects [7]. One of the most promising new approaches relies on liposome-based adjuvant systems. The approach adapted for so-called virosome adjuvanted vaccines is of particular interest, because they combine several properties known to contribute to immunostimulation and at the same time, due to the biocompatible nature of their constituents, are unlikely to produce unwanted side effects, such as cytotoxicity or unspecific inflammation at the site of injection. The mechanisms underlying the adjuvant properties of virosomes and the validation of the concept with two licensed vaccines are described in the second part of this chapter. In addition, novel routes of administration (i. e., mucosal vaccination) have been investigated for the induction of more effective immune responses at the site of first contact between the pathogen and its host. Due to the nature of the mucosa – mucociliary clearance in the respiratory tract, low pH and digestive enzymes in the gastrointestinal tract, and a general protective coating of mucus – this potentially advantageous route of immunization presents inherent obstacles. As described in the first part of this chapter, adjuvanted vaccines can be used to significantly enhance immune responses after mucosal administration. Fig. 10.1 (continued) recognized as an attack signal for antigen-specific cytotoxic CD8+ T cells (7). The starting point for intracellular antigen processing is the cytoplasm, where proteosomal degradation yields peptide fragments, which are subsequently transported into the endoplasmic reticulum (ER), loaded there onto MHC-I molecules, and finally presented on the cell surface. The humoral arm of the adaptive immune response depends primarily on activation of circulating immature B cells as a result of antigen binding to and cross-linking of the immunoglobulin chains

expressed on their surfaces (10). For further activation and full differentiation into antibody-secreting plasma cells, costimulatory signals are required from CD4+ T-cells and APC (11), both locally at the site of infection and in the lymphatic tissues, which in turn depend on MHC II presentation of processed antigen. The cytokine profile secreted by the accessory cells involved (APC and T cells) determines the type and kinetics of antibody maturation and the generation of memory B cells.

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10.2 Mucosal Adjuvants 10.2.1 Introduction

Most infectious agents are restricted to the mucosa or need to cross mucosa to cause disease. The administration of vaccine antigens by the parenteral route induces antigen-specific immune responses that are essentially restricted to the systemic compartment. In contrast, immunization by the mucosal route triggers immune responses at both systemic and mucosal levels. Thus, mucosal vaccination is considered the most appropriate immunization regime to stimulate local responses at the level of the pathogens’ port of entry. This can promote protection not only against disease, but also against infection (i. e., colonization) and leads to reduced pathogen shedding, thereby reducing the likelihood of horizontal transmission from infected to susceptible individuals. However, the mucosal epithelium constitutes a natural barrier to invading organisms; a barrier from which vaccine antigens are rapidly cleared (e. g., rapid degradation, peristaltic motility, poor penetration). Mucosal sites may also exhibit relatively strong tolerance to a large variety of antigens [8, 9]. Consequently, the use of special delivery strategies and vaccine formulations, such as mucosal adjuvants, is essential to breach this immunological barrier. The term ‘mucosal adjuvant’ has been quite broadly defined. Some of these so-called mucosal-adjuvant molecules correspond to chemically defined entities that exhibit intrinsic adjuvant activity and have been obtained by purification from biological sources or chemical synthesis. Recent advances in the field of mucosal vaccinology have resulted from enhanced comprehension of the interplay among the components of the immune system, greater understanding of the interactions between pathogens and the mucosal immune system, and improved knowledge of the biological effects triggered by adjuvant molecules. Considering that the use of a particular adjuvant can strongly influence the quality of the immune responses elicited against a given vaccine antigen, it is crucial to understand the mechanisms of adjuvanticity of candidate molecules. A large part of the research on vaccine adjuvants has been focused on the development of strategies that preferentially induce either a Th1 or a Th2 type T-cell helper response. However, since the quality of the induced immune responses is characteristic of a given adjuvant–antigen combination, it is extremely difficult to predict the results that will be obtained with a new antigen. This is particularly true for those antigens exhibiting immunomodulatory properties by themselves. The characteristics of the different types of mucosal adjuvants are discussed further below, using specific examples to highlight the advantages and limitations of the candidate molecules. 10.2.2 Families of Mucosal Adjuvants

Mucosal adjuvants cover a broad range of structures, as summarized in Table 10.1. Leader candidate molecules and their main derivatives are discussed below.

10.2 Mucosal Adjuvants Tab. 10.1 Types of mucosal adjuvants. Bacterial toxins

Cholera toxin (CT) CT derivatives: CT atoxic mutants, CTB subunit heat-labile enterotoxin (LT) LT-derivatives: LT atoxic mutants (LTK63, LTR72, LTR192G) chimeras: LT/CT, CTA1-DD

Bacterial lipoproteins, mucopeptides, lipopolysaccharides (LPS)

MALP-2 muramyl dipeptide (MDP) MPL-A (LPS derivative) lipopeptides derived from Gram-negative bacteria

Oligodeoxynucleotides (ODN)

CpG-ODN non-CpG-ODN

Anti-viral drugs and derivatives

imidazoquinolines adamantanes (amantadine and rimantadine), adamantyl dipeptide (AdDP)

Mineral salts and chemical adjuvants

aluminum hydroxide, aluminum phosphate, calcium phosphate saponin (QS21)

Host-derived adjuvants

cytokines (GM-CSF, IL-10, IL-12, etc.)

Previously, based on results obtained in animal models and partially shown in humans, researchers have assumed that stimulation of the common mucosal-immune system (CMIS) would induce an immune response at any remote mucosal-effector site [10, 11]. This has been superseded by the concept of compartmentalization of the CMIS. Stimulation of the various mucosal-inductive sites results in an uneven distribution of immune responses at the various effector sites [12]. Accordingly, the most effective means of inducing an immune response at a specific effector site is localized stimulation, or at least stimulation at a mucosal-inductive site related to the effector site in terms of lymph drainage [13]. Bacterial enterotoxins and their derivatives are among the first molecules to have been used as mucosal adjuvants. They are characterized by the presence of an A moiety with enzymatic activity and a B moiety that mediates toxin binding to the target cells. Cholera toxin (CT) and the closely related Escherichia coli heat-labile toxin (LT) are powerful adjuvants when coadministrated with soluble antigens by the mucosal route. They have been extensively used in preclinical research; however, their use has been limited to specific strategies in humans due to uncertainties concerning their toxicity [14, 15]. Various approaches (e. g., mutation, deletion, generation of chimeras) have been pursued to detoxify these molecules [13, 16–20]. These results led to the identification of nontoxic mutant derivatives, which retain adjuvanticity, at least in animal models. However, recent studies in the mouse indicate that even these derivatives or the B subunit alone may lead to potential non-target effects, such as retrograde transport of adjuvant and antigen to the olfactory nerves and olfactory bulb when such vaccines are administered by the nasal route [21].

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Several components from Gram-positive and -negative bacteria have also been explored for their activity as mucosal adjuvants. The monophosphoryl lipid A (MPL), a derivative of lipopolysaccharide (LPS), retains much of the immunostimulatory properties of LPS without the inherent toxicity [22]. The muramyl dipeptide (MDP) is derived from bacterial cell-wall peptidoglycan and exhibits a variety of biological activities. To improve bioavailability and decrease its toxicity, various derivatives have been generated. MDP-Lys18 shows antiviral activity and exhibits a potent effect as a mucosal adjuvant [23]. On the other hand, the N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) moiety of peptidoglycan has good bioavailability when administered by the oral route [24]. In addition, the fibronectin binding protein I of Streptococcus pyogenes is an efficient mucosal adjuvant able to improve cellular, humoral, and mucosal responses when coupled to or coadministered with antigens administered by the nasal route [25, 26]. Many reports have demonstrated the capacity of microbial lipoproteins to induce cytokine secretion [27, 28]; these studies also demonstrated that the lipid moiety of those molecules is responsible for this activity. A lipoprotein of Mycoplasma fermentans can activate monocyte macrophages at picomolar concentrations. A 2-kDa synthetic derivative, MALP-2 (macrophage-activating lipopeptide), retains the stimulatory capacity of the native lipopeptide [29]. In addition, cellular and humoral immune responses, both at systemic and mucosal levels, against soluble antigens were significantly improved when coadministered with MALP-2 by the intranasal route (Figure 10.2) [30]. The high potency, well-defined chemical structure, and lack of immunogenicity of this compound render it particularly attractive for vaccine development. Studies in the field of peptide-based vaccines have also shown the advantages associated with administration of lipidated antigenic peptides by the mucosal route [31]. Bacterial DNA, but not eukaryotic DNA, exhibits direct immunostimulatory effects on immune cells [32]. These effects are mainly due to the presence of unmethylated CpG motifs, which are generally absent in mammalian DNA. Various base combinations have immune stimulatory effects, some of which were defined as optimal (GACGTT, GTCGTT [33]). Interestingly, some non-CpG-ODN also present adjuvant effects when codelivered with antigens at mucosal sites [34]. Mechanisms of cytokine synergy are essential for vaccine protection against viral challenge [35]. In immunoprophylactic or therapeutic interventions, cytokines can be directly used as natural adjuvants, as an alternative to cytokine-inducing adjuvants. Parenteral cytokine treatment could lead to unwanted toxicity; however, their mucosal delivery results in low, but still biologically active, cytokine levels in serum [36]. Therefore, various cytokines have been used to accomplish a general immune activation or to promote a distinct pattern of immune response in preclinical settings [36, 37]. Various molecules with antiviral activity can also potentiate immune responses. Derivatives of these molecules are now considered to be a novel class of mucosal adjuvants. For example, the imidazoquinolines facilitate the clearance of genital warts by inducing the expression of proinflammatory cytokines [38, 39]. These properties can be used to improve conventional (protein-based) as well as DNA vaccines. On the other hand, adamantanes (amantadine and rimantadine) have been used for many

10.2 Mucosal Adjuvants

Lung IgA control

control

Vagina IgA Serum IgG Cell proliferation

ß-gal + MALP

ß-gal + MALP

ß-gal + CTB

ß-gal + CTB

ß-gal

ß-gal

10000

1

10

100

100000

1000

1000000

1000000

Ratio versus control group Fig. 10.2 b-gal-specific immune responses after intranasal immunization of mice with 50 mg b-gal mixed with 0.5 mg MALP-2 or 10 mg CTB as adjuvant. The results are expressed as the ratio of the mean signal of the b-gal-immunized mice group ( n = 5) to the mean signal of control mice that received PBS. Standard deviations are indicated by horizontal lines.

years as antiviral drugs. They inhibit viral uncoating by blocking the proton channel activity of the influenza A viral M2 protein. However, their use was limited by their lack of activity against influenza B, inherent toxicity, and rapid development of resistance [40]. Adamantylamide dipeptide (AdDP) is a nontoxic compound obtained by linking the L-alanine-D-isoglutamine residue of MDP to amantadine [41]. Recent studies have demonstrated that AdDP is an effective immunoadjuvant with an adequate safety profile when administered by the oral route in rabbits and mice [42]. A last group of immunostimulatory adjuvants is composed of mineral salts and saponins. QS21 is a nontoxic derivative of saponin, the highly purified complex triterpene glycoside isolated from the bark of the Quillaja saponaria Molina tree [43, 44]. This adjuvant has been included in several clinical trials, when the antigens were delivered by the parenteral route. However, QS21 is also under extensive investigation for mucosal use, particularly in combination with other adjuvants. Aluminum salts still remain the standard adjuvants licensed for human use. Interestingly, these cationic compounds also work as an absorption enhancer for nasal drug delivery [45] and have been included in various mucosal vaccine prototypes.

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10.2.3 Administration Strategies

Attenuated or inactivated microorganisms, proteins, or polypeptides, as well as DNA, can be used for mucosal vaccination. Vaccine efficacy depends on the antigen itself (inherent immunogenicity), the specific route of administration, the mucosal adjuvant added to the preparation, and the final formulation. Mucosal administration of purified proteins or peptides is usually accompanied by an important loss of immunogenicity. The presence of degrading enzymes and the poor penetration of antigens through the mucosal barrier constitute the major obstacles for the development of efficacious peptide- or protein-based mucosal vaccine formulations. Thus, major features of any mucosal adjuvant are its capacity to prevent antigen clearance, as well as the ability to promote rapid and efficient antigen uptake by APC. 10.2.3.1 Direct Admixing of Antigen and Adjuvants Simple admixing of the mucosal adjuvant with the antigen constitutes the most desired strategy, due to highly efficient, consistent manufacturing. As stressed above, although the existence of a common mucosal immune system [46] is generally accepted, it seems that antigen-specific B cells preferentially home into mucosal effector sites that are anatomically related to the immunization site. Despite this compartmentalization, effective mucosal responses in distant mucosal areas have been also obtained, depending on the inductive site stimulated through vaccination (e. g., better mucosal responses in the genitourinary tract after nasal than oral vaccination). Thus, in addition to selection of the appropriate mucosal adjuvant, the efficacy of immunization depends to a great extent on the administration route. In fact, it was recently demonstrated that the lipopeptide MALP-2 [30] can be used to stimulate efficient mucosal responses against the HIV-1 Tat protein in the genitourinary tract when both are coadministered by the intranasal route [47]. Only a few successful peptide-based vaccine formulations have been developed. Simmons et al. [48] exploited LT and LTK63 in an attempt to elicit CD8+ CTL responses against peptides from the M2 protein of the respiratory syncytial virus after intranasal vaccination. Induction of specific CTL responses depended on using the mucosal adjuvant. A new vaccination approach, DNA vaccination, has been developed in the past decade. It consists of the use of eukaryotic expression vectors encoding vaccine-relevant antigens rather than the purified antigen itself. However, only a few studies have demonstrated the efficacy of DNA vaccines when they are administered by the mucosal route. Etchart et al. [49] showed that induction of hemagglutinin-specific CTL responses was enhanced by coadministration of the mucosal adjuvant CT or cationic lipids. To further increase the efficacy of vaccine formulations administered by the mucosal route, combinations of adjuvants belonging to different families of molecules have been coadministered with the antigen, leading to synergistic effects [50–52]. However, variable effects have been observed in terms of the dominant Th pattern of the immune response elicited [51, 53]. This approach might facilitate the rational de-

10.2 Mucosal Adjuvants

sign of vaccines against specific diseases by coadministration of selected adjuvants, which promote the quality of immune response required to achieve protection. However, the use of complex adjuvant mixtures would represent a real challenge to the registration of a vaccine. 10.2.3.2 Covalent Linkage of the Adjuvant and Antigen or Adjuvant Incorporation into other Mucosal Delivery Systems The covalent linkage of antigen and mucosal adjuvant has been described as a powerful strategy to improve the efficacy of mucosally delivered antigens [54, 55]. Here, in addition to its biological activity, the adjuvant can also assume the role of a carrier and a stabilizing moiety. The linkage can be achieved by chemical coupling or genetic fusion. Covalent coupling has been largely used with CT, LT, and their derivatives [54]. This strategy seems particularly attractive for small antigenic molecules, such as peptides [56, 57]. In the specific example of DNA vaccines, CpG adjuvant motifs can directly be integrated into the plasmid or the DNA sequence of interest. Several studies have demonstrated the benefit resulting from combination of mucosal adjuvants with other antigen-delivery systems, such as liposomes, virosomes (see second part of the chapter), microparticules, or ISCOMs [58, 59]. One particular approach for an intranasal influenza vaccine included the use of virosome-adjuvant technology. This subunit vaccine contained LT as mucosal adjuvant and evoked promising results in preclinical [60] and clinical trials [61, 62]. Additional studies have demonstrated that ISCOMs containing a fusion protein encompassing the peptide OVA (323–339) linked to the chimeric derivative of CT, CTA1-DD, were highly immunogenic when given by the oral or nasal route [59]. On the other hand, preliminary studies on biocompatible microspheres gave more controversial results. Oral coadministration of antigen-containing particles with CT triggered similar responses to those observed when unprotected antigens were mixed with the adjuvant [63]. Live attenuated carriers (bacteria or viruses) can also be used as delivery systems for recombinant antigens. This strategy combines the invasive capacity of the carrier, the specific targeting of APC at the inductive site, and the immunostimulatory potential of bacterial components, such as LPS and CpG DNA motifs, with the delivery of the foreign antigen [64]. More recently, nonviable biological particles, so-called bacterial ‘ghosts‘, have also been exploited for a similar purpose [65, 66]. A further option consists in combining live or inactivated biological vectors with mucosal adjuvants. For instance, recombinant strains expressing cytokines or costimulatory molecules were more efficient than non-expressing strains [67, 68]. Coexpression of an antigen with CTA2 and B protein subunits in Salmonella also resulted in an improved immune response in preclinical studies [68]. Thus, these approaches expand the capacity to modulate the immune responses elicited by mucosal antigen-delivery systems. 10.2.3.3 Adjuvant in Prime–Boost Vaccination Strategies Recent studies have investigated the possibility of eliciting desired immune responses at systemic and mucosal levels by combining different strategies and tech-

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nology platforms for antigen delivery in prime–boost vaccination protocols [51, 70]. In such immunization protocols, administration routes can be combined by giving a parenteral or mucosal prime followed by a mucosal or parenteral boost, respectively. In some studies, adjuvants that are also amenable to mucosal use have been employed for parenteral administration, such as CpG [51]. Prime–boost strategies seem particularly efficient for inducing mucosal immune responses at proximal and distal locations [71]. In this context, using the nasal route offers several advantages. Indeed, the local microenvironment is less aggressive to the antigen preparation than that at other mucosal sites, and nasal immunization results in the broadest dissemination of effector cells to distant mucosal sites of the CMIS. Studies performed using the hepatitis B surface antigen administered with CpG, CT, or aluminum salts as adjuvants have shown that efficient immune responses can be induced by using prime–boost protocols in which parenteral and mucosal (intranasal) vaccinations were alternated [51]. We recently obtained excellent humoral and cellular immune responses against viral antigens by combining parenteral vaccination using protein or DNA with intranasal vaccination using protein and MALP-2 as an adjuvant (unpublished data). 10.2.4 Interaction of Mucosal Adjuvants with the Innate Immune System

A better understanding of the underlying mechanisms of adjuvanticity is a prerequisite to fully exploit the potential of novel immune stimulants to fine-tune elicited responses, as well as to comply with regulatory requirements for vaccine licensing. Elucidation of the chemical nature of the adjuvant molecules and identification of their ligands constitute critical parameters to be defined. This knowledge would also allow rational screening for novel molecules with similar or improved biological activity. A good understanding of the interactions between the innate and adaptive immune systems plays a pivotal role in this process (Figure 10.1). The human immune system has evolved to recognize entities that represent a danger to the host. The innate immune system recognizes danger signals, thereby leading to the activation of specific cells bridging the innate and adaptive immune systems. In this context, the innate immune response not only provides the first line of defense against infection or altered-self agents, but also determines the nature of the acquired immune response. Innate immune recognition relies on receptors that bind conserved motifs (pattern-recognition receptors), such as the various Toll-like receptors (TLR) and the complement system [72]. This basic knowledge provides us with essential clues to identify the putative receptors for microbial-derived mucosal adjuvants. For example, imidazoquinolines are both TLR7 agonists [73, 74] and ligands of TLR8 [75]. On the other hand, unmethylated CpG binds to TLR9 [76], and the heterodimer TLR2/TLR6 corresponds to the receptor of the lipopeptide MALP-2 [77, 78]. Studies dealing with the functions of TLR suggest that they can discriminate between different structures, thereby leading to the stimulation of distinct responses [79]. The TCR-mediated signaling pathways have not yet been completely elucidated [79, 80]. However, the activation and translocation of NF-kB constitute central relay

10.2 Mucosal Adjuvants

points within the signaling cascade, which can be achieved by MyD88-dependent or independent pathways. The activation of this nuclear factor is critical for innate immunity, being required for the production of proinflammatory cytokines (e. g., IFNa, TNFa, IL-6, IL-12). The distribution of TLR in mucosal tissues is only partially known [81, 82]. However, it is clear that TLR are expressed on key APC, such as DC and macrophages. The early stages of an immune response stimulated by mucosal adjuvants play a key role in the subsequent polarization of the adaptive immune response. Thus, the choice of the most appropriate mucosal adjuvant for a given vaccine antigen depends to a large extent on the quality of the immune response required for protective immunity (e. g., neutralizing antibodies, CTL, Th1 versus Th2 response). The differential activation of APC during the initial phase of the immune response has a pivotal role in the subsequent development of naive T cells [83]. In vitro studies have demonstrated that mucosal adjuvants can affect the maturation and activation of DC and macrophages [29, 32, 73, 83–86]. Additional studies have also shown that they can exert a mitogenic effect on B-cells [32, 38, 87–89]. The observed up-regulation in the expression of MHC class II, costimulatory (CD80, CD86), and adhesion molecules correlates with improved antigen-presentation functions. Activated APC also release cytokines, promoting a local microenvironment favoring T-cell priming. Thus, mucosal adjuvants allow the lowering of the antigen concentration threshold required for activation of immune cells, thereby allowing an efficient T-cell priming even when APC express little of the MHC class II molecules. The observed expression of chemokines and chemokine receptors also suggests that the use of mucosal adjuvants leads to increased cellular trafficking [90–92]. In this context, nasal application of MALP-2 leads to up-regulation of activation markers on APC isolated from the nasal-associated lymphoid tissues and to macrophage recruitment [30]. Recent studies suggest that different APC do not respond identically during infection, particularly with respect to their effects on naive T-cell polarization [93]. A comparison of the cytokine profiles of infected cells revealed that DC, but not macrophages, express the Th1-promoting cytokine IL-12. Accordingly, functional in vitro studies of mucosal adjuvants will have to address specific cellular populations from the target mucosal tissues. 10.2.5 Conclusion

The efficient prevention of mucosal transmission of infectious microorganisms is the primary goal of mucosal vaccines. Several strategies and tools have been developed to achieve this aim, such as the use of mucosal adjuvants. Available data suggest that, by combining different molecules and/or approaches, it would be possible to generate efficient and predictable immune responses. However, only a few compounds have been identified so far which exhibit this property. In addition, many molecules do not exhibit a safety profile allowing their use in humans, so that most studies have been restricted to experimental animal models. Thus, there is a crucial need to identify novel adjuvants, as well as to assess the efficacy of the most promis-

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ing candidates in human trials. A better understanding of the rules governing innate and adaptive immune responses, as well as unravelling the structure and function of the mucosal immune system will play key roles in achieving these aims.

10.3 Virosomal Technology 10.3.1 Introduction

The detailed production of virosomes was recently discussed [94]; here, it is sufficient to describe the structure of virosomes as reconstituted empty influenza virus envelopes, devoid of a nucleocapsid and of the genetic material of the source virus. Ultrastructurally, virosomes are spherical, unilamellar vesicles with a mean diameter of ~150 nm and short surface projections of 10–15 nm. In contrast to liposomes, virosomes contain functional viral envelope glycoproteins, e. g., influenza virus hemagglutinin (HA) and neuraminidase (NA) intercalated in their phospholipid bilayer membrane. The properties of virosomes enable stimulation of both the MHC I and the MHC II antigen-presentation pathways, depending upon antigen formulation (see also Figure 10.1). Antigens can be incorporated into virosomes [95], adsorbed to the virosome surface [96], or integrated into the lipid membrane [96], via a hydrophobic domain or via lipid moieties cross-linked to the antigen [97] (Figure 10.3). Antigens linked to the surface of virosomes are partially proteolysed within the endosome of APC upon endosomal fusion. This results in presentation of antigen-derived peptides in the context of MHC class II molecules. APC stimulation of specific CD8+ T cells by class I MHC-associated peptides is only achieved if an exogenous antigen escapes the default pathway, as a result of fusion activity of the carrier virosome. In this situation, the antigen is delivered to the cytosol of the APC, where it is processed and transported to the endoplasmic reticulum for association with nascent MHC class I molecules [95]. The stimulation of both helper and cytotoxic T lymphocyte (CTL) responses is discussed in more detail below.

4

2

1

3

Fig. 10.3 Options for association of antigen to virosomes : incorporation (1), membrane integration via inherent hydrophobic domains (2) or via crosslinking to a lipid molecule (3), and adsorption to the virosomal surface via electrostatic interaction (4).

10.3 Virosomal Technology

10.3.2 Adjuvant Properties of Virosomes

Virosomes have been successfully used as a carrier system for a variety of non-influenza antigens [99–101]. Physical association between the virosome carrier and the antigen of interest is a prerequisite for the full adjuvant effect of virosomes [96, 100]. In the context of the definition of an adjuvant mentioned in the Overview of this chapter, virosomes fulfil all major characteristics associated with vaccine adjuvants [1, 2]: (1) their virus-like structure provides regular, repetitive antigen presentation to B lymphocytes [102]; (2) they allow for partial antigen protection from extracellular degradation and depot effect [96]; (3) the presence of fully functional, fusion-active, influenza HA envelope proteins anchored in the virosomal membrane enables receptor-mediated uptake and intracellular processing of the antigen by, e. g., DC, leading to MHC I and II presentation and ultimately to costimulation of CD8+ and CD4+ T cells [101, 103]. 10.3.2.1 Virosome Structure and Immunopotentiation The majority of infectious agents, including viruses, bacteria, and parasites, expose highly ordered repetitive antigenic epitopes on their surfaces. Activation of B cells is enhanced by repetitive antigen presentation [102]. The repetitively arranged epitopes present on viral envelopes efficiently cross-link immunoglobulins expressed on the surface of immature B cells, resulting in a T cell independent activation. This indicates that B cells determine foreignness and are therefore activated by antigen organization [104]. Virosomes and natural viruses share structural similarities with regard to antigen presentation (Figures 10.1 and 10.4). The significance of this structural resemblance is manifested in the way in which virosomes mimic the viral induction of an immune response, providing the integral benefits of virosome technology in stimulating immune responses.

1 3 2

4

5 6

APC

B-Cell

6

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CD8+

Fig. 10.4 Virosomal adjuvant functions: (1) Antigen is protected from extracellular degradation, resulting in a depot effect; (2) the virus-like repetitive structure of antigens activates specific B cells to differentiate into antibody-producing plasma cells; (3) antigen is taken up by APC via receptor-mediated endocytosis and processed for presentation in the context of MHC I (4) and MHC II (5), which in turn is required for activation of antigen-specific CD8+ and CD4+ T lymphocytes, respectively. MHC I presentation depends on the pH-induced fusion activity of HA incorporated in the virosomeand leads to activation of specific CD8+ T cells. (6) Activated CD4+ T cells secrete soluble factors (cytokines), which modulate and define the humoral and cellular immune responses.

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10.3.2.2 Depot Effect Protection of the antigen from extracellular degradation allows efficient delivery of immunogenic molecules such as proteins to the cytosol of target cells and may also lead to prolonged exposure of the antigen for immunopotentiation. Prolonged exposure of antigens forming a depot effect has been claimed to be important for costimulation-dependent immune responses [3]. Post-vaccination antibody production depends on antigen retention on follicular DC within the draining lymph node [105]. Therefore, after a primary immune response, the persistence of antigen in the lymph node or injection site is likely to contribute to the duration of the immune response [106]. A depot effect has been shown with biotinylated virosomes in the mouse, by using 125 I-labeled streptavidin as a model antigen [96]. The results of this study showed that 28 days after vaccination, significant amounts of the streptavidin coupled to virosomes could still be detected at the site of injection. Slow release of peptide from a local depot represents an additional factor for optimal T cell priming in regional lymph nodes. Long-term clinical evaluation of a virosome-formulated hepatitis A vaccine showed consistent antibody profiles in vaccinees several years after vaccination [107]. A single vaccination induced high antibody after two weeks, and levels were retained 12 months later, at which time a booster dose was administered. 10.3.2.3 The Pivotal Role of Fusion-active Virosomal Hemagglutinin The unique properties of virosomes are predominantly related to the fusion-active influenza HA anchored in their membranes, so that great care is taken during the manufacturing process to keep this molecule biologically active [94]. This viral glycoprotein confers structural stability and homogeneity to virosomal formulations. In addition, HA provides the fusion properties characteristic of virosomes, a mechanistic feature clearly distinguishing virosomes from other liposomal or proteoliposomal adjuvants and carrier systems. The structure of influenza HA is related to that of other viral membrane fusion proteins [108, 109] and was the first such structure to be solved [110]. The molecular mechanism by which the envelope glycoprotein HA of influenza virus induces membrane fusion has been intensively studied [reviewed in 112, 117]. Influenza HA is a homotrimeric integral membrane protein. Each monomer is composed of two polypeptide domains [113] that are processed through post-translational cleavage of HA into two subunits, HA1 and HA2, which remain attached by a disulfide bond [114]. The HA1 globular head contains a receptor site that enables binding to the numerous sialic acid residues present on human cells, including APC [115, 116]. The fibrous subunit HA2 possesses a fusion peptide (amino-terminal peptide) and is anchored in the virosome membrane. Under neutral pH conditions, HA1 confines the HA2 subunit in an inactive, metastable state in which the fusion peptides are constrained by a network of hydrogen bonds [117]. A shift in pH from neutral to acidic results in a conformational change of the HA that exposes the hydrophobic domains of HA2 and triggers fusion with a target membrane. During natural influenza virus infection, such membrane fusion occurs between the viral

10.3 Virosomal Technology

membrane and the phagolysosomal membrane and results in release of the viral genetic material into the cytoplasm of the infected cell. Under artificial conditions, in the absence of a target membrane, exposure of influenza HA to pH 5 at 37 °C inactivates its fusogenic capacity [118]. The specific uptake and intracellular processing of virosome-associated antigen in APC, particularly DC, is conditioned by the action of the influenza HA. As stressed above, HA not only mediates attachment to sialic acid residues on the cell surface, followed by receptor-mediated endocytosis; but after acidification of the endosome through fusion with lysosomes, HA is also responsible for the pH-induced fusion of virosomal and phagolysosomal membranes. At this point, the antigen associated with virosomes may be degraded in the late lysosome and presented in the context of MHC II, according to the default pathway for an exogenous antigen. Alternatively, due to the HA-mediated membrane fusion with the acidified endosome, the antigen can also be released into the cytoplasm, from where it is channeled into the classical MHC I presentation pathway for endogenously synthesized antigens [reviewed in 119]. In fact, virosome-encapsulated antigens were shown to be efficiently delivered to the cytoplasm of DC in vitro [96] and to induce a strong CTL response in mice [120], two characteristics that strictly depended on the presence of a fusion-active influenza HA in the virosomal membrane. It is conceivable that the method of formulation, e. g., encapsulation as opposed to adsorption to the surface, may influence the preferred presentation pathway, MHC I or MHC II, to some extent. Further studies are required to fully explore the extent to which the type of virosomal antigen formulation can be used to direct the pattern of the immune response induced. Additional immunopotentiating effects have been proposed for influenza HA. Indeed, studies have provided evidence for the stimulation of peritoneal B-lymphocytes by HA via a so-called B-cell ‘superstimulatory’ antigen effect [121]. The implications are that the superstimulatory properties of the influenza virus glycoprotein activate, not only conventional B2 cells, but in addition a B-cell subset, so-called B1 cells, that represents a formidable first line of defense against invading pathogens. The potency of B1 cells in establishing an immediate immune response against antigens is reflected by their increased susceptibility to cross react with ‘third party’ antigens [121]. In addition, B-cell superstimulatory influenza viruses of the H2-subtype may induce B-cell proliferation by a Ca2+-independent protein kinase C (PKC)-activating mechanism [122]. 10.3.2.4 Effect of Pre-existing Immunity to Influenza Virus When virosomes are used as an antigen carrier system in a vaccine, the resulting immune response is also directed against the influenza glycoproteins, as observed for a virosomal hepatitis A vaccine [100]. Large proportions of vaccinees have preexisting immunity against influenza at the time of immunization with a virosomal vaccine, as a result of prior contacts with influenza viruses. Thus, the question of interference of preexisting immunity with the immune response to a virosomal vaccine is justified. Preclinical studies aimed at studying the possibility of such interference have shown that preexisting antibodies against the influenza glycoproteins HA and NA at the time of immunization do not interfere with the adjuvant function of virosomes,

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but rather enhances the immune response against virosome-bound antigen [100]. This presumably occurs via facilitated opsonization, which allows for more efficient uptake of antigen-loaded virosomes by APC. 10.3.3 Validation of the Virosomal Vaccine Concept

The virosome adjuvant technology platform is well tolerated and highly effective at immunopotentiation for a number of antigens. The initial virosome-adjuvanted vaccine for hepatitis A was launched in 1994, and a subsequent trivalent virosome-adjuvanted vaccine against influenza was licensed in 1997. During development of a novel hepatitis A vaccine, the initial clinical work demonstrated that the selected hepatitis A virus (HAV) antigen, RG-SB, was immunogenic in humans when administered as an aluminum-hydroxide-adsorbed formulation. The formalin-inactivated RG-SB strain was then incorporated into a virosomeadjuvanted vaccine for comparison trials. Comparison of virosome-formulated vaccine, soluble (no adjuvant) HAV antigen, and aluminum-adsorbed HAV antigen with respect to immunogenicity and tolerability after intramuscular administration showed that (Table 10.2): . All recipients (100 %) of the virosomal vaccine were seroprotected (620 mIU mL –1) after 14 days compared with 32% of those receiving soluble antigen and 71% of those receiving aluminum-adsorbed vaccine. . Antibody titers were higher after administration of the virosomal formulation than after the other two formulations [123]. Furthermore, the virosomal vaccine was significantly better tolerated than the aluminum-adsorbed vaccine in terms of local pain and swelling/induration. Subsequent work showed that a single dose of 500 RIA units of HAV antigen is sufficient to elicit a protective immune response lasting for at least 12 months. A large field efficacy study in Nicaragua, an area of high endemicity of HAV, demonstrated the safety and protective capacity of the virosome-adjuvanted HAV vaccine [124]. The re-

Tab. 10.2 Seroprotection after vaccination with three formulations of hepatitis A antigen [123]. Seroprotection day 14 day 28 day 180

GMT (mIU mL – 1) day 14 day 28 day 180

100 %

100 %

100 %

92

283

1550

Aluminum-based (n = 10)

71%

100 %

100 %

43

267

604

Soluble (n = 28)

32 %

100 %

100 %

22

160

622

Type of vaccine Virosomal (n = 40)

All subjects received 1000 RIA units of HAV antigen as a single dose.

10.3 Virosomal Technology

sults showed 100 % (p = 0.00007) protection from week 6 to month 15 in vaccinees who were seronegative at baseline. Mathematical modeling of the follow-up data from the Nicaragua trials, in which subjects were followed for several years after a booster vaccination performed one year after primary vaccination, indicated an extensive period of protection provided by the virosome technology [125]. The results of this study suggested a median duration of protection of 55.5 years. After 25.3 years, 95 % of the vaccinees should have a projected titer above the minimum protective level (defined as 10 mIU mL–1). If the higher titer of 20 mIU mL –1 is used, the median duration of protection is 46.8 years, and 95 % of subjects should be protected after 21.5 years. Based on these results, the virosome-adjuvanted HAV vaccine is registered to be protective for at least 20 years. Clinical trials comparing the virosome-adjuvanted hepatitis A vaccine against a traditional aluminum-adjuvanted vaccine have also been undertaken [126]. This study showed clear benefits in terms of local tolerability to the virosome vaccine. Studies have also compared the tolerability of virosome-formulated vaccines with other technology platforms and adjuvants. The excellent local tolerability to the trivalent virosome-adjuvanted influenza vaccine, consisting of a mixture of three types of monovalent virosomes, each harboring the HA and NA glycoproteins from one of the three strains recommended yearly by the World Health Organisation (WHO), has also been shown by comparison with an alternatively adjuvanted subunit vaccine and a vaccine based on whole influenza viruses [127, 128]. The immunogenicity of the virosome-adjuvanted influenza vaccine versus conventional influenza vaccines has been shown to be superior, with a statistically significant improvement in comparison to other vaccines: a 64-fold increase in anti-HA antibodies in elderly people [129]. Importantly, as with the virosomal hepatitis A vaccine [96], no evidence of an anti-phospholipid response was obtained in vaccinees [130]. 10.3.4 Conclusion

The increasing demand for safer and more effective adjuvants for modern vaccines has led researchers to investigate a broad array of adjuvant candidates, very few of which have resulted in viable concepts. Virosome-adjuvanted vaccines, which may include inactivated virus, recombinant proteins, synthetic peptides, or DNA-encoded antigens, have shown great potential for the development of prophylactic and therapeutic vaccines. The optimal antigen presentation provided by virosomes results in elicitation of highly efficient humoral and cellular immune responses. In addition, the well-defined components constituting virosomes, combined with a lack of preservatives or detergents, result in excellent tolerability. These conclusions are supported by the fact that virosome-adjuvanted vaccines against both hepatitis A and influenza are already on the market. These vaccines are registered in most EU countries and are distinguished by an excellent safety and tolerability record.

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Part IV Classical and Novel Vaccination Strategies: A Comparison

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11 Classical Bacterial Vaccines Thomas Ebensen, Claudia Link, and Carlos A. Guzmán

11.1 Bacterial Vaccines: Introductory Remarks

The successful control of bacterial infections depends on two opposing forces: on the one in-hand, host-specific and unspecific defense mechanisms, which are designed to restrict bacterial survival and multiplication; on the other hand, the coordinated expression of bacterial virulence factors, which are responsible for their subversion and/or circumvention. The host immune system provides two different lines of defense against infectious agents. The first nonspecific defense line is given by the innate immune system, and the pathogen-specific adaptive immune response constitutes the second. Cellular components of the innate immune system also represent the critical bridge between these two responses (e. g., dendritic cells). Vaccination constitutes the most cost-efficient strategy for preventing bacterial diseases through the stimulation of pathogen-specific adaptive responses, which are able to protect against subsequent challenge. Most bacterial pathogens are either restricted to the mucosae or need to transit across them to cause disease. Thus, elicitation of a local mucosal immune response after vaccination represents a clear advantage. In fact, the stimulation of a potent local response at the site in which the first line of defense is laid allows promoting protection against both disease and infection (i. e., colonization), thereby reducing the risk of pathogen transmission to other susceptible hosts. Human vaccination against bacterial diseases has been practiced with success for many years. Despite this success, however, human society continues to suffer millions of deaths and multi-billion dollar economic losses annually due to bacterial diseases. This is in part due to the lack of vaccines against many diseases, as well as to the fact that not all available vaccines are completely satisfactory in terms of immunogenicity, efficacy, and safety. In addition, not all efficacious vaccines are used properly. Anti-vaccine movements in developed countries and economic constraints in the developing world prevent optimal vaccine use. To reduce these losses, there is a critical need to develop new, better, safer, and cheaper vaccines, which are able to promote long-lasting immune responses. Various strategies have been exploited to develop antibacterial vaccines (Figure 11.1). Whole-cell vaccines are produced by inactivating the virulent organisms

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Fig. 11.1

Main vaccination strategies.

(e. g., by chemicals, heat). Alternatively, bacteria can be attenuated in such a way that the organisms are not able to cause disease in their natural hosts, but are still able to induce protective immunity. Thus, attenuated organisms are not able to produce clinical disease, but they retain the ability for transient colonization and/or self-limiting replication. These attenuated bacteria can also be used as live carriers (i. e., bacterial vectors) for the delivery of heterologous antigens from other pathogenic organisms (see Chapter 15). It is also possible to use organisms from a related but nonpathogenic species, as an alternative to attenuated organisms. However, it is essential that they share enough similarity to promote a cross-protective immune response. In other cases, mixed approaches have been established, such as the use of a microorganism from a different species that has been attenuated (e. g., attenuated Mycobacterium bovis BCG to protect against tuberculosis). Purified subcellular components from pathogenic bacteria, which are critical targets for the elicitation of protective responses, can also be used to develop subunit vaccines. Subunit vaccine components can be inactivated toxins, capsular polysaccharides, structural proteins, or nonstructural components. The used antigens can be native or obtained by recombinant DNA technology. Subunit vaccines usually exhibit a better safety profile than whole-cell vaccines. However, they are often less immunogenic, have higher production costs, and demand expensive storage procedures. A new approach has emerged in the vaccinology field in the last decade, the socalled DNA vaccines. This approach consists in the use of eukaryotic expression vectors encoding the antigen of interest, which are delivered to the vaccinees. Then, the biosynthetic machinery of the eukaryotic cells is responsible for synthesis of the vaccine-specific antigens. More recently, attenuated bacteria have been successfully exploited as a delivery system for DNA vaccines against both infectious agents and tumors [1]. The availability of different mutants and highly sophisticated expression tools, as well as the possibility to coadminister immune modulators (e. g., cytokines) allow predictable immune responses to be triggered, according to the specific needs.

11.2 Inactivated Vaccines

11.2 Inactivated Vaccines

Many vaccines against human or animal infectious diseases are composed of whole organisms, which have been inactivated to prevent in vivo multiplication, but retain their immunogenicity. Many of them have been empirically designed, without having a clear understanding of either the protective antigens or the immune protective mechanisms. Furthermore, these vaccines are generally associated with a high incidence of side effects. 11.2.1 Methods of Inactivation

Whole-cell vaccines are usually inactivated by heating and/or treating with chemicals, and the final vaccine preparation does not usually undergo further purification. Formaldehyde was the reagent first used for chemical inactivation, but glutaraldehyde and phenol have also been used. However, this procedure may affect the immunogenic epitopes present in the antigens. The use of colicin E2, a potent DNA endonuclease that enters target bacterial cells without disrupting cellular integrity, has also been proposed for bacterial inactivation [2]. A special inactivation approach is based on the use of phage lysis systems. The controlled expression of PhiX174 gene E in Gram-negative bacteria results in the formation of a transmembrane tunnel through the cell envelope complex [3]. The resulting so-called bacterial ghosts have intact envelope structures, but are devoid of cytoplasmic contents. This process was successfully exploited in a variety of Gram-negative bacteria, including Escherichia coli, Salmonella spp.,Vibrio cholerae, and Actinobacillus pleuropneumoniae. The well-preserved surface structures of bacterial ghosts stimulate efficient immune responses when administered by various routes. This seems to be due, at least in part, to the specific targeting of antigen-presenting cells, in which they promote maturation through the potent danger signal delivered by their structural components (e. g., lipopolysaccharides). Ghosts can be not only used as vaccines against diseases caused by the inactivated microorganisms, but also as a technology platform for antigen delivery. In this approach, they can be directly loaded with antigens or, alternatively, recombinant antigens can be expressed before lysis. 11.2.2 Advantages and Limitations of Inactivated Vaccines

Efficient bacterial inactivation leads to vaccine candidates with an extremely good safety profile, which can be administered even to immune-deficient individuals. However, nonreplicating organisms are generally less immunogenic than live bacteria, and multiple doses are required (Table 11.1). On the other hand, all virulence factors can be expressed by the strain used in the preparation of the vaccine, whereas key virulence factors may be missing in the attenuated vaccine strain. However, in vitro cultivation of bacteria does not necessarily lead to the expression of all virulence

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11 Classical Bacterial Vaccines Tab. 11.1 Comparison of live and killed whole-cell vaccines. Live/attenuated vaccines

Killed/inactivated vaccines

Attenuated microorganisms are used Replicate in the host Single or few doses are required Smaller number of microorganisms Less stable No adjuvants are required Can be delivered by natural infection route Stimulate antibody and cellular responses Promote cellular and humoral memory Sometimes spread from vaccinated to unvaccinated individuals Variable safety in immunocompromized patients Shorter shelf-life Easy transport and storage Higher development costs

Virulent bacteria can be used Do not replicate Multiple doses are required Larger number of microorganisms More stable Adjuvants often required Generally given by parenteral route Induce good humoral but poor cellular responses Stimulate weaker cellular memory No spread to unvaccinated individuals Excellent safety in immunodeficient patients Usually longer shelf-life Easy transport and storage Lower development costs

factors, whereas live vaccines are capable of eliciting immune responses also against in vivo expressed antigens. The reactogenicity of inactivated and live vaccines is usually greater than that of other types of vaccines (e. g., subunit vaccines). Vaccines may loose efficacy as a result of accumulated mutations in circulating strains of pathogens. This risk is lower for inactivated than for subunit vaccines, due to the presence of all potential antigens from the pathogen. However, the presence of multiple antigens may result in reduced vaccine efficacy, due to the suppression of immune responses against sub-dominant antigens by the immune dominants. In contrast to live vaccines, in which the immune response closely resembles that against natural infection, the immune responses against parenterally administered inactivated vaccine are limited to the systemic compartment. In addition, humoral rather than cellular responses are stimulated. Finally, inactivated vaccines are not shed, thus they are unable to infect unvaccinated individuals. On the one hand, they do not lead to herd immunity. On the other hand, they do not represent a risk for immunosuppressed contacts. Finally, the development costs of inactivated vaccines are usually lower than those of live or subunit vaccines. However, quality control (e. g., potency tests, killing tests) of inactivated vaccines may be very difficult.

11.3 Live Vaccines

Live vaccines lead to a self-limited asymptomatic infection, thereby stimulating similar immune responses to those observed after natural infections. Therefore, this

11.3 Live Vaccines

kind of vaccine closely matches some of the criteria for an ideal vaccine, since it stimulates long-lasting protection with minimal reactogenicity after a single or a few doses. 11.3.1 Attenuation

Attenuation of the microorganism eliminates its disease-causing capacity and can be obtained by biological or technical manipulations (e. g., multiple passages through unusual hosts, growth at suboptimal conditions, chemical mutagenesis). In the past, attenuation was performed by empirical techniques. However, the explosive development of our knowledge in the fields of microbial pathogenesis and recombinant DNA technology allows the identification of molecular targets for attenuation and thereby enables rational and precise construction of vaccine strains containing welldefined deletions in these genes. In strains generated by recombinant DNA technology, the attenuation should be sufficient to limit pathogenicity without risk of reverting to the wild-type phenotype. Thus, it is desirable to introduce at least two independent attenuating mutations. The attenuation should not rely on a fully functional host immune system. Furthermore, it must not be reversible by diet or by host modification of diet constituents, including the host's resident microbial flora. Finally, it is also necessary that the attenuation does not induce a persistent carrier state in vaccinees. 11.3.2 Advantages and Limitations of Live Bacterial Vaccines

Live vaccines have several advantages and disadvantages in comparison to subunit vaccines (Table 11.2). Although live attenuated vaccines replicate in the host, subunit or inactivated vaccines are unable to replicate. Therefore, attenuated vaccines usually

Tab. 11.2 Comparison of live and subunit vaccines. Live vaccines

Subunit vaccines

Live/attenuated microorganisms Replicate in the host Variable safety Can spread to unvaccinated individuals Potential risk of reversion Potential risk to immunosuppressed patients Usually more reactogenic Stimulate humoral and cellular responses May require fewer doses Generally long-lasting protection Lower production costs Easy administration logistics

Subcellular components of microorganisms Do not replicate in the host Excellent safety Not transmissible to contacts No risk of reversion No risk to immunosuppressed patients Usually less reactogenic Trigger mainly humoral responses Multiple doses are required Periodic boosters required Higher production costs More complicated/expensive administration logistics

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promote long-lasting immune responses, which mimic those obtained after natural infections. However, the presence of preexisting or cross-reactive immune responses against the vaccine strain may affect its overall efficacy. Live vaccines are less stable and may be easily damaged or destroyed by heat or light. However, cold storage is required for all vaccines registered up to now. Depending on the degree of attenuation and the specific vaccine, multiple doses may be required. The risk of reversion to the wild type phenotype, which was certainly an issue with the undefined first-generation attenuated vaccines, is now made negligible by the introduction of multiple well-defined independent deletions in second-generation vaccines (see Chapter 15). However, live vaccines can generally be considered more reactogenic. This also depends on the route of administration, since oral attenuated vaccines against typhoid fever are less reactogenic than parenteral ones. A specific issue to be taken into consideration is the potential risk associated with administration or transmission (i. e., shedding) to immunodeficient individuals. In addition, a careful and exhaustive evaluation of the potential impact of environmental release, including duration and rate of shedding, as well as the risk of horizontal gene transfer, needs to be carried out. A clear advantage of attenuated vaccines is the fact that they have lower production costs than subunit vaccines. In addition, technology transfer to developing countries is facilitated by the simplicity of the upstream and downstream processes. They are also associated with much simpler and less expensive administration logistics when they are given via the mucosal route. The mucosal route also provides better safety, since parenteral vaccination is an important cause of spreading of HIV and hepatitis in the developing world.

11.4 Vaccines for Human Bacterial Diseases

Bacterial infections play a major role in human disease. Although the development and use of anti-infective agents has resulted in reduced morbidity and mortality, increases in antibiotic resistance complicate the clinical management of infected patients. Furthermore, the implementation of therapeutic approaches does not reduce the human suffering and costs associated with disease. Vaccines have played a major role in the fight against bacterial diseases that have cost mankind heavily in terms of human lives, such as diphtheria, tetanus, whooping cough, meningitis, and tuberculosis. Thus, it is necessary to foster the use of available vaccines and to develop new ones against those diseases for which no vaccine is yet available. 11.4.1 Anthrax (Bacillus anthracis)

Anthrax is primarily a disease of herbivorous animals acquired via the ingestion of Bacillus anthracis spores. Humans occasionally acquire the disease through contact with infected animals or contaminated animal products via the skin (95 %) or by ingestion or inhalation. Without treatment, the cutaneous forms of anthrax have 20 %

11.4 Vaccines for Human Bacterial Diseases

mortality, whereas gastrointestinal and pulmonary anthrax reach mortality rates of 25 %–75 % and more than 80 %, respectively. The emerging threat of bioterrorism has awakened the general interest in both the pathogenesis of B. anthracis and vaccine development. Factors contributing to the pathogenesis process are the plasmid (pXO1 and pXO2) -encoded capsule components, the protective antigen, the lethal factor (LF), and the edema factor (EF) [4]. The capsule helps the bacterium to evade immune clearance, and the toxins are responsible for inducing edema and shock. Anthrax was the first bacterial disease for which vaccination was successfully demonstrated. In 1881 Pasteur observed protection in sheep after injection of a heat-attenuated B. anthracis strain. In 1937 the Sterne vaccine strain was developed, which is nonencapsulated (cured from pXO2) but retains the toxins [5]. The worldwide implementation of this vaccine has contributed to the successful control of the disease in livestock and wild animals. However, it cannot be used in sensitive animals, due to its residual virulence. More recently, several live vaccine candidates lacking EF and LF were developed [6]. However, they were considered not suitable for human use, since they still retain a certain degree of virulence [7]. In 1970 an anthrax vaccine was licensed for human use, which consists of a sterile filtrate of microaerophilic cultures of a nonencapsulated avirulent strain [8]. 11.4.2 Cholera (Vibrio cholerae)

Cholera is a serious water- and foodborne epidemic disease caused by V. cholerae, which has killed millions of people and continues to be a major health problem worldwide. Bacteria colonize the small intestine and produce an exotoxin, cholera toxin (CT), consisting of an enzymatically active subunit A and five identical B subunits. CT disrupts the function of ion pumps, leading to changes in the net flows of sodium, chloride, and water, and resulting in massive diarrhea, electrolyte imbalance, and circulatory collapse. According to the lipopolysaccharide (LPS), two serogroups are circulating (O1 and O139). O1 organisms are classified as classical or El Tor biotypes, according to the phenotype. V. cholerae O1 El Tor biotype is the predominant cause of cholera worldwide; however, a minority of cholera cases are caused by V. cholerae O139 [9]. Convalescents develop a protective immune response, as demonstrated by both volunteer studies and patients follow-up [10, 11]. Serological responses against LPS and CT are observed after a cholera episode, and can block colonization and neutralize CT. The first recorded attempt to develop a vaccine against cholera was in the late 1880s. This killed whole-cell vaccine was injected into the bloodstream, but failed to elicit protective immunity and was associated with high rates of side effects. However, a phenol-killed V. cholerae Ogawa and Inaba vaccine administered by parenteral route is licensed, which stimulates high titers of vibriocidal antibodies in 50 %–90 % of vaccinees and shows an efficacy of about 50 % for about 6 months [12]. To obtain a good local immune response in the gut, an oral vaccine based on heatkilled classic Inaba and Ogawa and formalin-killed El Tor Inaba and classic Ogawa,

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coadministered with 1 mg of the CT B subunit (CTB), has been developed and showed 60 %–85 % efficacy in humans [13]. Another oral vaccine is based on a genetically attenuated V. cholerae O1 classical Inaba strain (CVD 103-HgR), contains a deletion in the CT enzymatically active subunit, and has been marked with a gene coding for mercury resistance that permits differentiation between recombinant and wild-type circulating isolates. This vaccine was licensed in Switzerland in 1994 and is now also available in several other countries. Placebo-controlled trials in several countries have shown the safety and immunogenicity of a single dose of CVD 103HgR, even in HIV-infected individuals. Side effects, such as mild nausea, abdominal cramping, and diarrhea, were rarely observed. A single dose conferred 95 % and 65 % protection against V. cholerae classical and El Tor, respectively, in volunteers in challenge studies [14, 15]. Another candidate vaccine is the V. cholerae O1 El Tor Inaba Peru-15 strain, which was obtained by incorporating a series of genetic deletions and modifications [16]. A seroconversion rate of 97 % for vibriocidal antibodies was observed, and none of the volunteers showed moderate or severe diarrhea after challenge with V. cholerae O1 El Tor Inaba strain. None of theses vaccines is expected to confer protection against V. cholerae O139. However, candidates based on these approaches against O139-expressing V. cholerae are being developed. 11.4.3 Enterotoxigenic Escherichia coli

Enterotoxigenic E. coli (ETEC) is the most common cause of traveler’s diarrhea, which is acquired by ingestion of contaminated food or water. Whole-cell vaccines, which mimic natural infections, seem to promote longer immunoprotection and are being evaluated in clinical trials. A nonreplicating whole-cell anti-ETEC vaccine was prepared by treating the enterotoxigenic E. coli strain H-10407 (O78:H11, CFA/I), which produces heat stable and heat labile (LT) toxins, with colicin E2 [2]. After two oral doses, 77.3 % and 86.4 % of the volunteers showed anti-CFA/I and anti-LT sIgA responses, respectively, and 90 % showed antibodies to either CFA/I, LT, or both [17]. On the other hand, a formalin-killed oral ETEC vaccine expressing the most common colonization-factor antigens (i. e., CFA/I, CFA/II, and CFA/IV) and recombinant CTB was given to volunteers in Sweden. An increment in IgA-secreting cells and in the levels of intestinal sIgA against CTB and various CFA components were detected in the majority of volunteers after two doses [18]. Oral vaccination with a killed ETEC plus CTB vaccine in Egyptian children evoked a fourfold rise in antitoxic IgA and IgG titers in 93 % and 81% of the vaccinees. The vaccine was also safe and immunogenic in 2–12-year-old children [19]. Besides the use of whole-cell E. coli vaccines, other approaches have been pursued to stimulate efficient anti-ETEC immune responses, such as the use of purified fimbriae, toxoids, or carrier organisms expressing ETEC antigens.

11.4 Vaccines for Human Bacterial Diseases

11.4.4 Plague (Yersinia pestis)

Yersinia pestis is the etiological agent of plague, a disease that has caused over 200 million human deaths [20]. Plague is primarily a disease of rodents, but it can also affect humans. Thee main forms of plague occur in humans: bubonic, septicemic, and pneumonic. The common form is bubonic plague, which arises following a bite from a flea that previously fed on an infected animal. The most-feared form is pneumonic plague, which is transmitted from person to person via droplets and has mortality rates of up to 100 %. To prevent the disease, both killed whole-cell and live attenuated vaccines have been used [21]. Killed Y. pestis bacteria have been used as veterinary vaccines since 1897, and the first killed vaccine for human use was developed in 1946. Various methods have been exploited for inactivation, including formaldehyde and heat treatment [22]. The Commonwealth Serum Laboratories are currently producing a heat-killed Y. pestis vaccine (strain 195/P), which is given subcutaneously in three doses over a period of 2 months. This vaccine is mainly used in individuals who may be exposed to the pathogen, e. g., veterinarians, researchers, and persons who are deployed to work in areas where the disease is endemic. However, 10 % of immunized individuals show side effects such as malaise, headache, fever, and lymphadenopathy. In addition, no clinical trials have demonstrated the efficacy of killed whole-cell vaccines, and some studies suggest that killed vaccines do not provide protection against pneumonic plague. A live attenuated strain of Y. pestis (EV76) was derived from a fully virulent strain by in vitro passages. The vaccine has been in use since 1908, especially in the former Soviet Union and the French colonies [21]. Studies in mice indicate that this vaccine provides protection against bubonic and pneumonic plague. However, the safety of this vaccine in humans is questionable. In recent years we have seen a renewed interest in the development of efficient and safer vaccines against this old pathogen, due to the potential risk of illegitimate use of Y. pestis as a biological weapon. 11.4.5 Shigellosis (Shigella species)

Shigellosis is a diarrheal disease, which is transmitted via contaminated food and water or through person-to-person contact. As few as 10 organisms can cause infection, leading to diarrhea or dysentery with frequent mucous bloody stools and severe abdominal cramps. Shigellosis is caused by four main species, S. flexneri, the most common in endemic areas; S. dysenteriae type 1, most frequent in overcrowded areas and associated with large epidemic outbreaks; S. sonnei, more active in developed countries; and S. boydii. Infections caused by S. dysenteriae type 1 can also lead to severe complications, such as hemolytic-uremic syndrome. Between 1966 and 1997, the annual number of Shigella episodes worldwide was estimated to be 164.7 million, 69 % of them being children under the age of 5 [23]. Approximately 500 000 deaths occur each year due to this disease.

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Soon after isolation of S. dysenteriae in 1898, heat-killed cultures were tested to assess their potential as vaccines. However, there is still no vaccine available to prevent Shigella infections. In 1950 the first efforts to develop an attenuated vaccine strain were undertaken. The strains T32-ISTRATI, which was isolated after 32 passages on nutrient agar, and S. flexneri 2 a SmD, which was dependent on streptomycin, yielded promising results, but they were associated with side effects [24, 25]. Subsequent efforts were focused on the use of a hybrid E. coli K-12 containing the invasion plasmid that codes for somatic antigens of S. flexneri 2 a (E. coli EcSf2a-2 hybrid). More recently, work has addressed the development of recombinant strains containing mutations in the genes responsible for the biosynthesis of essential aromatic compounds (e. g., aroA, aroD) and/or virulence factors (icsA, iuc, virG, sen, set, stxAB). These mutants undergo only minimal intracellular replication or are deficient in intracellular and cell-to-cell spread. Promising results were obtained in clinical trials with volunteers after vaccination with some of these candidates. Finally, subunit vaccines, such as O antigen coupled to a carrier protein (conjugate vaccines), vesicles of Neisseria outer membrane proteins and O-polysaccharide (proteosome vaccines), and noncovalent complexes of O-polysaccharide and ribosomal particles (ribosomal vaccines) also gave encouraging results in preclinical and clinical studies [26–28]. 11.4.6 Tuberculosis (Mycobacterium tuberculosis)

Mycobacterium tuberculosis remains a major health problem worldwide, with about 2 million people dying of tuberculosis each year [29]. M. bovis Bacillus Calmette– Guérin (BCG), is the only available vaccine against tuberculosis and constitutes the most widely used live bacterial vaccine in the world. The original strain was attenuated by performing >200 successive in vitro passages over 13 years [30]. This strain was then distributed to several laboratories worldwide, where it was produced and maintained. Of the vaccines currently in use, more than 90 % belong to 4 main strains: the French Pasteur strain 1173 P2, the Danish strain 1331, the Glaxo strain 1077, and the Tokyo strain 172. Despite the WHO’s attempts to standardize production and vaccine characteristics, the concentration ranges from 50 000 to 3 million bacteria per dose. Some vaccines (Pasteur 1173 P2 and Danish 1331) induce strong immunogenicity in animal models, whereas the strains Glaxo 1077 and Tokyo 172 stimulate weak responses. Side effects differ from strain to strain and are predominantly related to infection or to errors in achieving intradermal inoculation (e. g., local reactions, lymphadenitis, osteitis). Recently, the BCG strains have been carefully characterized [31] and shown to lack expression of certain antigens present in M. tuberculosis, which might be important for protective immunity. Therefore, BCG is the most controversial vaccine in current use [32]. It is generally accepted that BCG protects children from meningeal and miliary tuberculosis. However, protection of adults from the most common form of the disease, pulmonary tuberculosis, varies in different studies from 80 % in the UK to 0 % in South India [33]. This variable efficacy can be due to many reasons, such as vaccine strain, vaccine quality, host genet-

11.4 Vaccines for Human Bacterial Diseases

ics, nutrition, exposure of the vaccine to UV, infection incidence, and clinical study protocols. Fundamental questions need to be addressed concerning the rational design of novel tuberculosis vaccines, preclinical and clinical tests, and future implementation in the field (see Chapter 21). 11.4.7 Typhoid Fever (Salmonella enterica serovar Typhi)

Typhoid fever affects about 17 million people worldwide each year, with approximately 600 000 deaths. The disease is caused by Salmonella enterica serovar Typhi, after ingestion of contaminated food or water. Typhoid fever and infections caused by other serovars in humans and animals are a serious medical and veterinary problem (see section 11.5.5). Thus, the development of vaccines against this pathogen has constituted an important focus of research. Whole-cell inactivated or attenuated vaccines, as well as subunit vaccines, have been used with variable results [34]. However, most licensed typhoid vaccines confer only about 70 % protection, show poor efficacy in young children, and are not used for routine vaccination. The first parenteral whole-cell typhoid fever vaccine was introduced 1896 in England. Using heat, alcohol, acetone, or formalin, different whole-cell vaccines were constructed and tested in field trials [35]. These vaccines conferred 51%–88 % protection to children and young adults, lasting for up to 12 years. However, inactivated vaccines often lead to local and systemic side effects, such as fever (6 %–30 %), headache (10 %), and severe local pain (up to 35 %). This type of vaccines also requires regular boosts; thus, they are rarely included in public health programs. The first human trials using attenuated strains against salmonellosis were made in the early 1970s with a streptomycin-dependent mutant of serovar Typhi [36]. However, no significant protection was conferred by this vaccine. Then, a live-attenuated vaccine strain (Ty21a) was developed by chemical mutagenesis. This strain is administered by the oral route and carries, among others, the galE mutation, which renders bacteria extremely susceptible to lysis in the presence of galactose. Ty21a constitutes one of the few licensed attenuated bacterial vaccines for human use and the only live vaccine against serovar Typhi. Two different formulations of Ty21a live oral typhoid vaccine have been commercialized. The liquid formulation provides better protection than enteric-coated capsules. Three doses of Ty21a in liquid formulation stimulated 77 % protection over three years and 78 % over five years of follow-up [37]. Ty21a caused minimal side effects, with nausea being the only adverse event appearing more frequently in vaccinees than in the control group. A post-marketing surveillance study showed only 743 spontaneous reports of adverse effects in more than 38 million vaccinees. On the other hand, a parenteral subunit vaccine based on purified capsular polysaccharide (Vi antigen) was shown to provide 64 %–72 % protection over 21 months [38]. A conjugate of Vi with nontoxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA) showed enhanced immunogenicity in adults and also in children 5–14 years of age and stimulated a booster response in 2–4-year-old children. Interestingly, an efficacy of over 90 % in 2–5-year-old children was reported after intradermal immunization with the Vi-rEPA conjugate vaccine [39]. The im-

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proved knowledge of bacterial physiology, pathogenesis mechanisms, and host immune response has allowed developing a new generation of live attenuated vaccine strains. They have been generated by introducing defined nonreverting mutations into genes coding for metabolic or virulence functions. These vaccines have given promising results in preclinical and clinical studies (see Chapter 15). 11.4.8 Tularemia (Francisella tularensis)

Tularemia, caused by Francisella tularensis, is a primary disease of a wide variety of wild mammals and birds, but it can also be transmitted to humans. Type A strains of F. tularensis found in North America are more virulent than type B strains found in Asia, Europe, and North America. Human infections can be caused by as few as 10 bacteria, which enter the host via the skin, mucous membranes, gastrointestinal tract, or the lungs [40]. The high disease incidence in the United States and Russia during the first half of the 20th century led to an intensive research aimed at the development of efficient vaccines, because it was observed that previously infected individuals were protected against reinfection. However, vaccine candidate strains inactivated by heat, acetone, or phenol treatment did not show a satisfactory efficacy in animal studies or human volunteers [41, 42]. During the same period, the Soviet Union developed attenuated strains, which were used to immunize millions of individuals living in tularemiaendemic areas by intradermal route until 1960. Attenuation was obtained by repeated subculturing in media supplemented with antiserum. One of those attenuated strains was brought to the United States in 1956, F. tularensis LSV, and was routinely used to protect technical personal against laboratory-acquired infections [43]. F. tularensis has been considered a potential biological weapon since 1932. Due to the current danger of bioterrorism, the LSV vaccine is currently under review by the US Food and Drug Administration to assess its future potential as a vaccine [40]. 11.4.9 Whooping cough (Bordetella pertussis)

Whooping cough is a highly contagious respiratory infection caused by the Gram-negative bacillus Bordetella pertussis. This pathogen still remains an important killer of children, although there has been a global decline in incidence, consistent with the overall increase in immunization. In 2001 over 12 million cases of pertussis were estimated to occur worldwide, which led to more than 285 000 deaths [44]. After the isolation and propagation of the causative agent in 1906, major efforts were focused in the development of a vaccine. In 1914, a whole-cell ‘whooping cough vaccine’ was listed in the USA [45]. Over the following decades, several whole-cell vaccines were generated, which consist of chemically- or heat-killed B. pertussis cells. In 1942, the inactivated whole-cell B. pertussis vaccine was combined with diphtheria and tetanus toxoids, leading to a triple vaccine (DTP) that is still in use and confers more than 80 % protection. The incidence of whooping cough has been greatly reduced by mass immunization with DTP.

11.5 Veterinary Bacterial Vaccines

Whole-cell vaccines were frequently associated with local side effects (pain, redness, swelling). Some children also showed transient fever, protracted inconsolable crying, hypotonic–hyporesponsive episodes, and seizures [46]. Additional studies showed that the vaccine does not cause sudden infant death syndrome, spasms, or epilepsy and that the risk of developing an acute encephalopathy was less than 1 : 100 000 [47–50]. Thus, the benefits associated with whole-cell vaccines clearly outweigh the risks of side effects, and vaccine-mediated neurological damage or death has never been conclusively proven. However, increasing concern about adverse effects has led to a reduction in public vaccine acceptance and a consequent increase in the incidence of the disease. Thus, efforts were made to develop acellular subunit vaccines against whooping cough based on key virulence factors (pertussis toxoid, pertactin, filamentous hemagglutinin), which are now in the market (see Chapter 12). These vaccines show much lower rates of adverse effects and have replaced whole-cell vaccines in developed countries. However, due to their lower costs, wholecell vaccines are still used in developing countries [51]. One of the major advantages of subunit vaccines is that they can be also used in older children and adults. Thus, they are particularly useful for boosting. This is particularly important, because both natural infection and vaccination with whole-cell or acellular vaccines induce protection against reinfection only for a limited time (about 5–15 years). Adolescents and adults become gradually susceptible, as demonstrated by the increased incidence of atypical whooping cough cases in these age groups [52].

11.5 Veterinary Bacterial Vaccines

Bacterial pathogens cause a wide range of diseases in animals and have a tremendous economic impact. In addition, individuals with occupational or chronic exposure can acquire zoonotic diseases (e. g., veterinarians, farm workers, butchers, pet owners). Thus, animal diseases should be combated both to improve the quality of animal products and to prevent human disease. The use of antibiotics does not constitute a valid alternative, due to both actual regulations for human food and the increased risk of emergence of antibiotic resistance. Therefore, vaccination is the best alternative for tackling this problem. The use of vaccines allows reduction of (1) the clinical symptoms and the consequent economic impact of veterinary diseases, (2) the risk of carrier development, and (3) bacterial shedding and horizontal transfer to susceptible hosts. Since the early 1900s, massive research efforts have been invested in preventing bacterial diseases in animals. Incomplete knowledge of the causative agents and limited information about clearance mechanisms rendered this task difficult. However, by the end of the 20th century the use of bacterial vaccines has led to a significant reduction in the incidence of infectious diseases in the veterinary field [53]. Thus, there are many examples of veterinary diseases for which vaccines were successfully implemented. Interestingly, the number of inactivated or attenuated classical bacterial vaccines is consistently larger in the veterinary than in the human

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field. This is due, at least in part, to broader margins in terms of acceptable side effects and to the critical need of keeping costs low. Furthermore, combined formulations containing different bacterial and/or viral components are the rule rather than the exception. Although the presentation of a fully comprehensive report of all veterinary vaccines goes beyond the scope of this chapter, several examples are given below. The focus is on those diseases that have the biggest impact, with a particular emphasis on those for which classical bacterial vaccines have been introduced into the market. 11.5.1 Infections Caused by Bordetella and Pasteurella Species

Symptomatic and asymptomatic infections caused by Bordetella bronchiseptica are extremely widespread in the veterinary field, with infection rates that may reach 80 %– 100 % in intensively reared animals. This microorganism produces a variety of pathological syndromes and predisposes infected animals to viral or bacterial superinfections (e. g., Pasteurella multocida Type D and Mycoplasma hyopneumoniae in pigs). Pigs infected with B. bronchiseptica and toxigenic strains of P. multocida can develop atrophic rhinitis [54]. The dermonecrotic toxin released by P. multocida affects osteocytes and osteoblasts, promoting bone resorption and leading to systemic manifestations, and is responsible for progressive atrophic rhinitis. The signs of the disease appear by 8–12 weeks of age and progress throughout the growing period. Infected animals show severe atrophy of the nasal turbinate bones, accompanied by lateral deviation or shortening of the nose. A British survey on abattoir pigs showed that 92 % of herds were infected, with 36 % of animals exhibiting atrophic changes. This may result in a 5 %–15 % reduction on live weight gain (25–40 g/day), which in turn may lead to an annual loss of output or resource wastage of 1–4 million £. Thus, parenteral vaccines against atrophic rhinitis, which are based on inactivated B. bronchiseptica in combination with P. multocida Type D dermonecrotic toxoid alone or together with chemically inactivated Pasteurella, are widely used in pigs. Vaccinated animals and their progeny develop high titers of antibodies against B. bronchiseptica and P. multocida dermonecrotic toxoid. Both sows and gilts and their piglets are vaccinated, leading to reduced bacterial colonization, toxin neutralization, better growth, increased average daily weight gain (ADG), better snout scores, and reduced mortality [55]. In some cases, these vaccines are combined with inactivated Erysipelothrix rhusiopathiae to prevent erysipelas in sow herds. In addition to inactivated B. bronchiseptica vaccines for pigs, field studies have been performed with live attenuated prototypes, which stimulate immune responses that can reduce morbidity rates relative to unvaccinated controls [56]. Vaccines against Bordetella spp. and Pasteurella spp. have also been developed for other animal species. Among them, attenuated B. bronchiseptica vaccines, for intranasal administration to dogs to prevent infectious tracheobronchitis (kennel cough), are generally combined with other antigens (e. g., attenuated canine parainfluenza virus). Live attenuated vaccines against the poultry pathogen Bordetella avium were introduced into the market to protect turkeys against rhinotracheitis.

11.5 Veterinary Bacterial Vaccines

Pasteurella pneumoniae is estimated to be the top killer of cattle in the United States, and Pasteurella haemolytica is the main contributor to shipping fever, costing millions of dollars annually in deaths and treatment costs. Thus, attenuated P. multocida strains are combined with P. haemolytica in formulations for cattle. Viral components, such as rhinotracheitis virus, bovine respiratory syncytial virus, parainfluenza, and bovine diarrhea virus are added to the basic formulations to cover a broad range of pathogens. Infections of domestic and wild birds with P. multocida lead to fowl cholera, a contagious, widely distributed disease with high morbidity and mortality. The disease is typically characterized by sudden septicemia and fibrinous pneumonia; however, chronic and asymptomatic infections also occur. P. multocida subspecies multocida is the most common cause of fowl cholera, although subspecies septica and gallicida may also cause a fowl cholera-like disease [57]. P. multocida is classified into 16 serotypes based on its lipopolysaccharide antigens, and the strains most frequently causing fowl cholera are A:1, A:3, and A:4 [58]. Live attenuated and inactivated (e. g., Cholervac-PM-1) P. multocida vaccines have been introduced into the market. Whole-cell inactivated vaccines (bacterins) can provide protection, but only against the homologous serotype. On the other hand, live attenuated vaccines can protect under in vivo conditions against heterologous serotypes. However, attenuated vaccines retain a low level of virulence, and these strains can be implicated in infection outbreaks. For the prevention of fowl cholera, the most commonly used live vaccines are the P. multocida M-9 and PM-1 strains. Current experimental studies revolve around identification of the specific cross-protective antigens that might be incorporated into vaccine formulations, such as the outer membrane protein Oma87, a type4 fimbrial subunit (PtfA), and a transferrin binding protein (Tbpl) [58]. Although conclusive evidence concerning a potential role of the capsule in virulence is still missing, the genes involved in capsule biosynthesis have been characterized as potential attenuation targets [58]. More recently, attenuated auxotrophic P. multocida aroA derivatives have been generated by recombinant DNA technologies and are able to protect against homologous and heterologous challenges in chickens [59]. 11.5.2 Brucellosis (Brucella spp.)

Brucellosis is a worldwide distributed zoonotic disease caused by Brucella spp. Infective cells can persist in the environment for weeks, and dried preparations can retain virulence for years. B. melitensis commonly infects goats and sheep and is particularly virulent in humans, whereas B. abortus, B. ovis, and B. suis preferentially infect cattle, sheep, and swine, respectively. The disease can be transmitted to humans by consumption of raw animal products or by direct contact with infected animals or their carcasses, through skin lesions or by inhalation. Because of the serious economic and medical consequences of brucellosis, consistent efforts have been invested to develop vaccines. However, no human vaccine is currently available against brucellosis, and the disease is mainly controlled by animal vaccination. Antibodies and cell-mediated immunity can influence the course of the

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infection. However, cell-mediated responses (e. g., CTL) are required for clearance of intracellular bacteria [60, 61]. These responses are best stimulated by using live vaccines or by multiple injections of protective antigens with adjuvants. Only a few effective candidates have been identified so far, that reduce abortion but not necessarily infection [62]. Live vaccines retain some virulence and, depending on the administered dose, abortions may occur during pregnancy. The strain B. abortus 19 has been the most widely used to prevent brucellosis in bovines. However, the efficacy differs according to the age, dose, route of administration, and disease prevalence in vaccinated herds. The strain was originally isolated from cow milk in 1923, and, after being kept at room temperature, it was found to be attenuated. The live attenuated B. melitensis strain Rev.1 can stimulate protection against B. melitensis in sheep and goats. This strain also protects rams against infection with B. ovis. Strains 19 and Rev.1 can be horizontally transferred from vaccinated to unvaccinated animals and change to a rough form, since they persist in some animals. Another disadvantage is that the vaccine strains are fully virulent to humans, and many accidental infections have been reported. Other attenuated strains have not been widely adopted, such as a mucoid derivative of B. suis (M vaccine), the B. abortus 104-M vaccine, B. suis strain 2, and B. melitensis strain 5, which were used as oral vaccines in China [63]. The smooth vaccine strains 19 and Rev.1 stimulate anti-O-chain antibodies, thereby preventing differentiation between vaccinated and infected animals. Therefore, efforts were addressed towards the development of a marker vaccine that would not interfere with eradication programs. The smooth B. abortus strain 45/20 was isolated in 1922 and became rough after 20 passages in guinea pigs. This strain was protective but tended to revert to the virulent form. Thus, it was used as an inactivated preparation with adjuvants based on water and oil emulsions. However, this vaccine was discontinued because its efficacy varied greatly from batch to batch and was associated with severe local reactions. B. abortus strain RB51, which lacks the Ochain, was very stable after multiple in vitro and in vivo passages [64]. Its efficacy after subcutaneous vaccination was similar to that of strain 19, but so far it has not led to deleterious side effects [65, 66]. Thus, strain RB51, which also induces protection in swine against B. suis and in goats against B. melitensis, is replacing strain 19 in several countries. Oral vaccination with RB51 stimulates protective immunity in mice and cattle [67, 68]; hence, a practical approach to wildlife immunization seems possible. Prevention of human brucellosis is best achieved by control or eradication of the disease in animals, combined with adequate heat treatment of potentially contaminated food products. However, in some countries this is not feasible. Therefore, attempts have been made to protect humans by vaccination. This has been reinforced by the renewed interest in Brucella resulting from its potential as a biological weapon; however, the results obtained have been generally disappointing. A derivative of strain 19 (19-BA), which was administered by scarification in the former USSR, stimulates short-time protection but leads to severe reactions [69]. Strain RB51 shows minimal pathogenicity in humans, but is resistant to rifampicin, which makes it unsuitable for human use.

11.5 Veterinary Bacterial Vaccines

11.5.3 Porcine Pleuropneumonia ( Actinobacillus pleuropneumoniae)

Actinobacillus pleuropneumoniae is the etiologic agent of a highly contagious porcine pleuropneumonia, characterized by fibrinous, hemorrhagic, and necrotic lung lesions [70]. The disease shows a morbidity of 15 %–30 % and is associated with high mortality rates in acute outbreaks (100 % of affected pigs) and a decrease in the ADG of up to 35 %. So far, 15 serotypes have been identified. Different vaccines have been introduced into the market; some are based on inactivated bacteria (bacterins), and other formulations are based on purified and detoxified Apx toxins I, II, and III (subunit vaccines). Many of these vaccines are combined with other agents, such as chemically inactivated E. rhusiopathiae or Haemophilus parasuis. Natural or experimental infection with live bacterial cells seems to offer the greatest level of protection against reinfection, suggesting that a live strain would be the ideal vaccine candidate. Several attenuated A. pleuropneumoniae strains have been constructed and conferred various degrees of protection against disease; these included temperature-sensitive, metabolic, nonencapsulated, and nontoxigenic mutants [71–74]. More recently, an attenuated mutant deficient in the biosynthesis of aromatic compounds (HS25 aroQ) has been safely delivered to pigs [75]. 11.5.4 Diseases Caused by Mycoplasma spp.

Mycoplasma spp. cause upper and lower respiratory tract infections in various animals species. These microorganisms contain the smallest bacterial genomes, lack cell walls, and are strictly dependent on the host for survival. Mycoplasma hyopneumoniae is a main player in the porcine respiratory disease complex, leading to significant economic losses. The prevalence of enzootic pneumonia ranges from 30 % to 80 % in different countries. M. hyopneumoniae also creates a port of entry for other pathogens. This phenomenon, known as Mycoplasma-induced respiratory disease (MIRD), aggravates the seriousness of the clinical signs and increases economic losses. MIRD can considerably affect the feed efficiency index (up to 14 %) and the ADG (up to 17.4 %). This situation results in an increase in the fattening period, which in turn leads to decreased rotation rates, increased labor and feeding costs, and reduced annual production. Infections spread from the infected sow to its litter and from older to younger susceptible pigs. Commercial vaccines have been available since 1991. They are based on inactivated and adjuvanted bacteria and are administered to baby or feeder pigs, as well as to breeding stocks. Some of the vaccine preparations have been combined with antigens from other pathogens, such as inactivated E. rhusiopathiae or H. parasuis, the etiologic agent of Glasser disease (i. e., polyserositis). Mycoplasma gallisepticum is also an important pathogen that causes chronic respiratory disease in chicken. Vaccination with killed or living vaccines is an option for controlling infections in poultry flocks when hygienic measures fail. Live attenuated M. gallisepticum vaccines include the strains F, ts-11, and 6/85. The F-strain (ad-

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ministered in drinking water or by aerosol) reduces the decline in egg production but has residual virulence. On the other hand, strain ts-11 is less virulent and immunogenic, but still provides effective long-term protection [76]. Temperature-sensitive mutants of M. gallisepticum (TS100) were used to immunize newly hatched chickens and protected them from developing air-sac lesions [77]. Mycoplasma agalactiae is the causal agent of contagious agalactia in small ruminants. The disease, which is more frequent in the Mediterranean region, north Africa, and the Middle East, is characterized by mastitis, arthritis, and pneumonia. Killed organisms are usually administered in combination with adjuvants. Inactivation with phenol seems to confer better protection than formalin- or heat-inactivation [78]. The lack of protection observed in the field may be due to infections with other Mycoplasma spp. Improved protection in comparison to killed vaccines was observed with live attenuated strains of M. agalactiae [79]. 11.5.5 Salmonellosis in Animals

Salmonella causes diseases characterized by acute or chronic enteritis and septicemia in many animal species, which are the principal reservoirs for human infections. Transmission to humans occurs by contaminated drinking water or food products (e. g., milk, meat, eggs), and their incidence has increased worldwide due to the intensification of livestock production. S. enterica serovar Typhimurium became the second-most-important cause of salmonellosis in humans. Since vaccination is compulsory for poultry meat producers, and EU regulations also require monitoring of broiler-breeder flocks and slaughter of infected flocks, efforts have been made to develop efficacious multivalent vaccines for poultry [80]. One of these vaccines consists of iron-restricted inactivated S. typhimurium and S. enteritidis. After intramuscular vaccination, chickens are protected against an oral challenge, but colonization still occurs [81]. Other mutants, e. g., dam, show better results with respect to protection and colonization [82]. The first live oral vaccine against S. typhimurium and S. enteritidis was launched by Lohmann Animal Health in 2003 (TAD Salmonella). Three doses administered into the birds’ drinking water give protection throughout the laying period. The efficacy of live vaccine candidates against Salmonella choleraesuis (e. g., Argus SC), which causes infections in pigs, has also been evaluated. Vaccinated pigs were able to maintain normal weight after challenge [83], and field studies demonstrated that this type of vaccine can lower the prevalence of Salmonella in swine herds. Salmonella gallinarum and S. pullorum cause fowl typhoid and pullorum disease in birds. The clinical manifestations include anorexia, diarrhea, hepatitis, splenitis, myocarditis, pneumonia, ophthalmitis, and high mortality due to septicemia. This leads to decreased egg production and fertility. Thus, inactivated and live attenuated vaccine strains have been developed for poultry [80]. Since 1950 attenuated derivatives of S. gallinarum strain 9 have been extensively assessed. One of them was attenuated by passage in a medium of low nutritional quality, leading to the so-called 9R fowl typhoid strain, which confers strong protection to adult chickens after intramus-

11.5 Veterinary Bacterial Vaccines

cular injection. However, this strain retains some virulence, may persist for many months, and can be transmitted through the egg. To obtain a more avirulent strain, an aro A mutant was developed, but it was less effective [84]. A single oral immunization with a S. gallinarum nuoG derivative reduced the mortality in 2-week-old chickens following challenge with virulent S. gallinarum from 75 % to less than 8 % [85]. Another approach was the intramuscular application of attenuated ‘phage type’ PT4 S. enteritidis. 11.5.6 Leptospirosis (Leptospira spp.)

Leptospirosis is one of the most widespread diseases in livestock and is caused by over 250 immunologically distinct serovars. Bacteria are spread by contact of skin or mucous membranes with contaminated products. Infected animals contribute to further spreading of the disease by actively shedding bacteria. Infections may be asymptomatic or symptomatic (e. g., fever, icterus, hemoglobinuria, renal failure, infertility, abortion, and death). Of the distinct pathogenic serovars, only a small number have been isolated from domestic animals. It is remarkable that only moderate cross-immunity between serovars is observed. Cattle, swine, and horses are the most economically relevant affected species. Because leptospirosis in domestic animals is leading to significant economic losses and infected animals are a potential source of human infection, vaccination with polyvalent vaccines have been widely implemented. Certain occupational groups, such as farmers, sewer workers, and meat workers, have special risks of acquiring the disease. Outdoor leisure activities can also lead to exposure to infection. Inactivated polyvalent vaccines containing different Leptospiras (e. g., grippotyphosa, hardjo, icterohaemorrhagiae, canicola, pomona) are commercially available for preventing abortions and stillborns in livestock and pets. Most of them have been combined with components against other pathogens, such as E. rhusiopathiae, parvovirus, Campylobacter, bovine rhinotracheitis virus, bovine diarrhea virus, and bovine respiratory syncytial virus. 11.5.7 Other Commercially Relevant Animal Diseases

Vaccines that are able to prevent other diseases in livestock and pets have also been developed and implemented in the field. Among them are inactivated oil-adsorbed vaccines against Haemophilus paragallinarum, which is responsible for infectious coryza in chickens. This disease is characterized by swelling of the infraorbital sinuses, facial edema, oculonasal discharge, and sneezing and may cause significant losses in the poultry industry. Inactivated adjuvanted vaccines based on various strains of Fusobacterium necrophorum and/or Bacteroides nodosus are also available to prevent and treat chronic foot rot in sheep and acute foot rot in cattle. It is also possible to immunize against equine monocytic ehrlichiosis by using a whole-cell inactivated Ehrlichia risticii preparation. For pigs, killed vaccines against Streptococcus suis have also been developed. In addition, inactivated vaccines that are based on inactivated

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enterotoxigenic and enteropathogenic E. coli combined with toxoids are available for preventing post-weaning diarrhea. Chlamydia psittaci is responsible for respiratory infections in cats, leading to serous conjunctivitis, sneezing, and nasal discharge. Vaccinated cats have mild clinical disease and are protected from severe clinical disease after challenge exposure. Due to the frequent emergence of adverse effects (e. g., lethargy, anorexia, lameness, and fever), the use of vaccines is frequently restricted to cats at risk of exposure. Live attenuated vaccines may cause atypical reactions in about 3 % of vaccinated cats. Finally, vaccines have been also developed to prevent bacterial infections in fish. A live attenuated vaccine is used to prevent the enteric septicemia caused by Edwardsiella ictaluri, which costs millions of dollars to the catfish industry.

11.6 Conclusions

During the past 200 years, various vaccines have been developed and exploited to control major diseases. In many instances, however, the exact mechanisms of action of successful vaccines were not fully understood. A new generation has emerged, of well-defined recombinant and/or subunit vaccines that exhibit a considerably improved safety profile. However, classical bacterial vaccines have not only played a critical role in the efficient control of infectious diseases in the past, they are still instrumental for the prevention of infections in both humans and, particularly, animal populations.

Acknowledgements

We thank Joan Plana Duran (Fort Dodge Veterinaria S. A.), Guido Dietrich (Berna Biotech Ltd.), and Lothar Staendner (GBF) for helpful discussions and critical reading of the manuscript. This work was supported in part by grants from DFG (GU 482/2–1 and GU482/2–2), EU (QLK2-CT-1999–00310), and BMBF (PathoGenoMik 601III4–1/1VIIZV-21) to CAG.

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31. Behr, M. A.,Wilson, M. A., Gill,W. P. et al. Science 1999, 284, 1520–1523. 32. Collins, H. L. and Kaufmann, S. H. Lancet Infect Dis 2001, 1, 21–28. 33. Colditz, G. A., Brewer,T. F., Berkey, C. S. et al. JAMA 1994, 271, 698–702. 34. Garmory, H. S., Brown, K. A. and Titball, R. W. FEMS Microbiol Rev 2002, 26, 339–353. 35. Engels, E. A., Falagas, M. E., Lau, J. et al. BMJ 1998, 316, 110–116. 36. Levine, M. M., Dupont, H. L., Hornick, R. B. et al. J Infect Dis 1976, 133, 424–429. 37. Levine, M. M., Ferreccio, C., Abrego, P. et al. Vaccine 1999, 17, S22–S27. 38. Klugman, K. P., Gilbertson, I. T., Koornhof, H. J. et al. Lancet 1987, 2, 1165–1169. 39. Lin, F. Y. C., Ho,V. A., Khiem, H. B. et al. N Engl J Med 2001, 344, 1263– 1269. 40. Dennis, D. T., Inglesby,T. V., Henderson, D. A. et al. JAMA 2001, 285, 2763– 2773. 41. Coriell, L., King, E. and Smith, M. J Immunol 1948, 58, 183–202. 42. Kadull, P., Reames, H., Coriell, L. et al. J Immunol 1950, 65, 425–435. 43. Burke, D. S. J Infect Dis 1977, 135, 55– 60. 44. WHO World Health Report, 2002. 45. Gordon, J. and Hood, H. Am J Med Sci 1951, 222, 333–361. 46. Stewart, G. T. Lancet 1977, 1, 234–237. 47. Hoffman, H. J., Hunter, J. C., Damus, K. et al. Pediatrics 1987, 79, 598–611. 48. Melchior, J. C. Arch Dis Child 1977, 52, 134–137. 49. Baraff, L. J., Shields,W. D., Beckwith, L. et al. Pediatrics 1988, 81, 789–794. 50. Miller, D.,Wadsworth, J., Diamond, J. et al. Dev BiolStand 1985, 61, 389– 394. 51. Wilson, C. B. and Marcuse, E. K. Nat Rev Immunol 2001, 1, 160–165. 52. Campins-Marti, M., Cheng, H. K., Forsyth, K. et al. Vaccine 2001, 20, 641– 646. 53. Makela, P. H. FEMS Microbiology Reviews 2000, 24, 9–20. 54. Elias, B., Albert, M.,Tuboly, S. et al. J Vet Med Sci 1992, 54, 1105–1110. 55. Riising, H., van Empel, P. and

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12 Subunit Vaccines and Toxoids Maria Lattanzi, Giuseppe Del Giudice, and Rino Rappuoli

12.1 Introduction

An ideal subunit vaccine is a vaccine that contains those antigen(s) essential for the microbial pathogenesis and virulence against which it is important to induce a protective immune response. Historically, detoxified bacterial toxins were the first subunit vaccines ever developed. This was made possible by the discovery in the 1920s that formaldehyde treatment was able to detoxify tetanus and diphtheria toxin and by the demonstration that animals immunized with the toxoids were protected against challenge with the wild toxin [26, 69]. Since then, the development of new subunit vaccines has been focused towards more and more purified antigens, like native and recombinant proteins, either alone or conjugated with bacterial poly- or oligosaccharides. These highly purified proteins are much more characterized at the molecular level than the first old vaccines, which contained impurities from the pathogen or from the purification process.

12.2 Toxoids

Toxins are virulence factors that are produced by a pathogen and secreted into extracellular space. Sometimes, the release of a potent toxin can induce clinical manifestations of a disease, even in the absence of an invasive infection, as with diphtheria and tetanus. Therefore, both diseases can be prevented solely by the presence of toxin-neutralizing antibodies, which can be induced by active immunization with nontoxic forms of the toxin or be provided by passive immunization. The discovery of an effective method to detoxify tetanus and diphtheria toxins by formaldehyde treatment, made in 1924 by Ramon in France [69, 70] and Glenny and Hopkins in England [26], allowed the introduction of mass immunization that led to the almost complete elimination of both diseases from developed countries. For these two vaccines, the titers of serum antibodies to antitoxin correlate well with protection. Diphtheria and tetanus vaccines are presently still prepared by the method described

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by Ramon and are mostly used in combination with other antigens (whole-cell or acellular pertussis, inactivated polioviruses, Haemophilus influenzae type b capsular polysaccharide, hepatitis B surface antigen) for infant immunization. Tetanus vaccines combined with a low dose of diphtheria toxoid are used to boost immunity in adults [71].

12.3 Subunit Vaccines: Conventional Vaccinology Approach 12.3.1 Polysaccharide Vaccines

Host defense against encapsulated bacteria depends on the phagocytic uptake and killing of bacteria by polymorphonuclear neutrophils. The presence of anticapsular polysaccharide IgG antibodies triggers the direct complement-mediated killing and/ or uptake of bacteria by phagocytes through opsonization. Moreover, the observation that mutants without the capsule are nonpathogenic, due to their high susceptibility to serum complement, led to the use of the capsular polysaccharide as the antigen of choice for the development of subunit vaccines [27, 28]. Purified capsular polysaccharides have been used to develop vaccines against Neisseria meningitidis group C (MenC), group A (MenA), group Y (MenY), and group W135 (MenW135) [39, 52], against 23 serotypes of Streptococcus pneumoniae [101], against Haemophilus influenzae type b [95], and against Salmonella typhi using the Vi polysaccharide [63]. All these vaccines have several disadvantages. Capsular polysaccharides are T-independent antigens: they can induce only transient antibody responses (mainly of IgM and IgG2 isotypes) mainly in adults. Their immunogenicity and efficacy are very poor or absent in children, especially those below two years of age. Moreover, polysaccharides do not induce any immunological memory, and repeated immunizations not only are unable to increase specific antibody titers, they can sometimes even provoke tolerance in adults [29]. In meningococcal meningitis, polysaccharide vaccines have proved useful for control of epidemic MenA disease in adults in Africa, but they are a poor public health tool for prevention of epidemics by routine childhood vaccination [35]. Many studies have suggested that the vaccines do not substantially affect carriage of serogroup A meningococci 1 year after vaccination and that they do not provide herd immunity [35, 96]. With S. pneumoniae, the efficacy of the 23-valent polysaccharide vaccine against pneumonia and invasive diseases is still questionable [101]. There is agreement, however, that this vaccine fails to decrease the carriage rates in children and adults [13]. The solution to the issues related to polysaccharide vaccines was found by conjugating the polysaccharides with the toxoids. This procedure converts T-independent antigens into T-dependent antigens by providing a source of appropriate T-cell epitopes (present in the carrier protein) able to prime carrier-specific T cells, which provide help to B cells for the production of sugar-specific antibodies. This technology

12.3 Subunit Vaccines: Conventional Vaccinology Approach

has been successfully applied to the development of conjugate vaccines, which now represent a powerful tool replacing the plain polysaccharide vaccines. 12.3.2 Recombinant DNA Technology for Subunit Vaccines

To develop a subunit vaccine (which consists only of one or a few pathogen components) a superior knowledge and a more advanced technology is needed than to develop a killed whole-cell vaccine. For these reasons, with the important exception of diphtheria and tetanus toxoids, which even today are manufactured with an 80-year-old technology, subunit vaccines are the newest vaccines to be developed and introduced into routine immunization programs. This was possible thanks to the development of modern molecular biology and recombinant DNA technology in the late 1970s, which represented a real revolution in vaccinology. Despite the introduction of these new technologies, one of the major issues for the development of subunit vaccines is the identification of protective antigens from a given microorganism. With a conventional biotechnological approach, the pathogen is first studied to identify the key factors in pathogenesis and immunity, and the identified factors are then produced in large scale by recombinant DNA. Several steps are needed in this approach, to develop a new vaccine. First, the pathogen is grown under laboratory conditions, to identify its individual components. Each component is produced in pure form and then tested for its ability to induce immunity. This approach takes time, allows only one antigen to be taken into consideration each time, and, significantly, achieves identification only of those components that are expressed or can be purified in large quantities. Unfortunately, the most abundant proteins are not necessarily the most suitable vaccine candidates, and the genetic tools required to identify the less available components may be inadequate or not available. Moreover, it can also happen that antigens that play an important role during infection in vivo are not equally well expressed in vitro. Therefore, this conventional biotechnological approach can require decades to develop a vaccine, and sometimes it can fail completely. Once a suitable antigen is identified, it needs to be produced in genetically engineered prokaryotic or eukaryotic vectors and then purified as a recombinant protein. Although successful in several cases, the conventional vaccinology approach took a long time to provide vaccines against those pathogens for which the solution was simple. In marked contrast, this approach cannot produce a product for those complex pathogens, bacteria and parasites, that do not have evident immunodominant protective antigens. The conventional approach also means that vaccine development is not possible when the pathogen cannot be grown in the laboratory. However, despite these limitations, this approach has generated three very efficacious recombinant vaccines: the hepatitis B vaccine, the acellular vaccine against Bordetella pertussis, and the recombinant vaccine against Lyme disease.

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12.3.2.1 HBV Vaccine In the 1970s it was shown that the antibody response to the hepatitis B surface antigen (HBsAg), which circulated in the bloodstream in large quantities, correlated with protection against disease. These discoveries led to the development of plasmaderived HBsAg subunit vaccines [47]. The application of recombinant DNA technology allowed the production of safer yeast- and mammalian cell-derived vaccines, which replaced the older vaccine. This represents the first recombinant subunit vaccine developed [93]. The recommended series of three intramuscular doses induce a protective antibody response in >90 % of healthy adults younger that 40 years of age. After age 40, the cumulative age-specific decline in immunogenicity drops below 90 %; however, by the age 60, 75 % of vaccinees develop protective levels of anti-HBs [77, 99]. In 1992, the Global Advisory Group of the World Health Organization recommended that all countries integrate hepatitis B vaccine into national immunization programs by 1997 [47]. By June 2001, 129 countries had included hepatitis B vaccine as part of their routine immunization programs, and by the end of 2002, 41 out of the 51 countries of the WHO European Region had implemented universal hepatitis B immunization [94]. The ultimate goal of hepatitis B vaccination programs is to decrease the incidence of HBV-related chronic liver disease and hepatocellular carcinoma. Recent studies in Taiwan have clearly demonstrated a reduction in the incidence of primary liver cancer in children born after the implementation of routine infant hepatitis B vaccination programs [41]: these results provide assurance that the strategy of routine infant immunization is a well-conceived public health practice that will benefit generations to come. In addition, these results show that vaccination against cancers due to infectious diseases is feasible. This may pave the way to development of vaccines against other cancers, e. g., cervical cancer by papilloma viruses [44], gastric cancer by Helicobacter pylori [16]. Efforts are under way to develop better HBV vaccines, the goal being to improve vaccine immunogenicity and simplify the delivery. Several strategies are being pursued: using novel adjuvants such as MF59 [37], the AS04 (MPL plus aluminum hydroxide) adjuvant system [19], or CpG oligonucleotides [14, 15]; adding other HBV protein sequences such as the preS sequence [5, 74]; or encapsulating the vaccine in control-released microparticles to achieve single-dose vaccination [86]. 12.3.2.2 Acellular Pertussis Vaccine A vaccine composed of whole, inactivated B. pertussis cells has been available for mass vaccination since the late 1940s [43]. This vaccine was very efficacious in preventing the disease, but the presence of severe adverse reactions, although never proved to be caused by the vaccine, caused a drop in vaccine compliance in the 1970s and stressed the need for a new, safer vaccine. Because pertussis toxin represents one of the major virulence factors of B. pertussis, several researchers developed acellular pertussis vaccines containing chemically inactivated purified PT. However, it is recognized that chemical treatment of PT with formaldehyde and glutaraldehyde is associated with significant reversion rates [53]. To overcome these problems, Pizza and coworkers applied genetic engineering to develop a mutant PT that had all the antigenic properties, without the toxic effects: this genetically

12.3 Subunit Vaccines: Conventional Vaccinology Approach

detoxified pertussis toxin is the first bacterial recombinant subunit vaccine ever developed [61]. The safety and immunogenicity of genetically inactivated PT has been tested in clinical trials, both in adult volunteers and in infants and children, as a monovalent mutant PT alone [64, 66], in association with FHA and pertactin [65, 67], and also with FHA and pertactin in association with diphtheria and tetanus toxoids (DTaP) [32, 56]. These trials showed that the various acellular vaccine formulations containing the nontoxic PT mutant were extremely safe and much safer than whole-cell pertussis vaccines. Furthermore, all formulations induced high titers of anti-PT neutralizing antibodies and very strong antigen-specific T-cell proliferative responses. Interestingly enough, about 5 to 6 years after the primary immunization schedule, this vaccine still exhibited an efficacy of about 80 %, and both antigen-specific antibody and CD4+ T-cell responses were still detectable at significant levels [20, 80]. 12.3.2.3 Lyme Disease Vaccine Early research demonstrated protection in hamsters given whole-cell preparations of Borrelia burgdorferi [40]. However, concerns about cross-protection and the potential reactogenicity and toxicity of a whole-cell preparation provided the impetus for pursuit of a recombinant vaccine [100]. Among several candidate proteins, outer surface protein A (OspA) was immunogenic and protective in a mouse model of infection [23, 100]. In December 1998, the US FDA licensed the first vaccine to prevent Lyme disease, which consisted of lipidated recombinant B. burgdorferi OspA, adjuvanted with aluminum hydroxide. Subsequently, in 1999, the Advisory Committee on Immunization Practices (ACIP) of the Centers for Disease Control and Prevention (CDC) recommended the vaccination for persons 15 to 70 years old who live in or visit high-risk areas and have frequent or prolonged exposure to ticks [1]. In a large phase III multicenter double-blind trial, the vaccine was administered on a 0-, 1-, and 12-month schedule. Efficacy for preventing symptomatic Lyme disease was 76 % after three doses and 49 % after two doses of vaccine. Efficacy for preventing asymptomatic disease was 100 % after the third dose and 83 % after the first two doses [85, 88]. The vaccine stimulates production of antibodies that are believed to destroy or inactivate the spirochetes in the midgut of the infected tick, preventing their transmission to the tick’s host [17, 18]. Because some patients with HLA-DR4 class II major histocompatibility complex (MHC) subtypes (0401 and 0404) and with elevated levels of antibodies to rOspA develop treatment-resistant arthritis after naturally acquired Lyme disease, some concerns were raised that Lyme arthritis could be induced by OspA-based vaccines. However, a recent comprehensive post-licensing study on serious adverse events of over 1.4 million doses of the Lime disease vaccine administered from December 1998 to July 2000 did not detect any unexpected or unusual patterns [45].

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12.4 The Future of Subunit Vaccine Development : The Genomic Approach

The dawn of the new millennium coincides with the beginning of the golden era of genomics. In the 1970s, sequencing a gene was impossible. Sequencing became possible in the 1980s, but approximately a year was necessary for a research team to sequence a gene of 2000 base pairs. Today, the situation is completely changed: the possibility of determining the complete genome sequence of a bacterium in a few months at low cost has allowed the sequencing of the genome of most bacterial pathogens in a short period of time (Table 12.1). Moreover, even higher organisms like parasites can be studied with this approach, collecting huge amounts of useful information. Powerful technologies such as genome sequencing, in silico analysis, proteomics (two-dimensional (2D) gel electrophoresis and mass spectrometry), DNA microarrays, in vivo expression technology (IVET), and signature tagged mutagenesis (STM) have revolutionized the way of studying microbial pathogenesis and vaccine design. The possibility to have access to the whole genome sequences of microorganisms has completely reversed the conventional approach to vaccine development. The genomic approach, also called ‘reverse vaccinology’ [72], does not start from microbiology, but from the comprehensive genomic sequence of the pathogen and, by strict computer analysis, it can predict those antigens that are most likely to be vaccine candidates. Knowledge of the genome of a microorganism makes unnecessary the cultivation of the pathogen itself, in marked contrast with the conventional approach. Analysis of the genome allows all the protein antigens to be equally visible at once, no matter which of them is more or less abundantly expressed in vitro and in vivo or in which phase of growth. This avoids handling dangerous pathogens and also offers the possibility of developing vaccines even for pathogens that cannot be cultured. Moreover, the process allows the identification, not only of all the antigens selected by the conventional biochemical, serological, and microbiological methods, but also the discovery of novel, unknown antigens [73]. In this sense, the genomic approach is much more ‘democratic’ than the conventional one, because all protein antigens can be selected, regardless of their condition, function, etc. The availability of suitable animal models of infection and knowledge of immunological correlates of protection, however, are still essential for identifying good vaccine candidates. Unfortunately, since good correlates of protection are infrequent, this may limit the speed and effectiveness of the approach. Another limit of the genomic approach is its inability to identify nonprotein antigens, such as polysaccharides, which are components of successful vaccines (see above), or to identify CD1-restricted antigens such as glycolipids, which represent new promising vaccine candidates, as in tuberculosis [82]. 12.4.1 When Theory Becomes Reality: The MenB Example

The most obvious approach to the development of a vaccine against N. meningitidis serogroup B (MenB) would be a conjugate vaccine using the capsular polysaccharide,

12.4 The Future of Subunit Vaccine Development: The Genomic Approach Tab. 12.1 Microbial genomes sequenced as of March 18, 2003 (www.tigr.org). Filum

Species

Strain

Archaea

Aeropyrum pernix Archaeoglobus fulgidus Halobacterium sp. Methanobacterium thermoautotrophicum Methanococcus jannaschii Methanopyrus kandleri Methanosarcina acetivorans Methanosarcina mazei Pyrobaculum aerophilum Pyrococcus abyssi Pyrococcus furiosus Pyrococcus horikoshii shinkaj Sulfolobus solfataricus Sulfolobus tokodaii Thermoplasma acidophilum Thermoplasma volcanium

K1 DSM4304 NRC-1 delta H DSM2661 AV19 C2A Goe1 IM2 GE5 DSM 3638 OT3 P2 strain 7 DSM 1728 GSS1

Bacteria

Agrobacterium tumefaciens Agrobacterium tumefaciens Aquifex aeolicus Bacillus halodurans Bacillus subtilis Borrelia burgdorferi Brucella melitensis Brucella suis Buchnera aphidicola Buchnera sp. Campylobacter jejuni Caulobacter crescentus Chlamydia muridarum Chlamydia pneumoniae Chlamydia pneumoniae Chlamydia pneumoniae Chlamydia trachomatis Chlorobium tepidum Clostridium acetobutylicum Clostridium perfringens Corynebacterium glutamicum Deinococcus radiodurans Enterococcus faecalis Escherichia coli Escherichia coli O157:H7 Escherichia coli O157:H7 Fusobacterium nucleatum Haemophilus influenzae Helicobacter pylori Helicobacter pylori Lactococcus lactis subsp lactis

C58 Cereon C58 Uwash VF5 C-125 168 B31 16M 1330 Sg APS NCTC 11168 CB15 strain Nigg AR39 CWL029 J138 serovar D TLS ATCC824 13 ATCC 13032 R1 V583 K12-MG1655 EDL933 VT2-Sakai ATCC 25586 KW20 26695 J99 IL1403

Human pathogen

Genome used for vaccine development

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

✓ ✓

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

✓

249

250

12 Subunit Vaccines and Toxoids Tab. 12.1 (continued) Filum

Species

Strain

Listeria innocua Listeria monocytogenes Magnetococcus sp. Mesorhizobium loti Mycobacterium leprae Mycobacterium tuberculosis Mycobacterium tuberculosis Mycoplasma genitalium Mycoplasma pneumoniae Mycoplasma pulmonis Neisseria meningitidis Neisseria meningitidis Nostoc sp. Oceanobacillus iheyensis Pasteurella multocida Porphyromonas gingivalis Pseudomonas aeruginosa Pseudomonas putida Ralstonia solanacearum Rickettsia conorii Rickettsia prowazekii Salmonella enterica serovar Typhi Salmonella typhimurium Shewanella oneidensis Sinorhizobium meliloti Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pneumoniae Streptococcus pyogenes Streptococcus pyogenes Streptococcus pyogenes Streptomyces coelicolor Synechocystis sp. Thermoanaerobacter tengcongensis Thermosynechococcus elongatus Thermotoga maritima Treponema pallidum Ureaplasma urealyticum parvum Vibrio cholerae El Tor Xanthomonas axonopodis pv. Xanthomonas campestris pv. campestris Xylella fastidiosa Yersinia pestis Yersinia pestis

CLIP 11262 EGD-e MC-1 MAFF303099 TN CDC1551 H37Rv lab strain G-37 M129 UAB CTIP MC58 group A Z2491 PCC 7120 HTE831 PM70 W83 PAO1 KT2440 GMI1000 Malish 7 Madrid E CT18 LT2 SGSC1412 MR-1 1021 COL Mu50 MW2 N315 2603V/R R6 TIGR4 MGAS315 MGAS8232 SF370 type M1 A3(2) PCC6803 MB4(T) BP-1 MSB8 Nichols biovar serovar 3 N16961 citri 306 ATCC33913 9a5c CO92 KIM

Human pathogen

Genome used for vaccine development

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

✓

✓

✓

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

✓ ✓ ✓

✓ ✓

✓

✓ ✓

12.4 The Future of Subunit Vaccine Development: The Genomic Approach

as has been done for the other major meningococcal serogroups. Unfortunately, a polysaccharide-based approach cannot be used for this strain, because the MenB capsular polysaccharide is identical to a widely distributed human carbohydrate ( a(2–8)N-acetyl neuraminic acid, or polysialic acid). This renders the polysaccharide poorly immunogenic in humans. Furthermore, the use of this polysaccharide in a vaccine may elicit autoantibodies [24, 36]. An alternative approach to vaccine development is based on surface-exposed proteins contained in outer membrane vesicles (OMVs). These vaccines have been shown both to elicit serum bactericidal antibody responses and to protect against developing meningococcal disease in clinical trials [89]. These trials have provided strong evidence that efficacious vaccines against MenB are feasible. However, the utility of these vaccines is limited by the sequence variability of major protein antigens, which therefore can elicit an immune response only against homologous strains, especially in children less than 5 years old [89]. The attempts to develop noncapsular, non-OMV vaccines using potentially conserved proteins have also failed so far. The proteins that have been investigated the most either are variable at the level of amino acid sequence, such as the transferrinbinding protein B (TbpB) [76] or are expressed by only about 50 % of the MenB strains circulating, such as the neisserial surface protein A (NspA) [50]. Therefore, although in some instances existing vaccines can be used, especially in people more than 5 years old, these conventional approaches seem far from providing a universal vaccine against this bacterium. Given all these problems, the genomic approach was applied to identify novel potential vaccine candidates able to induce protective immunity against all different MenB strains. This was made possible by the knowledge that, for meningococci, protection correlates with titer of antibodies able to kill bacteria in vitro in the presence of complement (bactericidal antibodies). This implied that screening of potential candidates was possible. The entire genome of the virulent strain MenB MC58 was sequenced and analyzed [91]. The computer analysis predicted 600 novel antigens, 350 of which were expressed in Escherichia coli, purified, and used to immunize mice. Sera were tested for bactericidal antibodies, and 85 novel surface-exposed antigens were found, 25 of which induced bactericidal antibodies. Some of the antigens induced bactericidal titers similar to those induced by efficacious OMV vaccines. Surprisingly, the antigens identified by the genomic approach were different from those identified by a conventional biotechnology approach. In fact, in addition to classical outer-membrane proteins with variable loops, many of the new selected antigens were lipoproteins or surface-exposed proteins with a globular structure and without transmembrane domains. Furthermore, some of these antigens are not abundant on the bacterial surface. The detailed analysis of nucleotide and amino acid sequences of the new antigens in a large number of strains led to the observation that most of these newly described proteins were very well conserved in strains representative of the circulating meningococcal population, thus suggesting the potential of their use in a universal vaccine against MenB [62]. Some of these conserved antigens, which can induce bactericidal antibodies in mice against a large panel of MenB strains, are now being developed for testing in phase I trials in humans.

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Because the genomic approach can identify new proteins regardless of their function, it is important to further characterize the new antigens and understand their role in the bacterial physiology. Some of the newly identified antigens, such as GNA33, NadA, GNA992, App, and GNA1870, have been further characterized biochemically and functionally. GNA33 is a lipoprotein highly conserved among all N. meningitidis serogroups and is surface-exposed on both noncapsulated and encapsulated bacteria. GNA33 shows 33 % identity to a membrane-bound lytic transglycosylase (MltA) from E. coli. Biochemical analysis confirmed that the molecule is a murein hydrolase of the lytic transglycosylase class, as it is capable of degrading both insoluble murein sacculi and unsubstituted glycan strands [38]. Recombinant GNA33 elicits a serum bactericidal response that is comparable to that obtained from the immunization with an outer membrane vesicle vaccine and confers passive protection against bacteraemia in infant rats by mimicking a surface-exposed epitope on loop 4 of porin A in strains with serosubtype P1.2 [30]. Epitope mapping of a bactericidal anti-GNA33 monoclonal antibody identified a short motif (QTP) present in GNA33, which is essential for recognition. The QTP motif is also present in the loop 4 of PorA and was also found to be essential but not sufficient for binding of the Mab to PorA [30]. NadA (neisseria adhesin A, NMB1994) induces strong bactericidal antibodies against both homologous and heterologous strains, suggesting that this protein can be a good candidate for a vaccine [11]. Sequence analysis revealed that NadA is homologous to YadA, a non-pilus associated adhesin of enteropathogenic Yersinia, and to UspA2, an ubiquitous protein involved in serum resistance of Moraxella catarrhalis. In spite of a low level of amino acid sequence homology, the three proteins show a well conserved secondary structure. These proteins have, in fact, a carboxyl terminal membrane anchor domain and an internal region with high coiled-coil probability. Interestingly, NadA forms high molecular weight oligomers that are extremely difficult to dissociate even after extensive boiling and treatment with detergents, and such oligomers are anchored to the outer membrane of meningococcus. Interestingly, the nadA gene is not ubiquitous in Neisseria strains, but it is present only in a subgroup of hypervirulent clonal complexes. When present, this antigen has a very well conserved primary structure, and only three defined alleles can be identified, perhaps suggesting recent acquisition of the gene. Furthermore, NadA can bind to human cells in vitro, suggesting a possible role of this novel MenB antigen in the pathogenesis of the disease [11]. GNA992 appears to be strictly related to two adhesins encoded by H. influenzae, Hsf and its allelic variant Hia, both involved in the formation of type b fibrils [87]. The elevated amino acid sequence similarity of GNA992 with Hsf and Hia (57 % and 51% identity, respectively) and the similar topology of the three proteins suggest that they could share a common role in the mechanism of adherence. The three adhesins have a modular structure and are composed of different numbers of repeats. The core of these repetitive units has been identified, and the conserved motif is present in other adhesive molecules of H. influenzae (HMW1), as well as in human proteins belonging to the family of cell adhesin molecules (CAMs), such as NB-2 [55], which also share a conserved folding. On the basis of these common features, GNA992 has been postu-

12.4 The Future of Subunit Vaccine Development: The Genomic Approach

lated to promote adherence of meningococcus to host cells by mimicking the cell–cell recognition phenomena that occur at the neural level [81]. By fluorescence-activated cell sorter (FACS) analysis and Western blots on outer membrane vesicles, GNA992 has been shown to be surface-exposed. Antibodies elicited by GNA992 are bactericidal against a subgroup of MenB strains, and this antigen is therefore being regarded as a possible component of a multi-component protein-based vaccine. App (adhesion and penetration protein) was described by Hadi and coworkers as a member of the autotransporter family and a homologue to the Hap (Haemophilus adhesion and penetration) protein of Haemophilus influenzae, a molecule that plays a role in the interaction with human epithelial cells [34]. Serruto and coworkers expressed App in E. coli to analyze the functional properties of the protein. They showed that the protein is exported to the E. coli surface, processed by an endogenous serine protease activity, and released in the culture supernatant. E. coli expressing app adhere to Chang epithelial cells, showing that App can mediate bacterial adhesion to host cells. The serine protease activity is localized at the amino-terminal domain, whereas the binding domain is in the carboxy-terminal region. The role of App in adhesion was confirmed also in N. meningitidis [84]. GNA1870 is a new surface-exposed lipoprotein of N. meningitidis that induces high levels of bactericidal antibodies. Sequencing of the gene in 71 strains representative of the genetic and geographic diversity of the N. meningitidis population showed that the protein can be classed into three variants. Conservation within each variant ranges between 91.6 % and 100 %, but between the variants the conservation can be as low as 62.8 %. Antibodies against a recombinant form of the protein elicit complement-mediated killing of the strains that carry the same variant and induce passive protection in the infant rat model. Therefore, this novel antigen is a top candidate for the development of a new vaccine against meningococcus [49]. 12.4.2 Further Applications of the Genomic Approach to Vaccine Development 12.4.2.1 Streptococcus pneumoniae Existing vaccines against S. pneumoniae are based on the capsular polysaccharide, either alone or conjugated with a carrier protein [101]. Although the conjugate vaccine is well tolerated and very efficacious against invasive disease caused by the seven serotypes covered by the vaccine [6], this type of vaccine has several potential limitations, including serotype replacement by strains that are not represented [46]. To circumvent these problems, the whole genome sequence was exploited: 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors were identified; 108 of these proteins were successfully expressed in E. coli and used to immunize mice, and 6 of them conferred protection against disseminated S. pneumoniae infection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection, providing a good base for the development of improved vaccines against S. pneumoniae [98].

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12.4.2.2 Staphylococcus aureus Despite the availability of potent antimicrobial agents, improved public health conditions, and hospital infection-control measures, S. aureus remains a major human pathogen. Indeed, the development of new antibiotic resistance and other epidemiological conditions have highlighted the need for an effective vaccine. The availability of the genomic sequence has allowed the development of a comprehensive approach for identification of the immunogenic proteins in this pathogen [22]. An approach based on genomic peptide libraries in combination with well characterized human sera was used. Small diversely sized peptide libraries, encoded by randomly fragmented genomic DNA, were generated, to ensure that all potential antigens encoded by the genome of the pathogen could be identified. S. aureus peptides were displayed on the surface of E. coli via fusion to one of two outer membrane proteins (LamB and FhuA) and were probed with sera selected for high antibody titer and opsonic activity. Magnetic cell sorting (MACS) was applied to screen these libraries: this step was able to determine the profile of antigens that are expressed in vivo and elicit an immune response in humans. More than 60 antigenic proteins were identified, most of which are predicted to be secreted or to be located on the cell surface of the bacterium. These antigens represent promising vaccine candidates for further evaluation [22]. 12.4.2.3 Porphyromonas gingivalis A process similar to the one used to develop a vaccine against MenB was used also for P. gingivalis, a key periodontal pathogen that has been implicated in the etiology of chronic adult periodontitis [92]. From a genomic sequence, 120 genes were selected by using a series of bioinformatics methods. The selected genes were cloned for expression in E. coli and then screened using a variety of P. gingivalis antisera before purification. This subset of recombinant proteins was then purified and used to immunize mice, which were subsequently challenged with live bacteria in a subcutaneous abscess model. Two of these recombinant proteins (showing homology to Pseudomonas sp. OprP protein) demonstrated significant protection in the animal model and therefore could represent potential vaccine candidates [78]. 12.4.2.4 Streptococcus agalactiae Streptococcus agalactiae, or group B streptococcus, is the leading cause of bacterial sepsis, pneumonia, and meningitis in neonates in the US and Europe [83]. The best protective known antigen in an animal model is the bacterium’s capsular polysaccharide [2]. Unfortunately, there are at least 9 different capsular serotypes and little or no cross protection among serotypes [3, 4, 42, 58, 59]. Therefore, reverse vaccinology was applied to develop a protein-based vaccine that could confer protection against all the different serotypes. The complete genome of a serotype V strain of S. agalactiae was determined and analyzed [90]. The genome is predicted to include 2175 ORFs, 650 of which were predicted to encode products exposed on the surface of the bacteria. Approximately 350 of these ORFs were successfully expressed in E. coli and their products used to immunize mice. By using the sera in ELISA and flow cytometric analysis against intact bacteria, it was demonstrated that 55 of these proteins are in fact measurably expressed on the surface of the bacterium. These

12.4 The Future of Subunit Vaccine Development: The Genomic Approach

new antigens are now being evaluated in in vitro and in vivo models for their capacity to protect against invasive infection by group B streptococcus. 12.4.2.5 Chlamydia pneumoniae C. pneumoniae is an obligate intracellular parasite with a complex biphasic lifecycle: an extracellular infectious phase characterized by a spore-like form, the elementary bodies (EB), and an intracellular replicative stage characterized by the reticular bodies (RB). The pathogen is a common cause of community-acquired acute respiratory infection and more recently has also been associated with atherosclerotic cardiovascular disease [31]. Because of the technical difficulties in working with C. pneumoniae and the lack of methods for its genetic manipulation, not much is known about the cell surface composition of the EB. To define the surface protein organization of C. pneumoniae, a reverse vaccinology strategy and proteome technology were combined [51]. The approach is based on six main experimental steps: (1) in silico analysis of the C. pneumoniae genome sequence to identify genes potentially encoding surface proteins (including outer and inner membrane and periplasmic proteins); (2) cloning, expression, and purification of selected candidates; (3) use of purified antigens to generate mouse immune sera; (4) analysis of sera specificity by Western blotting of total EB extracts; (5) assessment of antigen localization by FACS analysis on whole EBs; and (6) identification of FACS-positive antigens on 2DE maps of C. pneumoniae EB proteins. The results of this systematic genome–proteome approach represent the first successful attempt to define surface protein organization of C. pneumoniae and raise the possibility of finding suitable candidates for a purified vaccine. 12.4.3 The Genomic Approach to Parasite Vaccines

Despite many years of studies and several candidates that have reached Phase I or II clinical trials [75], we still are a long way from an efficacious vaccine able to combat malaria, the burden of which is increasing due to drug resistance and to worsening environmental and social conditions, mainly in Africa [33]. Development of a vaccine against malaria is a very hard task, for many reasons: immunity against malaria parasites is not only species-specific for the four species of human Plasmodia, but also specific for each stage of their development. Moreover, many parasite proteins exhibit polymorphism, which potentially limits the effectiveness of any vaccine not incorporating distinct variants of antigens. So far, antigen choice has been dominated by the arbitrary order in which antigens have been identified with the conventional vaccinology approach. The sequence of the Plasmidium falciparum genome recently published [24] is a great achievement for vaccine development, because it should allow selection of the very best vaccine candidates. Expression of genes predicted to be immunogenic as recombinant proteins or as DNA vaccines will eventually provide an effective vaccine against malaria. The task is not easy, since there is no small-animal model of malaria infection, which limits the possibilities of high-throughput screening of candidate

255

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12 Subunit Vaccines and Toxoids Tab. 12.2 Parasite genomes sequence d or being sequence d at TIGR as of March 18, 2003 (www.tigr.org). Parasite

Chromosome

Status or reference

Bugia malayi Entamoeba histolytica Plasmodium falciparum Plasmodium yoelii yoelii Plasmodium vivax Schistosoma mansonii Toxoplasma gondii Trypanosoma brucei

Whole genome Whole genome Whole genome Whole genome Whole genome Whole genome Whole genome I a/b II a/b III a/b IV a/b V a/b VI a/b VII a/b VII a/b IX a/b X a/b XI a/b Whole genome

In progress In progress 25 9 In progress In progress In progress Completed

Tripanosoma cruzi

In progress

In progress

antigens. However, in recent years there has been growing interest of government and nonprofit organizations in developing a safe and effective malaria vaccine, with an increased amount of funds allocated for this task [54]. The genomic approach is not limited to malaria: the genome of other relevant human parasites are being sequenced or have already been sequenced, including P. vivax, Trypanosoma spp., Schistosoma mansoni, and Brugia malayi (Table 12.2). 12.4.4 The Genomic Approach to Viral Vaccines

The approach to vaccines against viral diseases has usually been conventional: only structural antigens (envelope and core) have been usually considered. This despite the fact that viral genomes have a small size and have been available for several years. Hepatitis C Virus (HCV) can be considered the forerunner of the genomic approach applied to virus discovery and vaccine development. The virus cannot be cultured and it has never been observed by electron microscopy. Cloning and sequencing of the HCV genome were the only instruments for identifying the virus [11] and for studying the envelope proteins that were then shown to be protective in a chimpanzee model of infection [10]. Conventional vaccinology has been applied also to HIV vaccine development with poor results: the envelope glycoproteins (gp120, gp140, and gp160) and other structural proteins encoded by the gag gene have been intensively studied by traditional

12.4 The Future of Subunit Vaccine Development: The Genomic Approach

approaches. It is now clear that other proteins, which are either not part of the final viral particle or are present in such low quantities that they cannot be purified from the virus, for example, Tat, Nef, Rev, and Pol, can be protective in experimental animal models and may be tested also in humans [7, 8, 21, 57, 60]. Even in the complicated field of HIV vaccine development, the reverse vaccinology approach has not only provided new promising antigens, that would have been completely ignored by a traditional approach, but has also taught us an important lesson: even nonstructural, non-superficial proteins, and even naturally poor immunogenic antigens can be suitable vaccine candidates when they are correctly expressed and correctly delivered to the host. In March 2003 the WHO issued a global alert for a new illness designated ‘severe acute respiratory syndrome’ (SARS) [97]. Several hundred cases of severe atypical pneumonia of unknown etiology were reported in Guangdong Province of the People’s Republic of China beginning late 2002. Similar symptoms were detected in patients in Hong Kong, Vietnam, and Canada during February and March 2003. In mid-March 2003, SARS was recognized in healthcare workers and household members who had cared for patients with severe respiratory illness. Many of these instances could be traced through multiple chains of transmission to a healthcare worker from Guangdong province who visited Hong Kong, where he was hospitalized with pneumonia and died. In response to this outbreak, WHO coordinated an international collaboration that included clinical, epidemiological, and laboratory investigations and initiated efforts to control the spread of SARS. Attempts to identify the etiology of the SARS outbreak were successful during the third week of March 2003, when laboratories in the United States, Canada, Germany, and Hong Kong isolated a novel corona virus (SARS-CoV) from SARS patients. Unlike other human corona viruses, it was possible to isolate SARS-CoV in Vero cells. Evidence of SARS-CoV infection has now been documented in SARS patients throughout the world. SARS-CoV RNA has frequently been detected in respiratory specimens, and convalescent-phase serum specimens from SARS patients contain antibodies that react with SARS-CoV. This virus was purified, its RNA extracted, and the approximately 30 000-bp genome sequenced and characterized [48, 79]. Availability of the SARS virus genome sequence is important from a public health perspective. It will allow the rapid development of PCR-based assays for this virus, which will allow the diagnosis of SARS virus infection in humans and play an important part in a public health strategy to control the spread of this syndrome. In the future, this information will assist in the development of antiviral treatments, including neutralizing antibodies, and development of a vaccine to treat this emerging and deadly new disease [48]. These results represent the best example of the power of the new technologies applied to microbiology: within a few weeks from the appearance of the new disease, the etiological agent has been detected, and its full genome has been sequenced. The last time that mankind faced a new disease was in the early 1980s, with the appearance of HIV: at that time, only 20 years ago, it took two years to identify the etiologic agent of the infection.

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13 Engineering Virus Vectors for Subunit Vaccines Joseph Patrick Nkolola and Tomas Hanke

13.1 Introduction

The field of vaccinology has made significant strides in fighting a variety of infectious diseases by using the traditional approaches of attenuated or inactivated microorganisms, protein subunits, toxoids, or capsular polysaccharides [1, 2]. With the exception of general health care measures such as water sanitation, vaccines are the most efficient and cost-effective means of controlling infections, as illustrated by the eradication of smallpox and the control of diseases such as polio, yellow fever, and measles. Vaccines save about 3 million lives each year and an additional 3 million could be saved through improved vaccine coverage. Overall, vaccines are currently available against more than 25 infectious agents; however, many more pathogens remain on the vaccine wish list. Some of these agents are new and some are re-emerging, and for others, such as the human immunodeficiency virus (HIV) and Plasmodium, conventional vaccine technologies are still inadequate. The huge impact that infectious diseases still have on global public health, and indeed countries’ economies, underlines the urgent need for new, more efficient, and safer vaccines. Of the new approaches, recombinant subunit vaccines vectored by rationally manipulated virus vectors have shown promise and are likely to play an important role in vaccine development in 21st-century medicine. The past 20 years have seen a wide range of viruses explored for their ability to induce specific immunological responses against passenger proteins derived from foreign pathogens. Many vaccine vectors engineered from diverse viruses with distinct tropisms and gene expression strategies are currently in preclinical and clinical studies and have generated much hope in the fight against infectious and malignant diseases. Although each viral vector has different biological characteristics, they generally offer advantages over more traditional vaccine technologies, such as high expression of foreign immunogens, potential adjuvanting or immunomodulating effects, and the possibility of targeted delivery of proteins directly to the specialized cells of the immune system. These vectors closely mimic natural infection and thus present immunogens in their natural form (i. e., with correct conformation, glycosylation, and oligomerization), prolong the immunogen delivery, and induce potent

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CD8+ cytotoxic lymphocyte (CTL) responses through the introduction of immunogens into the major histocompatibility complex class I processing pathway. Each vector system is unique, and the ever growing list of them provides a flexibility to tailor vaccines for a particular disease. However, it is important to stress that the use of viral vectors is not without safety issues, all of which must be addressed before the vector can be used in humans and before their ultimate commercial application, especially if the target population is potentially immunocompromised. For vaccination to be successful, a certain copy number of the immunogen gene must be delivered to the target tissue so that sufficient amount of protein is synthesized and an appropriate immune response is induced, which then provides protection from infection and/or disease. In addition, an ideal vaccine should have an excellent safety profile with minimal risk of adverse reactions even in unscreened real world populations, should require only a single-dose administration, should induce protection for life, and should be cheap, stable, and easy to administer (e. g., orally). This chapter reviews the main types of recombinant viruses that are prominent in the field of vaccinology, the methodologies used in their generation, and their advantages and disadvantages.

13.2 Adenoviruses

Adenovirus-based vectors are among the most widely used viral vectors in preclinical studies of vaccines. Adenoviruses are nonenveloped double-stranded DNA viruses usually associated with upper respiratory tract infections. They efficiently infect and express their genes in a wide variety of cell types, including dividing and nondividing cells. The adenovirus genome is about 36 kbp long and is easy to manipulate with classical recombinant DNA techniques [4]. Although over 50 human adenoviral serotypes exist, recombinant adenoviral vectors (rAds) are primarily derived from serotypes 2 and 5 [5]. However, these vectors need to be administered in high doses because most people have preexisting vector immunity, resulting in a rapid neutralization of the adenovirus-vectored vaccines. Of note in current literature is a new recombinant adenoviral system based on serotype 35, which efficiently binds to and transduces cells that elicit a strong immune response, is not inactivated by the antiadenovirus antibodies that are present in the majority of individuals, and does not infect liver cells, which is an unwanted side effect of currently used vectors. As a consequence, it is expected that vaccines based on this system will result in strong immune responses, while using a 100-fold lower dose compared to current adenovirus vectors and improving overall safety and efficacy of the vaccines. 13.2.1 Replication Incompetent Adenoviruses

The first generation of recombinant Ad, and currently the most widely used, are replication-incompetent [6]. These rAds cannot replicate because of the deletion of the

13.2 Adenoviruses

essential viral E1 gene region containing two genes, E1 a and E1 b, which express several distinct proteins through alternative splicing (Figure 13.1). The removal of the E1 gene region creates room for the vaccine expression cassette and prohibits transactivation of viral genes required for viral replication. In addition to the E1 deletion, the viral E3 gene is dispensable for production of recombinant virus as well as the E4 gene, which reduces proinflammatory responses in vivo [6]. Adenoviral vectors without the E1 gene region have to be propagated in a cell line stably expressing a copy of the E1 region. Several cell lines, including human embryonic kidney (HEK) 293 [8] and human retinal cell 911 [9], have been generated that can propagate replication of E1-deficient rAds. The resulting rAd produced from these cell lines can be grown to titers as high as 1012 plaque-forming units per milliliter [4, 10]. After intravenous administration, most of the adenovirus vector is found in the liver, but direct injections can transduce most tissues [11]. Clinical application of rAd vectors has recently gained attention in the area of cancer immunotherapy in both ex vivo transduction of autologous tumor or dendritic cells and direct administration settings. This may be due in part to their efficiency in gene transfer but also because of their cellular toxicity and immunogenicity, which may actually enhance the antitumor response. Recent studies using replication-incompetent adenoviral vectors for disease prevention were encouraging. To this end, two recent reports in the nonhuman primate models of the Ebola virus and HIV [12, 13] have shown that priming with plasmid DNA followed by boosting with rAd augments responses compared with either vector alone [12]. When immunized animals in both studies were subsequently challenged with lethal doses of the virus they all showed resistance. In spite of the many positive attributes of replication-incompetent recombinant adenoviruses, some safety concerns about their acceptability as human vaccines remain to be resolved. During several in vivo studies with high virus inputs, inflammation and acute injury of infected tissues, and particularly the liver, occurred, causing hepatic necrosis and apoptosis [50]. To make these vectors more acceptable for clinical trials, there is a need to explore in depth the many factors involved in their use, including the molecular nature of the immune responses they invoke in the context of various genetic backgrounds, target organs, and routes of administration. This will, of course, require much longer development protocols, but any positive insights may make these vectors a more viable option for use as human vaccines. 13.2.2 Replication-selective Adenoviruses

Replication-selective or oncolytic adenoviruses have recently gained the attention of cancer vaccinologists [18]. These rAds are generated by selective growth and propagation in cells of solid tumors, which results in reduction of tumor mass without systemic toxicity (Figure 13.1). The most attractive feature of these viruses is that they can be propagated in selected unmodified cell lines, thereby facilitating the manufacturing process. To amplify the systemic and local potency of these vectors, techniques have been developed to equip these viruses with transgenes whose expression

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dl E3

(dl E4)

Replication incompetent Ag cassette

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ITR/psi +

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‘Stuffer’-Ag cassette- ‘stuffer’ dl E1A or E1B

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Replication selective Tissue-specific promoter

Fig. 13.1 Adenovirus (Ad)-based vaccines. A Schematic of genomic organization. Transcription of adenoviral genes occurs from both DNA strands, and expression is segregated temporarily, occurring either before (early genes E1–E4) or after DNA replication (late genes encoding structural proteins). B Configuration of replication-incompetent, helper-dependent, and replication-selective Ad-based vectors. Replication-incompetent vectors contain an antigen expression cassette substituted for the deleted E1A– E1B region. Typically, the E3 region is also deleted to accommodate larger insertions. Regions of E2 and/or E4 can be deleted to diminish expression of late viral genes. Helper-dependant vectors are deleted of all viral genetic information except the termini and the packaging sequence, which are required for vector propagation by helper systems. The antigen expression cassette is inserted into the deleted region together with a ‘filler’ sequence, so that the overall vector length is approximately the 36 kbp size similar to native Ad, to achieve efficient propagation. Replicationselective adenoviruses contain deletions in E1A or E1B, which render their propagation selective in cancer cells deficient in p53 or pRB function. Alternative ly, tissue-specific promoters can be substituted for the E1A promoter, so that productive virus infection occurs selectively in tumors having appropriate factors for activity of these promoters.ITR: inverted terminal repeats; psi: packaging signal. Adapted from [38].

cytokine

13.4 Poxviruses

is conditional on DNA replication, which preferentially occurs in cancer cells [19]. It is hoped that a combination of virus-induced oncolysis and cancer immunotherapy can enhance the extent and durability of the antitumor response.

13.3 Adeno-associated Viruses

Parvoviruses were initially discovered as a contaminant of an adenovirus preparation. At least 7 primate serotypes exist, of which the adeno-associated virus serotype 2 (AAV-2) has been studied the most. It has a 4680-bp DNA genome that is flanked by inverted terminal repeats (ITR). Because vaccine vectors need only ITRs and a packaging signal, recombinant AAV can accommodate 36 kbp of heterologous genetic material. In tissue culture, some AAV serotypes can integrate into the host cell chromosome. Integration in vivo is highly inefficient and has never been demonstrated in humans. Since no disease is associated with wild-type AAV infection, AAV make ideal candidates for vaccine vectors. However, human parvoviruses normally require helper viruses such as adenoviruses or herpes viruses for productive replication [14] and it has proved quite challenging to remove these helper viruses from purified AAV preparations. To circumvent this problem, an approach using the cre recombinase or loxP system to remove the packaging sequence from the helper virus has been developed, which greatly improves the production process [15]. Since the rAAV vector does not contain any parental virus coding sequences, the vector itself has not been associated with toxicity or any inflammatory response. It is generally produced by adding separate plasmids containing (1) the ITRs flanking the immunogen gene, (2) rep/cap genes encoding proteins required for viral genome replication and structural proteins, respectively, and (3) a helper adenovirus (Figure 13.2). Large-scale production of rAAV is labor-intensive but may become more commercially viable with the recent strides made in development of packaging cell lines and column chromatographic methods of vector purification [16, 17]. rAAV vectors are considered attractive vaccine vectors, essentially because of their abilities to accommodate large insertions, reduce vector-induced proinflammatory responses, and therefore prolong expression of the passenger immunogen. Thus, by use of rAAV vectors, the number of vaccinations required to deliver immunogenic antigens could be reduced.

13.4 Poxviruses

Poxviruses are large enveloped DNA viruses containing a linear double-stranded genome of 130–300 kbp. Genome and replication complexities have enabled these viruses to infect a wide array of hosts ranging from primates to birds. They are unique among DNA viruses in that their entire life cycle is restricted to the cytoplasm of the infected cell. As a direct consequence, the poxvirus genome can encode DNA and RNA poly-

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Fig. 13.2 Generation of rAAV vaccines. A Life cycle of wild-type AAV. Latency is established after AAV infection of the target cells, which may involve genome integration. AAV virions are rescued after subsequent infection with helper virus, which provides the sequences needed for viral replication in trans. B Production of rAAV vaccines. Permissive cells are cotransfected with a plasmid containing the transcription unit (blue) flanked by the AAV ITRs (green) and plasmid containing the AAV genome without the ITRs. Subsequent infection of these cells with a helper adenovirus enables rescue of rAAV virions, following the packaging of the ITR-containing plasmid, along with helper virus. Helper and rAAV virions can then be separated after heating at 56 °C by centrifugation on a cesium chloride gradient. Adapted from [3] (see colour plates page XXXVI).

13.4 Poxviruses

merases, transcription factors, and DNA biosynthesis elements, in addition to the structural proteins required for virion assembly. As is true for most DNA viruses, transcription is modular, in that early promoters are active before DNA replication, and intermediate and late promoters drive transcription after replication. During the poxvirus replication cycle, four particle forms are generated: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV). These particles differ in their content of envelope proteins. Early events in the vaccinia virus life cycle result in IMV particles being formed from noninfectious precursors (called crescents) within the cytoplasm of an infected cell. A small proportion of IMV can leave the cytoplasm on microtubules and subsequently become wrapped in a double layer of intracellular membranes derived from early endosomes or trans-Golgi network, to form IEV. Again using microtubules, IEV migrate to the cell surface where their outer membranes fuse with the plasma membrane, exposing an enveloped virion on the cell surface. A proportion of these particles are retained on the cell surface and are referred to as CEV, others are released and called EEV. The EEV form of vaccinia virus mediates long-range dissemination of virus, and CEV is important in cell-to-cell spread. It is the IMV that is routinely prepared for experimental recombinant vaccines. Poxvirus vaccine vectors have essentially developed from the Orthopoxvirus and Avipoxvirus genera. These vectors are probably the most-advanced, widely tested vaccine vectors, on the basis of data obtained from nonhuman primate and human clinical studies. Recombinant poxviruses can carry multiple large foreign genes, which are stably expressed at high levels, from the recombinant genome. Also, given the fact that poxviruses are relatively thermostable, vaccine vectors derived from them are attractive for application in developing countries where refrigeration facilities may not always be available. Two general strategies can be used to produce recombinant poxvirus vaccine vectors. The first and more commonly used method involves homologous DNA recombination within infected cells, using a shuttle plasmid that contains the passenger gene-expression cassette flanked by additional poxvirus sequences that direct recombination within the specified genome region [20, 21]. Recombinants can be identified by a variety of methods, including incorporation of reporter genes or drug-selection markers, recombination into the thymidine kinase (TK) locus, and selection in TK-negative cells. The second method of generating poxvirus vectors involves direct ligation of vaccine genes into selected restriction sites within the viral genome followed by transfection of the DNA into cells infected with a helper virus that contains a negative-selectable phenotype. 13.4.1 Mammalian Poxviruses

The first work elucidating the use of poxviruses as expression vectors made use of vaccinia virus [20, 21]. For clinical applications it is of paramount importance to ensure the highest levels of biosafety for any recombinant virus vaccine. Because vaccinia can cause severe disseminated disease in individuals with eczema or compromised immune systems, this prompted vaccinologists to try to develop vectors with

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better safety profiles than those already in use. Of the candidate attenuated vaccinia viruses, one well-characterized strain, modified vaccinia virus Ankara (MVA), has been established as a very safe vaccine vector. It was generated from the Ankara strain by ~570 serial passages in chicken embryo fibroblasts (CEF), which resulted in extensive genome deletions and a replication limited to CEF cells and a few mammalian cell lines [22]. Strikingly, the defect for virus growth in mammalian cells does not impair transcription from the viral promoters for expression of heterologous genes within the vector format. This is a desirable and attractive property if MVA vectors are to be used as effective vaccine vectors. The safety and immunogenicity of MVA in preclinical studies have been extensively documented. In addition, immunization of well over 100 000 individuals during the smallpox eradication campaign has demonstrated the safety of MVA in humans. Based on these facts, a new attenuated poxvirus, NYVAC, was developed as a virus vector for vaccine delivery. NYVAC was generated by a rational deletion of 18 genes from the Copenhagen vaccine strain’s genome, several implicated in virulence and host-range phenotypes [23]. Like MVA, NYVAC efficiently expresses heterologous genes as a vaccine vector. 13.4.2 Avipoxvirus Vectors

Avipoxvirus vectors offer an attractive alternative to poxvirus vectors, due to their limited host range, favorable safety profile of attenuation, and nonneutralization by preexisting vaccinia immunity in the vaccinee. The Avipoxvirus genus can replicate only in avian cells, and thus, when used to infect nonavian cells, avipoxvirus vectors initiate an abortive infection, resulting in expression of genes without subsequent viral DNA replication and production of progeny virus [24]. Initial strategies in the development of avipoxvirus vector involved the use of an attenuated poultry vaccine known as fowl pox virus (FPV), but this was subsequently replaced by canary pox because of the higher immunogenicity of canary pox [25]. An example of a commonly used canary pox vector is derived from the ALVAC virus, a plaque derivative of the KANAPOX vaccine strain [23].

13.5 Herpes Simplex Viruses

The herpes simplex virus type 1 (HSV-1) is an enveloped, double-stranded DNA virus with a genome of 152 kbp encoding more than 80 genes. It has a wide host range and can infect cells lytically or establish latency. Because of its intrinsic ability to cause latent infection (where disease is absent) in human hosts with normal immune status and to persist and be reactivated after primary infection, HSV holds promise as a potent vaccine vehicle. HSV-1 is in fact currently the most extensively engineered herpes virus for purposes of vaccination. Approximately half of the genes present in HSV-1 are nonessential for virus replication in tissue culture, providing

13.5 Herpes Simplex Viruses

multiple sites for gene insertion within the viral genome and making HSV-1 a highcapacity vector capable of harboring at least 30 kbp of heterologous genes [26]. There are three types of HSV-based vectors: recombinant HSV vectors, amplicons, and disabled infectious single-cycle (DISC) HSV. A brief description of each is given below. 13.5.1 Recombinant HSV Vectors

Recombinant HSV vectors are generated by the insertion of transcriptional units directly into the HSV genome via homologous recombination events. To achieve this, HSV genomic DNA is cotransfected into a permissive cell line together with a shuttle plasmid harboring a transcriptional unit containing the passenger vaccine gene. During mitotic division, homologous recombination occurs between HSV sequences within the shuttle plasmid and the viral DNA. Recombined viral genomes that have replicated are subsequently processed and packaged into new virus particles. Just as in other systems, recombinant virus is then separated from parental virus by several rounds of plaque purification. 13.5.2 Amplicon Vectors

Amplicon vectors are the second type of HSV vectors currently under investigation for vaccine application. They are based on plasmids/amplicons of approximately 15 kbp, which bear, in addition to the transcriptional unit of the passenger gene, only an origin of replication and a packaging signal. Recombinant amplicons are usually cotransfected into a cell line together with a packaging-defective helper HSV genome, which enables the amplicon DNA to be packaged into infectious virions. Replication of HSV DNA occurs via a rolling-circle mechanism enabling amplicon vectors to contain several copies of the plasmid [27] (Figure 13.3). In spite of their many advantages, such as large capacity for insertion of foreign genes, ability to cause latent infection, and a wide host range, two major problems have limited the use of HSV as vaccine vectors. First, vector toxicity has been linked to the host protein shut-off functions of some viral gene products and the direct toxicity of others [28]. Second, the production of helper-dependant recombinant HSV has proved quite challenging, due to difficulties in producing materials that are not contaminated with wild-type HSV helper (a similar scenario as described for AAV preparations). In the particular case of HSV vectors, this problem has been recently addressed by using helper virus genome plasmids devoid of packaging signals, propagated in bacteria as bacterial artificial chromosomes [29]. The primary advantage of this new system is that it guarantees an almost helper-free preparation of recombinant amplicons. It is important to stress that, until systems can be developed in which the helper DNA is completely devoid of sequences similar to that of the amplicon vector, recombination between the amplicon and helper DNA will occur, raising the possibility of contamination of vector stocks with unwanted recombinants, some

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Fig. 13.3 Generation of recombinant herpes simplex viruses(HSV) and HSV-derived amplicons. A Recombinant HSV vectors. HSV genomic DNA is cotransfected into permissive cells along with a plasmid containing the transcription unit (red) inserted in fragment of HSV genome (gray). During mitosis, homologous recombination occurs between the HSV genome sequences within the plasmid and HSV DNA. Both wild-type and recombined viral genomes are packaged into a mixed virus particle population, from which the recombinant viruses are isolated by plaque purification. B Amplicon-derivedHSV vectors. The amplicon plasmid containing the transcription unit

(red) and the viral cleavage and packaging site is cotransfected into permissive cells together with a helper virus genome, which contains all the regulatory and structural genes needed for viral growth, including the HSV hostshutoff protein, but is defective in packaging. Immediately after transfection, the amplicon plasmid undergoes DNA replication driven by helper proteins, which results in the generation of head-to-tail plasmid concatemers. These concatemers are then cleaved into genome unit-length moleculesand packaged into virus particles,which after concentration are ready for use. Adapted from [35] (see colour plates page XXXVII).

13.6 Retroviruses

of which might be replication-competent. In addition and as is true with other viruses, production systems dependant on transfection may be difficult to scale up and may not yield high virus titers. 13.5.3 Disabled Infectious Single-cycle HSV

Progress in the area of HSV vector development has highlighted a new vector known as disabled infectious single-cycle (DISC) HSV. This vector is designed to combine the safety advantages of inactivated vaccines with the immunogenic activity of live viral vaccines. The DISC HSV is based on either HSV-1 or HSV-2 and comprises live herpes simplex virus with the essential gene for glycoprotein H (gH) removed to disable the virus. DISC HSV can infect cells much the same as native HSV, and viral progeny are produced but are defective and cannot further replicate. These viral particles from the single cycle of DISC virus replication are nonpathogenic and are capable of inducing broad cytotoxic T-cell and humoral immune responses against multiple antigens. Several studies have shown the effectiveness of these vectors in preclinical [31] and clinical studies of cancer vaccines [52]. It is envisaged that in the coming years, DISC HSV vectors may hold promise as potent recombinant vaccine vectors.

13.6 Retroviruses

Retroviruses are lipid-enveloped single-stranded RNA viruses comprising a diploid genome of about 7–10 kb with four major ORF termed gag, pro, pol, and env. These gene regions encode for structural capsid proteins, viral protease/integrase, reverse transcriptase, and envelope glycoproteins, respectively. In addition, the retroviral genome contains a packaging signal and cis-acting sequences, termed long terminal repeats (LTRs), at each end, which play a role in transcriptional control and integration. Upon entry into target cells, the RNA genome is reverse-transcribed into linear double-stranded DNA and integrated into the host cell chromatin. Within the family of retroviruses, several candidates are being exploited for their potential use as vaccine delivery systems, namely, mammalian lentiviruses and avian C-type spumaviruses. Recombinant retroviral vectors are bioengineered such that they are devoid of all retroviral genes and contain only the LTRs and packaging signal sequences; as a result, propagation of recombinant retroviruses requires expression of the gag, pol, and env genes in trans. The use of recombinant retroviruses in vaccine development is fairly limited, due to several disadvantages. These include their ability to be integrated into the host genome, which could lead to oncogene activation or tumor-suppressor gene inactivation; limited insert capacity of about 8 kb; low titers; inactivation by human complement; inability to transduce nondividing cells; and the potential shutoff of transgene expression over time.

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Of the family members of retroviruses, lentiviruses are probably the most studied and understood. They are complex retroviruses that can infect and express their genes in both mitotic and post-mitotic cells. Replication-defective lentivirus vectors were originally derived from human immunodeficiency virus 1 (HIV-1), the most commonly known lentivirus [30]. This vector system used HIV and the envelope glycoproteins of other viruses to target a broad range of cell types. The technique used to generate recombinant lentivirus vector requires a triple-plasmid cotransfection in 293 cells. The first of the three plasmids has no packaging signal but provides structural viral genes in trans, with the exception of env and genes for several accessory proteins. The second plasmid harbors the gene for the pseudotyped envelope protein. The third plasmid carries the transcription unit of the passenger immunogen alongside the cis-acting sequences of HIV required for packaging, reverse transcription, and integration. Reversion to replication of this HIV-derived vector is highly unlikely, because two independent homologous recombination events would have to take place. An added safety feature of this system is the fact that viral sequences present on the three separate plasmids have been generated to have low sequence homology, thus making a recombination event highly unlikely. As an alternative to the parental HIV, hybrid vectors derived from nonhuman simian, equine, and bovine lentivruses have been also developed as an additional safety precaution.

13.7 Alphaviruses

Alphaviruses belong to the Togaviridae family and have a small single-stranded positive-sense RNA genome of approximately 12 kb, which is both encapsidated and polyadenylated. Alphavirus virions are small (60 nm diameter), spherical, and posses a lipid envelope through which 80 glycoprotein spikes project. Mature particle assembly occurs at the plasma membrane of infected cells, where the cytoplasmic nucleocapsids become enveloped by budding at the membrane previously modified by the insertion of two viral glycoproteins. The development of alphavirus vaccine vectors began with studies on defective interfering (DI) particles, which require helper virus for their replication and propagation. DI particles were used to identify the cis-acting sequences essential for replication and packaging of alphavirus RNAs: the 5´ terminal, packaging signal, and 3´ terminal regions. This was followed by development of a self-replicating RNA (replicon) expression vector using an infectious clone of modified Sindbis virus (SIN) [31, 32]. The development of alphaviruses as vaccine vectors is not as advanced as that of other vectors like adenoviruses or vaccinia viruses, but recent publications have elucidated their use as viable and potent vaccination agents. Although some alphaviruses, such as the eastern equine encephalitis virus, are serious human pathogens, others, including SIN and Semliki Forest virus (SFV), are not. Indeed, SIN and SFV have served as models for molecular studies of alphaviruses and, together with the Venezuelan equine encephalitis virus (VEE), form the core of alphaviruses

13.7 Alphaviruses

developed into safe and efficient vaccine vectors. Several features of alphaviruses make them attractive. They infect a broad host range (including humans) with mild or no symptoms. Seropositivity to alphaviruses is, at least in the mild climate, infrequent, reducing the chances of immune interference. Alphaviruses may be engineered or may posses lymph-node tropism that results in effective antigen presentation and induction of strong, balanced immune responses. They induce apoptosis of infected cells and, in any case, replication of their genome occurs exclusively within the host cell cytoplasm as RNA and therefore their genome cannot integrate or persist. Finally, alphaviruses provide transient but high levels of gene expression through amplification of mRNA. Three major groups of alphavirus vectors have been developed: full-length infectious particles engineered to contain a duplicated subgenomic promoter for antigen expression, replicons engineered for insertion of genes of interest downstream of the subgenomic promoter, and versions of the replicon vector launched from DNA plasmids. A brief description of each of these vector types is given below. 13.7.1 Full-length Infectious Clones

Development of viral expression vectors derived from SIN, SFV, and VEE was preceded by earlier work that was able to generate full-length infectious clones of each of these alphaviruses [32–34]. SIN and VEE full-length clones contain a second subgenomic promoter upstream or downstream of the complete structural protein-coding region, resulting in heterologous genes under its control being expressed at high levels. With this system, it is possible to generate high-titer virus preparations without multiple tissue culture passages. 13.7.2 RNA Replicons

Of the various types of alphavirus expression vectors, replicons are probably the most developed. These RNAs are self-replicating and can express heterologous genes cloned downstream of a subgenomic promoter at very high levels. Replicon vectors lack structural polyprotein genes, which are replaced with the heterologous gene; however, the 5´- and 3´-end cis replication signals, the nonstructural replicase genes, and a natural subgenomic promoter are maintained. The absence of genes for structural protein abolishes the replicon’s spread from cell to cell, because the progeny virus cannot be formed, but it does not affect the RNA amplification and high-level expression of the heterologous gene within the target cell. 13.7.3 DNA Plasmid Replicons

In an effort to increase the potency of DNA vaccines, some alphavirus vectors have been engineered to be launched completely from DNA plasmids. In these vectors, a

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nsP1 nsP2 nsP3 nsP4

Ag

Plasmid replicon

Figure 4. “Engineering Virus Vectors for Subunit Vaccines” Nkolola and Hanke

NUCLEUS

CYTOPLASM (+) strand m7G RNA

(-) strand RNA

Ag

5’

nsP1 nsP2 nsP3 nsP4

JR

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AAAn 3’

AAAn 5’

3’

m7G

Ag

m7G

Ag

AAAn

m7G

Ag

AAAn

AAAn

Fig. 13.4 Plasmid DNA-based alphavirus replicon. The positioning of replicon cDNA immediately adjacent to the polymerase II transcription site provides for in-vivo synthesis of a positivestranded RNA replicon with an authentic 5´ end. After cytoplasmic RNA transport, translation of the four nonstructural genes (nsP1–4) as a polyprotein that is post-translationally processed results in an active replicase complex,which programs high-level cytoplasmic RNA amplification through a negative-stranded RNA intermediate. The replicase also mediates transcription of additional positive-strandedRNA and an abundant subgenomic mRNA encoding the antigen. Alphaviruses can be delivered as RNA, DNA, or particle replicons. Adapted from [38].

eukaryotic promoter drives transcription of the replicon RNA, which exits the nucleus and begins to amplify itself and express heterologous genes (Figure 13.4). Studies have shown that for several antigens this system significantly increases DNA vaccine potency in comparison with conventional cytomegalovirus (CMV) promoterdriven expression [37]. Furthermore, because plasmid-based alphavirus replicons are simply a plasmid DNA, they have all the other advantages of DNA vaccines.

13.8 Polioviruses

13.7.4 Particle-based Replicons

Although the first recombinant alphavirus vaccines tested in animals used doublesubgenomic vectors, more recent studies have tended to concentrate on particlebased replicons. These vectors are single-cycle viruses and work by delivering antigen via ‘infection’ using natural receptors. Packaged SFV, SIN, and VEE particlebased vaccines induce robust humoral, mucosal, and cellular immune responses in animals. Packaging of replicon RNA into particles is essentially achieved by way of cotransfection of permissive cells with in vitro transcribed replicon RNA and ‘helper’ RNA encoding the structural proteins under the control of their native subgenomic promoter. Helper RNA lacks any replicase genes or packaging signal but is required to maintain the 5´- and 3´-end cis signals needed for coamplification with the replicon. This original technique using a single helper system had an unforeseen complication, in that it also generated notable levels of replication-competent virus due to RNA recombination. To try to eliminate this problem, a two-helper system was developed in which three mRNAs are cotransfected into the packaging: one mRNA with the packaging signal coding for the SFV polymerase and an immunogen, and two other mRNAs supplying the capsid and envelope proteins in trans structure [53]. Even more recently, technological advances in this area have generated stable alphavirus replicon particle packaging cell lines that have immense potential in large-scale commercial applications. These packaging cell lines harbor two integrated DNA cassettes, encoding the capsid and envelope glycoprotein genes in a split helper configuration. Studies using alphavirus replicon particles bioengineered for delivery to dendritic cells (DCs) have shown in both mouse models [54] and humans [55] that these replicons induce activation and maturation of transfected DC. This unique DC-inducing capability of particle replicons is a desirable property in any vaccine, because it may improve induction of the immune responses.

13.8 Polioviruses

The poliovirus belongs to the Picornaviridae family and is a small positive-stranded RNA virus with a 7.5 kb genome, which is encapsidated within a nonenveloped protein shell. Two classes of poliovirus vaccine were developed over four decades ago: the formalin-inactivated poliovirus vaccine (IPV) developed by Jonas Salk and the live attenuated oral poliovirus vaccine (OPV) developed by Albert Sabin. Both vaccines were able to elicit effective humoral immune responses that protected from poliomyelitis, but only OPV induced strong mucosal immunity and was able to prime cellular immune responses [39]. As a result, all poliovirus vectors have been derived from the highly characterized and attenuated Sabin vaccine strains. The poliovirus is an attractive live viral vector for several reasons. It produces long-lasting and herd

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immunity [39], it is very safe and very easy to experimentally manipulate [40], it has a proven safety and efficacy record in over one billion vaccinees [39], and it produces potent mucosal immune responses [41]. Given the favorable aspects and characteristics of the Sabin poliovirus vaccine, it is easy to see why it was envisaged that recombinant poliovirus expressing foreign antigens would be able to provide a convenient and safe vaccine vector system to induce protective immunity against other pathogens. Expression of poliovirus gene products occurs via a single long polyprotein that is post-translationally cleaved into the final products (Figure 13.5). Recombinant polioviruses have already been constructed that include a number of antigenic components from various infectious agents [42]. Early poliovirus vectors were essentially constructed as replicons, in which the virion coat protein genes VP2 and VP3 were replaced with a heterologous gene, by creating in-frame fusions with remaining portions of the capsid precursor protein P1. To release the expressed antigen from the large poliovirus polyprotein, the insert was flanked with engineered viral protease cleavage sites [43]. The actual technique by which replicon RNA is packaged into virus-like particles involves transfection of in vitro transcribed RNA into cells infected with helper poliovirus or with vaccinia virus recombinants expressing the intact poliovirus P1 region [44]. In an alternative approach, some poliovirus vaccine strains have been modified to express additional sequences as replication-competent vectors using the fusion protein approach coupled with the previously described

Proteolytic cleavage sites

Ag

Insertion

Substitution Cleavage site 5’ VPG

VP0

VP3 P1

VP1

2A

2B

2C

P2

Fig. 13.5 Construction of poliovirus-vectored vaccines. Replication-defective poliovirus replicons are constructed by replacing in-frame the virion coat protein gene sequences VP2 (portion of VP0) and VP3 with the antigen-encoding heterologous sequence flanked by protease cleavage sites. Replication-competent attenuated poliovirus vectors are constructed by insertion of the heterologous genes flanked by one or more protease cleavage sites at either the polyprotein amino terminus or the P1–P2 junction. Adapted from [38].

3A

3C P3

3D

An3’

13.9 Rhabdovirus Vectors

flanking protease cleavage sites. The major advantage of this alternative is that no helper strategy is required for production, because the generated vectors are replication-competent. Insertion of the heterologous gene(s) of interest at either the polyprotein amino terminus or the P1–P2 junction results in viable recombinant virus. Various studies have been undertaken using both replication-competent and replicon-based poliovirus vectors in mouse and primate models, with both vector platforms demonstrating immunogenicity. The capacity of poliovirus vectors to generate a strong mucosal immune response may be of particular importance in the fight against HIV-1, given that more than 90 % of the HIV type 1 (HIV-1) infections worldwide have occurred via sexual transmission [45]. Any strategy developed with the hope of controlling the current AIDS pandemic will have to include a vaccine that prevents the sexual transmission of HIV-1. Although poliovirus has many notable attributes that allow for its use as a vaccine vector, we must stress that its widespread use will probably not fully bloom until pertinent issues such as limitations in size and stability of heterologous gene inserts and preexisting vector immunity in the general population are addressed.

13.9 Rhabdovirus Vectors

Rhabdoviruses are negative-stranded RNA viruses with a genome of about 12 kb. Their genome encodes five monocistronic RNAs: the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), the single transmembrane protein (G), and the viral polymerase (L). Transcription of these gene products occurs in a sequential manner as the polymerase moves across the genome in a linear manner, encountering distinct intergenic transcription stop and start signals. Several members of the rhabdoviridae family have been singled out as potential vaccine delivery vectors: the rabies virus (RV) and vesicular stomatitis virus (VSV). In both wild-type versions of these viruses, pathogenicity has been primarily linked with the G protein. With RV, studies showed that when the arginine residue at position 333 (R333) in the G protein is mutated to glutamic acid (E333) or aspartic acid (D333), complete abolishment of pathogenesis is observed [48]. For VSV, a recombinant VSV expressing influenza A virus hemagglutinin (HA) that contained a truncation of the VSV G-protein cytoplasmic domain was completely attenuated in a small-animal model [49]. The implications of these and other findings are that generation of suitable rhabdovirus expression vectors for use in clinical studies will need the G protein to be inactivated. Development of rhabdovirus expression vectors has essentially utilized duplication of the transcription start–stop signals flanking either side of the heterologous gene and positioning this cassette into the intergenic region following the G gene [46, 47] (Figure 13.6). The initial infectious viral stock is subsequently used for high titer amplification of the recombinant virus preparation. Recombinant rhabdoviruses offer genetic stability of the heterologous gene insert during virus passage and induce potent immune responses while expressing different antigens in murine models [49].

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13 Engineering Virus Vectors for Subunit Vaccines stop-start

3’

N

P

M

G

Ag

stop-start

L

5’

Fig. 13.6 Generation of rhabdovirus-vectored vaccines. Construction of vectors is accomplished by insertion of the immunogen gene flanked by transcriptional start–stop signals on both ends into the intergenic region between the envelope glycoprotein (G) and polymerase (L) genes. (N, P, M, G, and L are rhabdovirus proteins). Adapted from [38].

13.10 Heterologous Prime–Boost Vaccination Strategies

First, induction of immunological memory involves an intricate network of interactions among a number of host cell types. Data are emerging that suggest that viruses with different biological properties stimulate different memory T cells or drive T cells into various stages of differentiation. Therefore, subunit vaccines vectored by different viruses will also differ in the T cell memory they stimulate. Second, as rule of thumb, the more attenuated the vaccine vector is, the less damage it causes and the less immunogenic it is likely to be. Therefore many vaccines, especially if they are replication-incompetent, require boosts for efficient induction of immune responses. However, multiple applications of the same vaccine render the boosts less efficient, due to the immune responses generated to the vector proteins themselves. Both the qualitative differences in induced T cell responses and the need to overcome antivector immunity may contribute to the increased immunogenicity of heterologous prime–boost regimens, which deliver the same passenger immunogen gene using different vectors. This combined approach is highly immunogenic for both humoral and cell-mediated immune responses.

13.11 Cell Lines Acceptable for Growing Human Recombinant Subunit Vaccines

All methodologies used to generate and grow recombinant viruses require the use of living cells. In view of the possibility of harmful elements that may be present in these cells and contaminate the vaccine preparations, it is of paramount importance to test and evaluate each cell line that is to be used to produce clinical-grade vaccines. Stringent rules have been laid down by regulatory authorities such as the Food and Drug Administration (FDA) in the United States [56], which can be applied to most if not all situations. The current approach to working with cell lines for vaccine production focuses on:

13.11 Cell Lines Acceptable for Growing Human Recombinant Subunit Vaccines

. Production, identification, and characterization of the cell substrate. . Validation of the manufacturing process for removal and/or inactivation of adventitious agents. . Testing the bulk and final product to assure safety. It is mandatory that the testing be performed in compliance with Good Laboratory Practice (GLP). The selection of tests depends on many variables, such as the nature of the cell line, manufacturing procedure, and the final product and its use. When a cell line has been identified as potentially suitable for vaccine production, its characterization requires a history and general characteristics of the cell line, use of the cell bank system, and quality control testing. A brief summary of each of these points is given below. In addition, there is often a need for validation studies regarding the inactivation and removal of adventitious agents by the manufacturing process. 13.11.1 History and General Characteristics of the Cell Line

The history of any cell line used for the production of recombinant virus vaccines should include: age, sex, and species of the donor for cell lines; the donor’s medical history; the culture history of the cell line, including methods used for isolation of the tissues from which the line was derived, passage history, media used, and history of passage in animals; and the results of previous identity tests and tests for all available adventitious agents. In looking at the general characteristics of the cell line, the growth pattern and morphological appearance of the cells should be determined and should be stable from the master cell bank to the end-of-production cells. If any specific markers may be useful in characterizing the cell line, these too are required to be characterized for stability. 13.11.2 The Cell Bank System

A cell bank system is primarily generated to assure that an adequate supply of equivalent cells exists for use over the entire life span of the product. In addition to providing a constant supply of starting material, the advantages of a cell bank system include a detailed characterization of the cell line and a decrease in the likelihood and increase in the detection of both cross- and adventitious-agent contamination of the cell line. 13.11.3 Quality Control Testing

Quality control of cell substrates used for production is an important part of the product quality control and covers areas such as cell culture media, management of cell cultures, and specific testing. Accurate records should be always kept of the composition and source of the cell culture medium, unprocessed and processed cell culture

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fluids, and tests for the presence of bacteria, fungi, and mycoplasma. In addition, there may also be a need to conduct other tests to screen for viruses as well as for tumorigenicity of cell lines. Finally, in the generation of cell lines acceptable for growing human recombinant viral vaccines, it may be necessary to validate the elimination of adventitious viruses. Therefore, when choosing a vector for a candidate subunit vaccine, the readiness and ease with which these recombinant viruses can be produced on a large scale and to GLP standards should be taken into consideration.

13.12 Conclusion

The list of recombinant viral vectors described in this chapter is by no means exhaustive, but has included most of the approaches that are currently in clinical trials or under advanced preclinical studies or that have the potential to be breakthroughs in the coming years as candidate vaccine vectors. It is important to stress, however, that many new viral (and nonviral) vectors that may not even have been discovered yet are likely to appear. No single vector system is optimal for all potential vaccine applications. It is more likely that alternative vector systems may be required for different diseases or that a combination of different vectors could stimulate the best suited antigen-specific immune responses for a given application. Since the viruses described in this chapter are amenable to many types of manipulations, there is no doubt that future advances in immunology and virology will increase the utility of these viral vectors as vaccine delivery systems.

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velopment of herpes simplex virus replication-defective multigene vectors for combination gene therapy applications. Gene Ther. 5 1517–1530. Leib D.A., Olivo P.D. (1993) Gene delivery to neurons: is herpes simplex virus the right toool for the job? Bioessays 15, 547–554. Laquerre S. (1999) Gene-transfer tool: herpes simplex virus vectors. In The Development of Human Gene Therapy, 173–208. Ed T Friedman. New York: Cold Spring Harbor Laboratory Press. Saeki Y., Ichikawa T., Saeki A., Chiocca E.A.,Tobler K., Ackermann M., Breakefield X.O., Fraefel C. (1998) Herpes simplex virus type I DNA amplified as bacterial artificial chromosome in E. coli: rescue of the replicationcompetent virus progeny and packaging of amplicon vectors. Hum. Gene Ther. 9, 2787–2794. Naldini L., Blomer U., Gallay P., Ory D., Mulligan R., Gage F.H.,Verma I.M.,Trono D. (1996) In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector. Science 272, 263–267. Xiong C., Levis R., Shen P., Schlesinger S., Rice C.M., Huang H.V. (1989) Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. Science 243, 1188–1191. Rice C.M., Levis R., Strauss J.H., Huang H.V. (1987) Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J. Virol. 61, 3809–3819. Liljestrom P., Lusa S., Huylebroeck D., Garoff H. (1991) In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6000-molecular-weight membrane protein modulates virus release. J. Virol. 65, 4107– 4113. Davis N.L.,Willis L.V., Smith J.F., Johnston R.E. (1989) In vitro synthesis of infectious Venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant. Virology 171, 189–204.

35. Stone D., David A., Bolognani F., Lowenstein P.R., Castro M.G. (2000) Viral vectors for gene delivery and gene therapy within the endocrine system. J. Endocrinol. 164, 103–118. 36. Rayner O.J., Dryga S.A., Kamrud K. (2002) Alphavirus vectors and vaccination. Rev. Med. Virol. 12, 279–296. 37. Leitner W.W., Ying H., Driver D.A., Dubensky T.W., Restifo N.P. (2000) Enhancement of tumor-specific immune responses with plasmid DNA replicon vectors. Cancer Res. 60, 51–55. 38. Polo J.M., Dubensky T.W. (2002) Virusbased vectors for human vaccine applications. Drug Discovery Today 7, 719– 727. 39. Sutter R.W., Cochi S.L., Orenstein W.A. (1999) Live attenuated poliovirus vaccines in S.Ploktin and W.Orenstein (ed.) Vaccines, 3rd ed. W.B. Saunders, Philadelphia, Pa., 364–408. 40. Melnick J.L. (1996) Enteroviruses: polioviruses, coxsackieviruses, echoviruses, and new enteroviruses in Fields Virology, 3rd ed., eds Fields B.N., Knipe D.M., Howley P.M., Chanock R.M., Monath T.P., Melnick J.L., Roizman B., Straus S.E. vol. I Lippincott-Raven Publishers, Philadelphia, Pa. 655–712. 41. Morrow C.D., Novak M.J., Ansardi D.C., Porter D.C., Moldoveanu Z. (2000) Recombinant viruses as vectors for mucosal immunity. Curr. Top. Microbiol. Immunol. 236, 255–273. 42. Andino R., Silvera D., Suggett S.D., Achacoso P.L., Miller C.J., Baltimore D., Feinberg M.B. (1994) Engineering poliovirus as a vaccine vector for the expression of diverse antigens. Science 265, 1448–1451. 43. Choi W.S., Pal-Ghosh R., Morrow C.D. (1991) Expression of human immunodeficiency virus type 1 gag and pol proteins from chimeric HIV-1-poliovirus minireplicons. J. Virol. 65, 2875– 2883. 44. Porter D.C., Ansardi D.C., Choi W.S., Morrow C.D. (1993) Encapsidation of genetically engineered poliovirus minireplicons which express human immunodeficiency virus type 1 gag and pol proteins upon infection. J. Virol. 67, 3712–3719.

References 45. UNAids and World Health Organization. (1998) Report on the global HIV/ AIDS epidemic. World Health Organization, Geneva, Switzerland.. 46. Mebatsion T., Schnell M.J., Cox J.H., Finke S., Conzelmann K.K. (1996) Highly stable expression of a foreign gene from rabies virus vectors. Proc. Natl. Acad. Sci. USA 93, 7310–7314. 47. Schnell M.J., Buonocore L.,Whitt M.A., Rose J.K. (1996) The minimal conserved transcription stop-strat signal promotes stable expression for a foreign gene in vesicular stomatitis virus. J. Virol. 70, 2318–2323. 48. Coulon P.,Ternaux J.P., Flamand A., Tuffereau C. (1998) An avirulent mutant of rabies virus is unable to infect motor neurons in vivo and in vitro. J. Virol. 72, 273–278. 49. Roberts A., Kretzschmar E., Perkins A.S., Forman J., Price R., Buonocore L., Kawaoka Y., Rose J.K. (1998) Vaccination with vesicular stomatitis virus expressing an influenza virus hemagglutnin provides complete protection from influenza challenege. J. Virol. 72, 4704– 4711. 50. Lieber A., He C.Y., Meuse L., Schowalter D., Kirillova I.,Winther B. and Kay M.A. (1997) The role of Kupffer cell activation and viral gene expression in early liver toxicity after the infusion of recombinant adenovirus vectors. J. Virol. 71, 8798–8807. 51. Franchini M., Abril C., Schwerdel C., Ruedl C., Ackermann M., Suter M. (2000) Protective T-cell-based immu-

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nity induced in neonatal mice by a single replicative cycle of Herpes simplex virus. J. Virol. 75 (1), 83–89. Rees RC, McArdle S, Mian S, Li G, Ahmad M, Parkinson R, Ali SA. (2002) Disabled infectious single cycleherpes simplex virus (DISC-HSV) as a vector for immunogene therapy of cancer. Curr. Opin. Mol. Ther. 4(1), 49–53. Polo J.M., Belli B.A., Driver D.A., Frolov I., Sherrill S., Hariharan M.J.,Townsend K., Perri S., Mento S.J., Jolly D.J., Chang S.M., Schlesinger S., Dubensky T.W. (1999) Stable alphavirus packaging cell lines for Sinbis virus- and Semliki Forest virus-derived vectors. Proc. Natl. Acad. Sci. USA 96, 4598–4603. MacDonald G.H.,Johnston R.E. (2000) Role of dendritic cells targeting in Venezuelan equine encephalitis virus pathogenesis. J. Virol. 74, 914–922. Gardner J.P., Frolov I., Perri S., Ji Y., MacKichan M.L., zur Megede J., Chen M., Belli B.A., Driver D.A., Sherrill S., Greer C. E., Otten G.R., Barnett S.W., Liu M.A., Dubensky T.W., Polo J.M. (2000) Infection of human dendritic cells by a Sindbis virus replicon vector is determined by a single amino acid substitution in the E2 glycoprotein. J. Virol. 74, 11849–11857. Center For Biologics Evaluation and Research, Food and Drug Administration (1993) Points to consider in the characterization of cell lines used to produce biologicals. http://www.fda.gov/cber/ gdlns/ptccell.pdf.

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Summary

DNA vaccines can induce protective cellular and humoral immune responses and have therefore been used during the past decade to develop vaccines against a variety of pathogens. Because current antiviral vaccines predominantly generate humoral immunity, DNA immunization may be especially useful to provide long-term protection against viral diseases that also require cellular immunity. A significant portion of published articles in the field of DNA vaccines deal with viral diseases, reflecting the need for better and alternative vaccination strategies against viruses. The success of DNA immunization depends on a variety of parameters, e. g., type of antigen, method of application, usage of adjuvants. Therefore, different strategies have been explored for modulating the induced immune response with respect to the requirements necessary to protect against a specific pathogen (e. g., induction of mucosal or cell-mediated immunity). This chapter provides an update on various aspects of antiviral DNA vaccine research for the period from the beginning of 2000 until the beginning of 2003. Previous reviews cover the literature before 2000 [1–4].

14.1 Effect of Antiviral DNA Vaccines in Mice

Genetic vaccines are under investigation against a number of human viral diseases. During the past two years research has focused on improving their efficacy, using viral infections in animal model systems. Table 14.1 summarizes the literature on DNA vaccines against viral infections for the period 2000 through the beginning of 2003 and is an update of previous overviews [2, 4]. During this period DNA vaccines have been described against many viral infections, such as bunyaviruses, including hantavirus and La Crosse virus; hepatitis D virus; flaviviruses, including Dengue virus type 1, Japanese encephalitis virus, Murray Valley encephalitis virus, central European encephalitis virus, and Russian spring/summer encephalitis virus; herpes viruses, including human cytomegalovirus and varicella zoster virus; and the toga-

290

14 Update on antiviral DNA vaccine research (2000–2003)

virus rubella virus. The efficacy of DNA vaccines was monitored by humoral or cellular immune responses or resistance to virus challenge. Table 14.1 summarizes the viruses, the specific gene(s) used for immunization, the model systems used, the responses, modes of application, and references.

Tab. 14.1 DNA vaccination to viruses (01/2000–03/2003). Virus

Antigen

Application

Host

Effect

Ref.

LCMV

NP

i.m.

mice

45

LCMV

NP

i.m.

mice

LCMV

NP

i.m.

mice

LCMV

NP

i.p.

mice

LCMV

NP

i.m.

mice

LCMV

NP

intragastrically mice

LCMV

GP33

i.m., i.d., peripheral lymph nodes

mice

DNA vaccination at birth resulted in rapid induction of antigen-specific CD8(+) T cells. Vaccination with plasmids expressing NP as an intracellular or secretory protein caused liver injury. Multiepitope DNA vaccines should be cocktail of plasmids, each with its own epitope, to allow maximal epitope dispersal among APCs. VV-based vaccines optimized prime–boost regimens. Described system for targeting proteins and minigenes to lysosomes. Mice vaccinated with vector expressing LCMV NP were protected against virus challenge. Immunization with DNA by direct injection, into a peripheral lymphnode enhances immunogenicity.

LCMV

NP

i.m.

mice

Protection from lethal and sublethal LCMV infections conferred well before the peak of CD8(+) T cell response.

54

Hantavirus envelope gene gun glycoproteins G1 and G2, N gene

55

Hantavirus NP

rhesus First immogenicity data for Hantavimonkeys, rus DNA vaccines in nonhuman prihamsters mates; Hantavirus M gene-based DNA vaccines could protect humans against the most severe forms of HFRS. i.m. mice Vaccination with DNA plasmids encoding NP combined with secretion signal. into leaf discs/ tobacco, Transgenic plants expressing Hantatuber discs potato viral proteins could be used for alterplants native vaccination strategies.

56

i.m.

58

Arena viridae

49

19

50 51 52

53

Bunya viridae

Hantavirus NP

Hantavirus NP

mice

DNA vaccination could be used for identification of highly immunogenic epitopes.

57

14.1 Effect of Antiviral DNA Vaccines in Mice Tab. 14.1 (continued) Virus

Antigen

Application

Host

Effect

Ref.

Hepatitis delta virus

HDAg

gene gun

woodchucks

Modification of the course of DNA immunization.

59

Hepatitis delta virus

HDAg

gene gun

woodchucks

DNA immunization with vectors expressing p24 could not induce protective immune response.

60

Hepatitis delta virus

large hepatitis D antigen

i.m.

mice

Large hepatitis D antigen DNA induced significant titers of anti-HDV antibodies.

61

HCV

E1, E2, NS3, i.m. NS4, NS5A and -B

mice

Revealed clustered organization of HCV immunogenic HLA.A2.1 epitopes and strategies to modulate their dominance.

62

HCV

E2

i.m.

chimpan- Titration in chimpanzees of diffezees rent HCV variants to provide wellcharacterized challenge pools.

63

HCV

E2 (cell-surface and intracellular)

i.m.

chimpan- DNA vaccine encoding cell surfacezees expressed E2 did not elicit sterilizing immunity in chimpanzees.

26

HCV

capsid, E1, i.m. E2, NS2, NS3

mice

Combination of naked DNA with nonreplicating canary pox booster encoding HCV.

35

HCV

envelope ge- i.m. nes E1, E2

buffalo rats

Addition of GM-CSF gene to envelope genes E1/E2 in a bicistronic plasmid augmented antibody and lymphoproliferative responses.

64

HCV

envelope E2 gene gun, protein, trun- i.m. cated form of E2 targeted to cell surface

rhesus macaques, mice

Targeting of E2 protein to cell surface increased its ability to induce humoral immune responses by DNA vaccination.

25

HCV

E2

i.m.

mice

DNA prime–protein boost induced stronger immune response than DNA or protein alone, evidenced by enhanced IgG2 a and CTL responses.

34

Dengue nonstructural i.m. virus type 1 proteins

mice

gp160 expressed from dengue virus replicons was considerably more toxic than GFP or gp120.

65

Dengue prememi.d., i.m. virus type 1 brane, envelope

rhesus Immunization protected 4 of 8 monkeys monkeys completely and 4 partially from developing viraemia.

66

Dengue prememi.d., i.m. virus type 1 brane/membrane, envelope

Aotus Immunization conferred complete nancymae or partial protection from viral chalmonkeys lenge.

67

Deltavirus

Flaviviridae

291

292

14 Update on antiviral DNA vaccine research (2000–2003) Tab. 14.1 (continued) Virus

Antigen

Application

Host

Effect

Ref.

Dengue prememsubcut., i.c. virus type 2 brane, envelope

mice

Induction of CD8 T-cell response specific for DEN-2 virus prM and E proteins protected against lethal dengue encephalitis.

28

Dengue prememvirus type 2 brane, envelope

i.m.

mice

Induction of neutralizing antibodies and memory B cells.

68

Japanese envelope encephalitis virus

oral

humans

JE vaccines including naked DNA, oral vaccination, and recombinant subunit vaccines.

69

Japanese envelope encephalitis virus

i.m., gene gun

mice

Adoptive transfer/antibody production by pE DNA vaccine.

70

preM, E, Japanese encephalitis NS1, NS2a virus

subcut.

humans

Double-blind clinical trial with attenuated recombinant poxviruses with JEV.

71

Japanese prememencephalitis brane, envelope virus

i.m.

mice

Single injection induced neutralizing antibody titers and prevented Japanese encephalitis.

72

Tick-borne envelope encephalitis (prime– virus boost)

i.m.

mice

Recombinant alpha virus RNA molecule elicited protection against 3 model virus diseases.

73

mice

Induction of antibody production and protective immunity.

74

Chimeric virus against WNV.

75

Tick-borne E-NS1, Eencephalitis NS3 virus West Nile virus

prM, E

Hepadna viridae HBV

HBsAg

i.m.

mice

DNA vaccines induced antigen-specific immune responses.

76

HBV

gB

i.m., i.d.

rhesus macaques, mice

5 juvenile macaques were immunized, developing antibodies to B virus, gB neutralizing activity.

77

HBV

surface and i.m. core antigens

mice

Modulation of antigen expression by Kazak's translation-initiation sequence.

78

HBV

HBsAg

i.v.

rhesus SHIV- and HBV-specific cytotoxicity macaques led to expansion of memory cells.

79

HBV

HBsAg

i.m.

chimpan- Immunization of chimpanzee HBV zees carrier using DNA followed by recombinant canary pox booster.

36

HBV

HBsAg

subcut.

mice

Dominant epitopes in human human D transgenic mouse model.

24

HBV

HBsAg

electro-poration

mice

Electroporation resulted in more rapid onset and more antibody formation.

80

14.1 Effect of Antiviral DNA Vaccines in Mice Tab. 14.1 (continued) Virus

Antigen

Application

Host

Effect

Ref.

HBV

HBsAg

i.d., i.m.

mice

Human desmin promoter/enhancer and CMV promoter induced comparable antibody and CTL responses.

81

Herpes viridae Human VP2 cytomegalovirus

chickens 2 herpes virus, complete protective immunity to chickens.

82

Human gB, pp65 cytomegalovirus

i.m.

mice

Mice inoculated at 3 sites.

83

Human pCMV/V-18 cytomegalovirus

i.v.

rats

Allograft tolerance in adult rat by pregraft donor-specific blood transfusion.

84

HSV-1

gB, gD

i.m., i.p.

mice

gD or gB plasmid alone or mixed with copolymers; immunized mice were protected.

85

HSV-1

gB

i.m., i.n., i.g.

mice

CCR7 ligands DNA administration; mucosal cotransfer provided significant increases of distal mucosal IgA responses; CCR7 ligands were useful adjuvants for genetic vaccination.

86

HSV-1

gB

i.m.

mice

Limited HSV-1 infection of sensitive ganglia.

87

HSV-1

gD

i.m.

mice

Immunity to HSV by gD-DNA immunization not strictly correlated with antibody levels.

88

HSV-1

gB

i.m.

rabbits

Local protein and systemic DNA administration elicited neutralizing antibody response.

89

HSV-1

gB

i.n.

mice

Value of CXC and CC chemokines in mucosal DNA vaccination.

HSV-1

gM

subcut.

mice

HSV-1 amplicon vectors appeared to be effective for cytokine-enhanced vaccination therapy of glioma.

90

HSV-1

gD

oral

lambs

Single oral exposure in utero to plasmid DNA encoding truncated form of glycoprotein D of bovine herpesvirus-1 induced detectable immune responses in 80 %.

91

HSV-1

gD

i.v.

mice

Targeting of herpes simplex virus type-1 glycoprotein D to liver by complexing it with an asialoorosomucoid DNA carrier system induced antigen expression in liver cells 6 days after immunization and influx of T cells.

92

9

293

294

14 Update on antiviral DNA vaccine research (2000–2003) Tab. 14.1 (continued) Virus

Antigen

HSV-2

Application

Host

Effect

Ref.

glycoprotein i.m., oral D

mice

Salmonella harboring pIL10; oral administration led to oral immunization.

7

HSV-2

gD (prime– boost)

gene gun

mice

Use of MVA and plasmid DNA vector dramatically enhanced immune response.

93

HSV-2

gD

oral

mice

Expression of plasmid was due to transcription of protein by eukaryotic nuclear process.

94

HSV-2

gD

i.m.

mice, guinea pigs

Guinea pigs were not protected by immunization with DNA encoding cytosolic gD2, mice were.

95

HSV-2

gD

i.m., i.d.

mice

Coinjection (GM-CSV) increased both humoral and cell-mediated responses.

HSV-2

gD

i.m.

mice

Despite weak expression in vitro restricted to myofibers, a myogenic DNA vaccine under control of a promoter derived from murine muscle creatine kinase gene induced virus-specific immunity that protectted HSV-2-infected mice from death.

96

HSV-2

gD

Coimmunization with lymphocyte function-associated antigen-3 (LFA-3) enhanced IgG-production, TH-proliferative responses, and survival rate.

8

6

Orthomyxo viridae Influenza virus

matrix protein

i.n., i.m.

mice

CD8 T cell populations induced by DNA/MVA prime–boost vaccination did not show enhanced capability to mediate protection in IFN-gammaindependent influenza challenge model.

42

Influenza virus

hemaggluti- i.m., i.n. nin (HA), HN

mice

Liposomal ISS-ODN coadministered with soluble antigen or liposomal antigen were up to 30 times more effective than formulations containing unencapsulated ISS-ODN in protecting against virus challenge.

97

Influenza virus

NP, matrix proteins

mice

Vaccination reduced replication of nonlethal H5N1 strain, protection against lethal challenge; DNA vaccination alone protected poorly against this highly virulent strain.

98

14.1 Effect of Antiviral DNA Vaccines in Mice Tab. 14.1 (continued) Virus

Antigen

Application

Host

Influenza virus

NP

i.m.

pigs

Influenza virus

HA

Influenza virus

HA

Influenza virus

M1, M2

Influenza virus

HA

Influenza virus

HA

Influenza virus

HA, NP

Influenza virus

HA

Influenza virus

HA

Influenza virus

HA

Effect

Fusion proteins M2eHBc and M2eNP induced antibody response against M2 e; M2eNP vaccine induced influenza virus-specific lymphoproliferation response. gene gun mice Attenuated murine caspase 2 and a chimera of murine caspase 2 prodomain and human caspase 3 strongly enhanced humoral and cell-mediated immune response to HA when coadministered with an immunogen DNA. gene gun pigs Coadministration of pIL-6 DNA did not significantly enhance immune responses to HA DNA vaccination or protect from challenge exposure. i.m., i.n. mice 70 %–80 % protection was observed in mice immunized with pME18SM plasmid followed by lethal infection with influenza viruses of the A/ WSN/33 and A/PR/8/34 strains. i.p. mice Inhibition period of anti-HA antibody in offspring born to dams immunized with DNA was shorter than that of offspring born to dams immunized with virus. gene gun pigs Administration of conventional inactivated influenza virus vaccine 4 weeks after a priming dose of HA DNA led to higher levels of virus-specific serum antibodies and protection from challenge virus infection. intratracheal baboons Neonatal DNA vaccination triggered (i.t.) virus-specific and neutralizing antibodies of titers and persistence, depending on vaccine dose. multiple injec- chickens Beneficial response was observed in tion sites birds boostered at 3 wk. of age, in birds given larger amounts of DNA, and with multiple injection sites. multiple injec- mice Neonatal mice coimmunized with tion sites HA-DNA and either IL-12 or IFNgamma-expressing DNA developed IgG2a-biased immune responses, regardless of inoculation method. i.m. mice Significant enhancement of cellular and antibody responses after electrotransfer for 1- and 10-mg DNA doses, respectively, but no effect at lower dose.

Ref. 20

99

10

100

101

102

104

105

11

106

295

296

14 Update on antiviral DNA vaccine research (2000–2003) Tab. 14.1 (continued) Virus

Antigen

Application

Influenza virus

HA, NA and i.m., NB, NP gene gun

Influenza virus

HA

Influenza virus

HA, NP

Influenza virus

HA

Influenza virus

HA

Influenza virus

HA

Influenza virus

neuraminidase from different subtype A viruses

Host

Effect

mice

HA and NA DNAs conferred complete protection against lethal challenge; both were used as vaccine components to provide effective protection against influenza B virus infection. i.n, i.t. chickens Birds challenged with dose of lethal parent virus were protected to different extents, depending on their age. i.v. pregnant Uptake of plasmid DNA by fetuses, mice protection against challenge by homologous influenza virus. i.m. Targeting HA-based DNA vaccine to APCs by fusion to CTLA-4 increased antibody response and reduced viral titers 100 fold after nonlethal viral challenge. electromice Electroporation with 10 mg HAporation, i.m., DNA, administered twice, induced mucosal strong antibody production, weak CTL response, and protection against lethal virus challenge; intranasal DNA immunization by electroporation provided reduced nasal virus titers. gene gun, mice APCs were detectable first in draini.m. ing lymph nodes and then in spleen of gene gun but not i.m. DNA immunized mice. gene gun, mice H3N2 virus NA-DNA conferred electro-poracross-protection against lethal chaltion muscle lenge with antigenic variants within same subtype did not protect against infection by different subtype virus (H1N1).

Ref. 107

108

48

27

109

110

111

Paramyxo viridae Measles virus

nucleocapsid oral (N) protein

Measles virus

H, F protein i.d., gene gun

Measles virus

H, nucleocapsid

i.d., i.m.

mice

Replication-deficient adenovirus construct expressing virus N protein elicited antibody and cytotoxic T cell responses. rhesus Protection correlated with levels of macaques neutralizing antibody and not with cytotoxic T lymphocyte activity. cotton DNA vaccination with both hemaggrats lutinin and fusion protein but not nucleocapsid induced T cell responses and antibody production and protected against experimental virus infection.

112

44

113

14.1 Effect of Antiviral DNA Vaccines in Mice Tab. 14.1 (continued) Virus

Antigen

Application

Host

Effect

Ref.

i.m.

mice

Challenge infection showed that RNA immunization elicited significant levels of protection against influenza A, RSV, tick-borne encephalitis virus.

73

Respiratory F- ,G-protein gene gun syncytial virus

mice

Complete protection against RSV infection was induced with DNA encoding fusion protein of RSV.

21

Respiratory G-protein syncytial virus

i.m.

mice, cot- DNA vaccination elicited balanced ton rats systemic and pulmonary Th1/Th2 cytokine responses, induced neutralizing antibodies, conferred protection against RSV infection, and did not provoke atypical inflammatory reaction.

114

Respiratory F-protein syncytial virus

Retroviridae HIV-1

env

oral (PLG)

mice

Intragastric administration of DNA encapsulated in PLG microparticles resulted in antigen expression in intestinal epithelium, type 1- and CTL responses in spleen and mucosal tissue, increased levels of IgA, and higher resistance to mucosal virus.

115

HIV-1

gp160

i.m., i.n.

mice

CMV promoter activation by 8-Br-cAMP increased immune response, as evidenced by elevations in IgG antibody titers, CTL activity, and DTH response.

116

HIV-1

env, gag

i.m.

rhesus Augmentation of antibody responmonkeys ses and tetramer-positive CD8+ T cells upon IL-2/Ig plasmid coadministration.

117

HIV-1

env, gag

i.m.

macaques CMV promoter is superior to LTR promoter in inducing humoral and cellular immune response.

118

HIV-1

env, rev

i.m.

humans

In a phase I study 300 mg of HIV env/rev DNA at 0, 4, 8, and 24 weeks induced antigen-specific lymphoproliferative responses and antigen-specific production of interferon-g and b-chemokine.

119

HIV-1

gag, env

electroporation

mice, guinea pigs, rabbits

Electroporation resulted in higher gag antibody titers and increased CD8+ T cell response.

80

HIV

multiepitope i.m.

mice

Semliki Forest virus was used as vector carrying HIV clade A for prime–boost regime.

120

297

298

14 Update on antiviral DNA vaccine research (2000–2003) Tab. 14.1 (continued) Virus

Antigen

Application

Host

Effect

Ref.

HIV-1

env

i.m.

mice, guinea pigs, rhesus monkeys

Immunogenicities of synthetic HIV1 env DNA prime and recombinant adenovirus boost vaccinations were evaluated.

41

HIV

gag

i.n.

mice

Similar effect to i.m. injection, strong boosting effect was observed in mice primed with DNA.

121

HIV

gag-pol, env-rev

i.v.

rhesus Plasmid vaccines supplemented by macaques IL-2 Ig cytokine gene adjuvants or boosted by recombinant MVA vectors expressing relevant SIV and HIV antigens prevented CD4(+) T-cell loss and lowered viral loads after pathogenic challenge.

13

HIV

structural, regulatory proteins

gene gun, i.m.

rhesus Chronically infected rhesus macamacaques ques were benefited by boosting in combination with antiretroviral therapy.

122

HIV-1

gp160

i.m, i.d, gene gun

mice

HIV-specific immune responses and protection were strongest in animals immunized with combination of subtypes A-, B-, and C-specific gp160 genes relative to subtype B only.

123

HIV-1

tat, rev, SFV-tat, SFV-rev

rectal

rhesus 3 different prime–boost regimes macaques were compared, no clear differences were shown.

124

HIV-1

SIVmac251

intravaginal

rhesus Intravaginal challenge of macaques; macaques monitored SIV- specific T cell responses in peripheral blood.

125

HIV

gag-pol, nef

intrarectal

rhesus Animals covaccinated multiple macaques times with Nef vaccine and pVpr/ Vpx plasmid suffered rapid and profound loss of CD4(+) T cells.

126

HIV

SIVmac239

intrarectal, i.d. rhesus Repertoire of immune responses macaques detected in peripheral blood after immunization and after infection substantially differed.

127

HIV-1

tat, gag, rev

intrarectal

rhesus Partial protection from virulent mumacaques cosal SHIV challenge detected only in prime–boosted macaques.

31

HIV

gag

i.m.

rhesus Vaccinated monkeys responded to macaques 25 epitopes and on average recognized a minimum of 2.7 epitopes.

15

HIV-1

gp-160

gene gun, i.d.

rhesus After prime–boost vaccination macaques SHIV-infected macaques were rechallenged with pathogenic SHIV89.6P; 4 of 6 animals showed normal CD4+ T-cell counts.

46

14.1 Effect of Antiviral DNA Vaccines in Mice Tab. 14.1 (continued) Virus

Antigen

Application

Host

Effect

HIV

SIVmac239

rectal

rhesus DNA prime–modified vaccinia virus macaques Ankara boost regimen immunized rhesus macaques against nearly all SIV proteins.

128

HIV

env

i.v.

rhesus Plasmid DNA or recombinant modimacaques fied vaccina virus Ankara vaccinated rhesus macaques, after pathogenic simian–human IV challenge, generated secondary CTL responses to subdominant epitopes.

18

HIV

gag, env, pol i.d.

rhesus Gag-Pol DNA priming and Gag-Pol macaques rMVA boosting to evaluate contribution of anti-Env immune responses to viral control.

39

HIV-1

tat

i.m.

mice

Novel class of cationic block (PEG/ poly(dimethylamino) ethylmethacrylate) copolymer for DNA delivery was safe and possessed required biological characteristics.

129

HIV

tat

i.d.

rhesus Role of Tat-specific CTL in controlmacaques ling pathogenic SIV mac239 replication after Ankara boost.

130

HIV

gag-pol-env

rectal

rhesus SIV-specific mucosal antibodies macaques were detected; 4 of 7 vaccinees were protected.

16

HIV-1

gag, env

i.p.

mice

Investigation of primary cellular immune responses elicited by recombinant vesicular stomatitis viruses.

131

HIV-1

gp-160

i.m.

humans

HIV-1 gp-160 plasmid DNA vaccine was safe and did not induce antiDNA autoimmune antibodies.

132

HIV-1

tat

i.d.

cynoTat vaccination induced specific Th1 molgus responses, including CTLs. monkeys

22

HIV-1

env, gag

i.m.

rhesus Mutation within immunodominant macaques Gag epitope after vaccination and viral challenge resulted in viral escape.

14

HIV-1

gag

rhesus Several vaccine vector delivery macaques systems were examined.

40

HIV-1

gag

nasal, i.m., rectal, vaginal

mice

133

HIV

gag

intranasal

macaques All macaques vaccinated with prime–boost regimen were protected from depletion and showed greatly reduced peak viral loads compared with controls.

Mice were protected against VV-Gag replication in ovaries after rectal or vaginal immunization.

Ref.

37

299

300

14 Update on antiviral DNA vaccine research (2000–2003) Tab. 14.1 (continued) Virus

Antigen

Application

Host

HIV-1

env

i.v.

macaques Vaccinated macaques had lower levels of peak viremia, rapidly cleared virus from periphery, and developed delayed seroconversion to SIV core antigen prime–boost.

38

HIV-1

env, gp120

i.n., intraperitoneally, i.m.

mice

Single dose of gp120 DNA vaccine vector afforded significant protection against vaccinia-env challenge.

134

HIV-1

nef, rev, tat

i.m.

humans

Efficacy of DNA plasmid combination in inducing cellular immune responses was analyzed; no clinical advantages or adverse effects were noted.

135

HIV-1

env, gag, gp120

i.m.

humans

T cell memory to HIV-1 antigens and anti HIV-1 CTL activity were assessed after administration of vaccina virus.

136

HIV-1

gag

i.p.

mice

Immunized mice developed cytotoxic T lymphocyte response against HIV-1 gag.

137

HIV-1

env

subcut.

mice

Combination of DNA vaccination, vector infection, and passive transfer of infected DCs induced strong immunity.

138

HIV

env

gene gun

mice

Fusion of Env and complement component (C3 d) in DNA vaccine enhanced titers of antibody to Env.

29

HIV-2

env, gag, pol i.v., rectal

cynoALVAC HIV-2 vaccine followed by molgus exposure to live HIV-2 induced cross monkeys protection against mucosal infection with SIVsm and seemed more efficient than immunization with live HIV-2 vaccine only.

139

HIV-1

gag, pol

i.m.

mice

23

HIV

tat

i.m.

cynoInoculation of pCV-tat prevented the molgus CD4(+) T cell decline; undetectable monkeys virus replication and negative virus isolation correlated with presence of anti-tat CTLs.

HIV

env/rev, gag/pol

i.m.

Effect

Gag-Pol fusion proteins stimulated cytotoxic T lymphocyte and antibody responses, Gag-Pol pseudoparticle did not elicit substantial Pol responses.

rhesus Coadministration of IL-2 and IFNmacaques gamma cDNA resulted in enhancement of antigen-specific T cell mediated immune responses.

Ref.

140

12

14.2 Effect of Antiviral DNA Vaccines in Larger Species Tab. 14.1 (continued) Virus

Antigen

Application

Host

Effect

Ref.

HIV-1

gag, env

i.m.

17

HIV-1

gp120

i.m.

HTLV-1

tax

intraperitoneally

rhesus Plasmid DNA vaccination led to macaques high-frequency CTL responses specific for both epitopes. mice B7–1 or B7–2 administered concurrently with plasmid DNA vaccine fully costimulated vaccine-elicited CTL responses. rats DNA vaccine with Tax effectively induced Tax-specific CTL activity; transfer of these immune T cells effectively inhibited in-vivo growth of HTLV-1-transformed tumor. mice

143

141

142

Rhabdoviridae Rabies virus glycoprotein i.n. G Rabies virus glycoprotein G Rabies virus CRV, DRV

Rabies virus glycoprotein G

Rabies virus glycoprotein G

Rabies virus glycoprotein G

Immunized mice developed antibodies and remained resistant to challenge with otherwise lethal dose. gene gun, i.m. monkeys Antibody titers of gene gun-vaccinated animals were higher than titers of i.m.-vaccinated animals. i.m. mice Mice immunized with CRV (novel combination RV) developed higher levels of RVNA than those immunized with DRV and were completely protected against peripheral and intracerebral RV challenge. gene gun, rhesus Post-exposure vaccination with i.d. macaques DNA or HDCV in combination with one-time treatment with HRIG protected 50 %–75% of monkeys. i.m., i.d., mice Post-exposure therapy, substituting gene gun a DNA vaccine for HDCV, did not compromise protection against rabies virus. gene gun, i.d., mice The adjuvant monophosphoryl lipid i.m. A increased primary, but decreased booster antibody response.

144

145

146

147

148

14.2 Effect of Antiviral DNA Vaccines in Larger Species

Several DNA vaccines have shown efficacy in small-animal models, especially mouse. In larger species, DNA vaccines were less effective. However, monkeys were completely protected from dengue virus type 1 and measles virus infection. Furthermore, immune responses were induced by HCV, central European encephalitis virus, Russian spring/ summer encephalitis virus, and HIV antigens. In particular, the efficacy of HIV DNA vaccines was studied and enhanced by coadministration of cytokines, costimulators,

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14 Update on antiviral DNA vaccine research (2000–2003)

and so-called DNA-prime-peptide boost strategies, which improved antibody and CD8+ T cell responses. Long-lasting antibody responses were generated in swine vaccinated with DNA coding for the premembrane and envelope proteins of Japanese encephalitis virus, and in dogs vaccinated with DNA coding for the rabies virus glycoprotein. Improvement of vector design and optimization of the quantitative and qualitative aspects of the immune response were approached by (1) modulating the mode of DNA application with respect to time, route of administration, and auxiliary means employed, (2) addition of genetic adjuvants, (3) modification of the plasmid design comprising the use of different promoters, CTL-epitope DNA vaccination, and attempts to target the expressed antigen to intracellular and extracellular compartments or the cell surface, (4) altering the DNA antigen carrier system, and (5) combining DNA with protein or other viral immunization strategies.

14.3 Genetic Adjuvants

Genetic adjuvants are vectors coding for a cytokine, a costimulatory molecule, or a ligand. Ertl and coworkers were the first to report the induction of profound imunomodulation by cytokine DNA codelivery [5]. In a rabies virus model, granulocytemacrophage colony-stimulating factor (GM-CSF) DNA strongly enhanced the antigen-specific B and T helper cell response. Meanwhile, cytokine DNA coadministration has proven efficacy in several other viral model systems. An important advantage over protein therapy may represent the low and long-lasting expression, which is a characteristic feature of genetic adjuvants and may be responsible for their effectiveness, feasibility, and low toxicity. Stimulation of antigen-presenting cells (APC) directs the immune response to the T-cell helper (TH)-1/TH-2 type by the production of an array of cytokines, comprising interleukin-12 (IL-12). IL-12 induces the differentiation of TH-0 to IL-2-, IFN-g-, and lymphotoxin-producing TH-1 type cells. Moreover, IL-12 activates natural killer cells (NK cells), which in turn also generate interferon-gamma (IFN-g. The main effector function of TH-1 type immune reactions represents the promotion of phagocytosis-mediated defense against infections. Then main effector cytokine is IFN-g, which enhances the microbicidal activity of phagocytes, the production of complement-fixing and opsonizing antibodies, and, in concert with IL-2, the proliferation and differentiation of CD8+ cells to CTLs. Delayed type hypersensitivity (DTH) responses are typical TH-1 type immune responses. In contrast, TH-2 type immune responses are characterized by IL-3, IL-4, IL-5, IL10, and IL-13 production. Thereby, TH-2 cells mediate allergic reactions and defense against helminthic and arthropod infections. Moreover, immune reactions of the TH-1 type are antagonized. Typical features of TH-2 immune reactions are IgE- and IgG-neutralizing antibody generation and activation of eosinophils. GM-CSF DNA showed substantial effectiveness against HSV-2 infections. Codelivery of DNA encoding GM-CSF with DNA encoding the HSV-2 full length glycoprotein D (gD) increased both the humoral and cellular immune response. A strong ac-

14.4 CTL-Epitope Immunization

tivation of IL-4-secreting cells was observed in the spleen and draining lymph nodes, together an increased number of IFN-g-secreting cells. A reduction of vaginal virus titer after intravaginal challenge was observed in mice coinjected with a GM-CSF encoding plasmid relative to those immunized with gD encoding plasmid only [6]. Oral coadministration of Salmonella harboring a plasmid encoding IL-10 together with Salmonella carrying a plasmid encoding HSV-2 gp D or B to mice led to severe lesions and death after virus challenge, compared to mice receiving the plasmid encoding gD only. [7]. Costimulation by coinjection of vectors coding for CD80 (B7.1) and HSV gp D induced a strong DTH reaction and an adjuvant effect upon viral challenge with HSV [6]. Coimmunization of a gp D plasmid vaccine with the cDNA of lymphocyte function-associated antigen-3 (LFA-3), an adhesion molecule present on antigen-presenting cells, which binds to CD2 on T cells, enhanced IgG-production, TH-proliferative responses, and survival rate upon challenge with HSV-2 [8]. Costimulation of plasmid DNA encoding gB with DNA coding for the conserved cystine residues (CC) chemokines, macrophage inflammatory protein 1beta (MIP1beta) and monocyte chemotactic protein 1 (MCP-1), biased the immunity to the Th2-type. Costimulation with DNA encoding CXC chemokine MIP-2 and the CC chemokine MIP-1alpha biased the immune responses to the Th1-type [9]. Coadministration of IL-6 DNA with a swine influenza virus hemagglutinin DNA vaccination did not lead to a significantly enhanced virus-specific antibody response [10]. Neonatal mice coimmunized with hemagglutinin DNA and either IL-12 or IFNgamma expressing DNA developed IgG2a-biased immune response. In contrast, there was no effect in adults. In neonatal and adult mice the Th1 genetic adjuvants also shifted the pattern of lymphokine production by recall splenocytes from a mixed response to exclusively IFN-g [11]. Coimmunization of rhesus macaques with plasmids encoding IL-2 or IFNgamma, combined with plasmids coding for HIV env/rev and SIV gag/pol, enhanced the antigen-specific T cell-mediated immune response. Coimmunization with plasmids encoding IL-2 did not lead to an enhanced immune response [12]. Plasmid vaccines encoding HIV gag-pol, env-rev proteins, supplemented by IL-2 Ig cytokine gene adjuvants or boosted by recombinant MVA vectors expressing relevant SIV and HIV antigens, prevented CD4(+) T-cell loss and lowered viral loads after pathogenic challenge [13]. One rhesus monkey immunized with env/gag plasmid DNA augmented by an IL-2-immunoglobulin plasmid and subsequently infected with SHIV showed undetectable plasma viral RNA. A single nucleotide mutation within an immunodominant Gag CTL epitope resulted in viral escape and finally in the monkey's death from AIDS-related complications [14].

14.4 CTL-Epitope Immunization

The importance of cellular immunity for vaccination against HIV and the ease with which DNA can be manipulated favor CTL-epitope immunization. Combination of epitopes may generate a broader immune response, providing a strategy for simulta-

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neous vaccination against multiple pathogens. Multiepitope DNA vaccination encoding 25 epitopes from the gag protein of HIV [15] induced an immune response biased toward more antigen-specific CD8(+) T cells. Multiple immunization of rhesus macaques with DNA encoding the SIV fusion epitope gag-pol-env led to a significant CTL response, but low-to-undetectable serum antibodies. SIV-specific mucosal antibodies and CTL were detected in rectal washes and gut-associated lymphoid tissues, respectively. Four of seven vaccinees were protected against infection [16]. Two additional studies tried to evaluate the benefit of immunization with both dominant and subdominant SIV epitopes by DNA vaccination of rhesus macaques. In the first study, immunization with the two epitopes SIV Gag p11C and HIV-1 Env p41A led to a high-frequency CTL response specific to both epitopes only in infected macaques vaccinated with the plasmid DNA [17]. The second study showed that immunization with a repertoire of viral epitopes greater than those typically generated in the course of viral infection rapidly induced a potent secondary CTL response to subdominant epitopes after a SHIV challenge [18]. Multi-epitope DNA vaccination against LCMV showed the strongest CTL response when every epitope was encoded by a separate plasmid DNA [19]. Epitope-specific CTL responses were also induced by DNA constructs encoding single epitopes or fusion proteins of influenza [20], respiratory syncytial virus [21], HIV-1 gag epitope [22], HIV-1 gag-pol fusion protein [12, 23], as well as the HBsAg of HBV [24].

14.5 Targeting DNA Vaccines to Cellular Compartments or the Cell Surface

In the past two years only a few studies on this vaccination strategy were ongoing. Targeting of the expressed antigen (1) to the cell surface, (2) to the extracellular compartment in general by the induction of secretion, and (3) to specific extracellular compartments had a profound impact on the immune response. Engineering a truncated form of the HCV envelope E2 protein directed the antigen to the plasma membrane and increased its ability to induce humoral immune responses [25]. Two chimpanzees vaccinated with this plasmid resolved a virus challenge early, whereas the control animal became chronically infected [26]. Targeting of APCs was accomplished by fusion of a HA-based influenza DNA vaccine to CTLA-4. CTLA-4 binds to B7–1 (CD80) and B7–2 (CD86) expressed on antigen-presenting cells. This construct increased the antibody response and reduced viral titers over a 100-fold after a nonlethal viral challenge [27].

14.6 DNA for Chimeric Antigens

The immunogenicity of epitopes may be improved by the use of chimeric DNA vaccine plasmids. These are composed of epitope-encoding DNA fragments inserted into genes, which function as DNA antigen carrier systems. The latter may modify

14.7

the immune response to the respective antigen. In one study, a chimeric yellow fever/dengue virus was constructed expressing the premembrane and envelope genes from dengue type 2 virus in a yellow fever virus genetic background. Immunized mice showed CD8 T-cell response and were protected against lethal dengue virus challenge [28]. In another study, mice were immunized with three different DNA plasmids encoding a fusion of a secreted form of HIV Env protein and the complement component C3d. Immunization with each plasmid accelerated the onset and the avidity maturation of antibody to Env [29]. A similar approach was performed by Barouch and coworkers to improve DNA vaccination against HIV. They constructed a plasmid expressing an IL-2/Ig fusion protein, which combined IL-2 functional activity with divalent avidity and a long in vivo half-life. Interestingly, coadministration of both the fusion protein and the IL-2/Ig expressing plasmid with the HIV immunogen augmented immune responses in mice and nonhuman primates [30]. Chimeric virus-like-particles consisting of multimerized HPV L1 proteins and fragments of SIV Gag p27, HIV-1 Tat and, Rev proteins were administered to macaques systemically and mucosally and led to antibody induction against HPV L1. Only macaques that received a priming vaccination with DNA encoding the same SIV and HIV-1 antigens prior to chimeric boosting showed significant but partial protection from a virulent mucosal SHIV challenge [31]. Antibody–cytokine fusion proteins, called immunocytokines, are also used to target cytokines into tumor microenvironment. Therapeutic efficacy has been shown in melanoma and neuroectodermal tumor models [32]. Finally, antipeptide antibody generation is often augmented by peptide fusion or chemical linkage to larger proteins, such as keyhole limpet hemocyanin (KLH), glutathione S-transferase (GST), or sparingly soluble nonantigenic protein (SSNAP) [33].

14.7 DNA-prime–Protein/Viral-boost Immunization

Prime–boost vaccination involves priming with DNA vaccines and boosting with either recombinant protein or attenuated recombinant viral vectors, including fowl poxvirus (FPV) and modified vaccinia virus Ankara strain (MVA). Prime–boost strategies aim at augmenting immune responses to pathogens that are not completely inhibited by DNA vaccination alone (e. g., HIV). DNA/protein boost strategies were employed for HCV and HIV vaccination and resulted in significantly enhanced specific antibody responses. Priming with DNA encoding the E2 protein of HCV and subsequent boosting with the respective recombinant protein induced a stronger humoral and cellular immune response than immunization with E2 DNA or protein alone, as evidenced by enhancement of E2-specific IgG2 a levels and CD8+ CTL responses. The immunological effects were further augmented by using two instead of one DNA priming [34]. Mice immunized with a combination of DNA encoding the HCV capsid and E1, E2, NS2, and NS3 genes with a nonreplicant canary pox booster containing the same genes showed enhanced CD8+ T cell response [35]. In another study, chronically HBV-infected chimpanzees

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14 Update on antiviral DNA vaccine research (2000–2003)

were immunized first with DNA followed by a recombinant canary pox booster. One week after the booster, HBV DNA declined more than 400-fold and remained undetectable by quantitative PCR for 186 weeks. Interestingly, the plasma levels of hepatitis surface antigen declined for only a short time [36]. Attenuated recombinant viral vectors, such as FPV and MVA, have significant advantages over alternative immunization strategies. They are replication-deficient, nonintegrating, stable, and relatively easy to prepare. Boosting with recombinant FPV favors the induction of cellular immunity, which is of primary importance for HIV defense. DNA priming is thought to focus the immune responses to the antigen expressed by the DNA vaccine, whereas the recombinant pox virus booster immunization is thought to boost the response, both by expression of higher levels of the antigen and by the immunostimulatory activity of a pox virus infection. In this regard, macaques primed with an env and nef deletion-containing SHIV proviral DNA followed by a single booster with a Gag-expressing Sendai virus showed protection from CD4+ T cell deletion and significantly reduced viral load after virus challenge. Vaccination with either the DNA alone or the Gag-expressing Sendai virus alone did not lead to a consistent protection [37]. In another study, macaques were immunized with a V2-deleted HIV-1 envelope using a DNA-prime–protein-boost regime to evaluate the protective potential of the induced neutralization antibodies during acute infection. Therefore, the macaques were depleted of their CD8+ T cells were and were challenged with pathogenic SHIV. The vaccinated animals had lower levels of peak viremia and rapid clearance of the virus from periphery and developed delayed seroconversion to SIV core antigens [38]. In a study by Amara and coworkers, priming macaques with Gag-Pol-Env-expressing DNA and boosting with GagPol-Env-expressing rMVA was compared with Gag-Pol DNA priming and Gag-Pol rMVA boosting to evaluate the contribution of an anti-Env immune response to viral control. After mucosal challenge with a pathologic SHIV, the Gag-Pol-vaccinated group developed severe, sustained loss of CD4+ T cells. Interestingly, most of the lost CD4+ T cells in that group were uninfected cells. They suggested that the rapid appearance of anti-Env antibodies in gag-pol-env-vaccinated macaques helped to protect uninfected CD4+ T cells from Env-induced apoptosis [39]. Shiver and coworkers compared several vaccine vector-delivery systems containing plasmid DNA vector and MVA encoding SIV gag. After viral challenge, the most effective responses were elicited by a replicon-incompetent Adenovirus type 5 vector [40]. In additional studies, the immune responses of DNA-prime–virus-boost immunized animals were evaluated [41, 42].

14.8 Age-dependent Effectiveness of DNA Vaccines

DNA vaccines are intended to be used in infants, who have an immature immune system. In neonates, low doses of antigen are frequently associated with suboptimal responses, but higher doses may induce tolerance. Comparable to self-antigens, DNA vaccine-encoded antigens are endogenously synthesized and presented via the

References

MHC-class I pathway. Consistent with this concern, a DNA vaccine encoding the circumsporozoite protein of malaria induced tolerance in neonates [43]. Live attenuated measles vaccine is safe and immunogenic when administered to children over the age of nine months. However, it is less efficacious in younger infants, due to the immaturity of their immune systems and to transplacentally acquired maternal antibodies. Younger infants are especially predisposed to measlesassociated fatalities. Alternative strategies for immunization of infants against measles are therefore desirable. However, further development has been complicated by the occurrence of a severe atypical form of the disease when individuals previously immunized with a formalin-inactivated vaccine contracted measles. In contrast, DNA vaccination raises hope for nontoxic immunization of infants against measles. In a rhesus macaque model, DNA vaccines encoding measles virus glycoproteins were protective and did not induce atypical measles [44]. Another study examined the immune response of mice that were DNA-vaccinated at birth against LCMV and showed that, at least for CD8+ T cells, neonates are not as immunodeficient as previously supposed [45]. Rasmussen and coworkers vaccinated newborn macaques by HIV-1 gp160-encoding DNA priming and protein-boosting, followed by SHIV challenge. Containment of infection was observed in 4 of 15 animals given DNA-priming–protein-boost vaccination and in 3 of 4 animals given gp160 boosts only. Rechallenge with homologous virus of 6 animals that contained the first challenge virus resulted in rapid viral clearance or low viral loads [46]. Recently, DNA vaccination also proved to be efficacious for fetal immunization. Antibodies, as well as cell-mediated immune responses, were generated when DNA was delivered into the amniotic fluid in the oral cavity of fetal lambs. Fetal vaccination may be of particular importance for the prevention of neonatal diseases such as HSV, HIV, HBV, CMV, group B streptococcus, hemophilus, and chlamydia infections [47]. Finally, in another study, liposome-encapsulated DNA encoding antigens from HIV-1 and influenza virus were administered i.v. to pregnant mice. Examination of plasmid DNA uptake by the fetus was observed at 9.5 days postconception. DNA vaccination of the offspring of immunized dams mounted stronger specific immune responses than controls and were protected against challenge by homologous influenza virus [48].

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sponses and protection against rabies virus 1 year after vaccination. Vaccine 2001, 20, 838–844. Biswas S, Reddy GS, Srinivasan VA, Rangarajan PN, Preexposure efficacy of a novel combination DNA and inactivated rabies virus vaccine. Hum Gene Ther 2001, 12, 1917–1922. Lodmell DL, Parnell MJ, Bailey JR, Ewalt LC, Hanlon CA, Rabies DNA vaccination of non-human primates: post-exposure studies using gene gun methodology that accelerates induction of neutralizing antibody and enhances neutralizing antibody titers. Vaccine 2002, 20, 2221–2228. Lodmell DL, Ewalt LC, Post-exposure DNA vaccination protects mice against rabies virus. Vaccine 2001, 19, 2468– 2473. Lodmell DL, Ewalt LC, Rabies vaccination: comparison of neutralizing antibody responses after priming and boosting with different combinations of DNA, inactivated virus, or recombinant vaccinia virus vaccines. Vaccine 2000, 18, 2394–2398.

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15 Live Recombinant Bacterial Vaccines Simon Clare and Gordon Dougan

Summary

The application of modern molecular techniques has generated renewed interest in live bacterial vaccines. Targeted mutagenesis can be used to construct genetically defined and attenuated derivatives of pathogens that can be exploited as live vaccines and as vectors for delivering heterologous antigens to the immune system. Such vaccines offer the potential for improved safety and quality control. Many candidate vaccines have been generated, and some of these have progressed to evaluation in the clinic. Problems have been encountered in translating data obtained from experimental models into successful candidate vaccines suitable for full development. This chapter focuses on candidate live vaccines based on enteric bacteria that are undergoing or close to clinical evaluation.

15.1 Introduction

Live vaccines have played a critical role from the very beginning of the practice of vaccination. Indeed, Jenner’s very first vaccination experiments utilized live viral vaccines based on poxviruses [1]. Live vaccines offer the potential advantage of mimicking aspects of natural infection and are readily recognized as ‘foreign and infectious’ by the immune system, a factor that can enhance their immunogenicity [2]. Live vaccines can be based on attenuated forms of a particular pathogen or on immunologically related microorganisms that are preferentially adapted to infect a host different from the one targeted for vaccination. Any microorganisms selected as a basis for live vaccine production must be able to establish a limited infection in the host targeted for vaccination but must also be unable to go on to cause full clinical disease. Hence, they must be attenuated with respect to the expression of full virulence. For many years the microorganisms selected for use as live vaccines were developed or identified by empirical approaches. They were attenuated by passage on laboratory medium or on an unnatural host animal or tissue culture cell line. For example, the tuberculosis vaccine strain Bacillus Calmette–Guerin (BCG) was isolated

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after many years of passage on laboratory medium. An example of a vaccine based on a microorganism that caused a limited but protective infection in an unnatural host is the smallpox vaccine based on vaccinia, which is a poxvirus poorly adapted for growth in healthy humans. A consequence of the empirical approach is that the basis of attenuation usually remains undefined. Pathogenic microorganisms harbor specific genes that contribute to virulence, and attenuated derivatives harbor one or more mutations in these so-called virulence-associated genes. By using modern molecular approaches, such as whole genome DNA sequencing, it is possible to identify some of the potentially attenuating lesions in existing live vaccine strains. A simplified example of this approach is the sequencing of the different polio virus vaccine strain genomes, which has led to the identification of specific point mutations that contribute directly to attenuation [3]. Using this, together with related technologies, we can retrospectively analyze well-known live vaccine strains in an effort to decipher their mechanisms of attenuation. This information is useful for safety and quality control. By identifying the number and type of attenuating lesions present in a particular vaccine strain, it will be possible to more accurately calculate the potential for reversion to virulence and the stability of the strain. However, we should bear in mind that such approaches may not enable us to identify all mutations that contribute to attenuation. Interestingly, comparison of different stocks of BCG located around the world has highlighted significant differences in their genomes [4]. Some batches harbor more mutations or different combinations of mutations than others. This is believed to be a consequence of the fact that BCG isolates were distributed across the world before careful batch-management methodologies were introduced into the vaccine production industry. In consequence, significant genetic drift occurred involving the accumulation of mutations as BCG was passaged in different centers. Thus, BCG-based vaccines around the world may differ significantly in terms of their immunogenicity and protective potential. This may in part explain the different results obtained in tuberculosis efficacy studies performed around the world. Although retrospective characterization of existing vaccines provides useful scientific information and is potentially of enormous practical benefit, it is important to use more rational approaches to develop live vaccines for the future. One approach would be to introduce specific, well-defined attenuating mutations into fully virulent pathogens. Here, specific genes essential for survival and the expression of virulence in a particular host would be targeted. ‘Clean’ genetic techniques would be used to introduce well defined mutations into these genes in situ in the pathogen genomes, using manipulation techniques that do not favor the accumulation of secondary mutations. In this way a series of defined attenuating mutations can be introduced into an individual genome. This approach has been referred to as ‘rational attenuation’, in that specific genetic loci are targeted for manipulation and these loci provide a basis for attenuation and ultimately vaccine development [5–11]. The theoretical advantages of this approach are many. Knowledge of the genetic nature of the attenuating lesions can facilitate more informed and accurate quality assurance and control of vaccines

15.1 Introduction

based on these attenuated strains. Mutations can be identified that cannot readily revert or favor accidental selection of less attenuated forms of the vaccine strains. Indeed, several attenuating mutations can be built up in individual vaccine strains to limit the potential for reversion to virulence to theoretical levels. Also, it might be possible to engineer-in mutations that still attenuate even in severely immunocompromised individuals, limiting the potential for clinical complications in vulnerable groups [12]. The concept of rational attenuation is not a new one, and many attempts have been undertaken to exploit this approach since the development of recombinant DNA techniques. Attempts to rationally attenuate viruses, bacteria, and even eukaryotic parasites have reached different levels of sophistication, and vaccines based on these technologies have been evaluated in the clinic. Since this is relatively new technology, it will be important to thoroughly evaluate how such candidate vaccines perform in the clinic. In this chapter we concentrate on the generation of vaccines based on rationally attenuated enteric bacteria that have reached the level of clinical evaluation (Table 15.1). Enteric bacteria exhibit different patterns of infection and pathogenicity (Figure 15.1). Hence, significantly different approaches are required to attenuate different pathogens. Molecular approaches are now being applied to decipher the mechanisms involved in the infection process and to understand how the immune response deals with different pathogens. Tab. 15.1 Some key examples of live recombinant vaccines that have been tested in humans. Bacterial strain

Mutations

Comment

Ref.

541Ty, 543Ty

aroA purE

32

EX492 CVD908

galE via aroC aroD

CVD908-htrA Chi3927 ZH9 Ty800

aroC aroD htrA crp cya aroA aroC ssaV phoP phoQ

Regarded as over-attenuated; poorly immunogenic. Caused a typhoid like infection. Good immunogenicity but bacteria found in the blood. Good immunogenicity into phase 2 trials. Good immunogenicity but reactogenic. Good immunogenicity; in phase 2 trails. Good immunogenicity.

Typhoid Salmonella typhi

30 31 36, 37, 39 31 4, 7 44, 45

Bacterial dysentery Shigella flexneri SF124 CVD 1207

aroD virG sen set guaA

Good immunogenicity but reactogenic. Good immunogenicity with some reactogenicity.

69–71 75

ctxA hlyA

Good immunogenicity, protective in volunteers but poor results in the field.

53

Cholera Vibrio cholerae CVD103-Hg-R

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15 Live Recombinant Bacterial Vaccines Immunological interaction

1. Commensal Colonisation

IgA, RNI Defensins Mucus

2. Attachment Attaching/Effacing (Vibrio cholerae, EPEC, ETEC)

M

3. Invasion (Salmonella, Shigella)

B

B T

T Lymph Nodes

4. Systemic spread (Salmonella Typhi)

Blood stream

Fig. 15.1 Patterns of intestinal colonization displayed by enteric pathogens.Distinct enteric pathogens exhibit different modesof pathogenicity after interaction with the gut. This diagram illustrates the typical types of enteric infection and the level of the gut tissueassociated with infection in a healthy human. EPEC enteropathogenic E. coli; ETEC enterotoxigenic E. coli (see colour plates page XXXVIII).

15.2 Early Efforts to Generate Recombinant Live Bacterial Vaccines

Enormous progress has been made in the past 20 years in our understanding of the molecular basis of infection caused by pathogenic bacteria. For example, a decade ago only a handful of genes involved in the pathogenesis of Salmonella infections had been identified. To date, over 100 genes that are involved in different aspects of Salmonella pathogenicity are known. Many of these virulence-associated genes are organized into discrete pathogenicity islands that harbor groups of genes with coordinated functions or expression patterns [13]. To date, there are ten different pathogenicity islands identified in Salmonella enterica and interestingly different S. enterica serovars can exhibit significant variation in the composition of some of these islands [14, 15]. Salmonella pathogenicity islands (SPI)-1 and SPI-2 are significant in that they encode Type III secretion systems involved in epithelial cell invasion and intracellular vacuole adaptation, respectively (Figure 15.2). The S. enterica type III secretion systems are molecular syringes designed to deliver packages of bacterial effector proteins that are injected into the cytoplasm of host cells to modify these cells to favor bacterial proliferation. Although many Salmonella genes are implicated in virulence, the vast majority have been identified by using the favored murine model of salmonellosis based on S. enterica serovars such as S. enterica serovar Typhimurium (S. Typhimurium). Because some S. enterica serovars, including S. enterica serovar Typhi (S. Typhi), the

15.2 Early Efforts to Generate Recombinant Live Bacterial Vaccines

Fig. 15.2 Interaction of Salmonella enterica with immune cells shown by confocal microsc opy. A. anti-Salmonella B. anti-LAMP-1 C. Merged image. Provided by Dr Liljana Petrovska, Imperial College London (see colour plates page XXXVIII).

cause of human typhoid, exhibit significant degrees of host restriction, it is important not to extrapolate data whole-sale from the mouse to other species. In other words, mutations that attenuate in the mouse may not attenuate in other host species or in other S. enterica serovars. Indeed, a number of genes, including htrA, have been identified for which this occurs for salmonellosis in cattle [16, 17]. A further caveat is that murine S. enterica infection does not mimic human gastroenteritis, normally associated with diarrhea, and it is not possible to model human gut disease in the mouse. The problem of host adaptation and the lack of suitable model systems is equally severe for many other bacterial diseases, including tuberculosis, shigellosis, and cholera. In any of these models it is also impossible to accurately measure the potential of strains to induce reactogenicity. So, taken together, all of these factors illustrate the enormous importance of clinical studies. The fact that so many different genes can be involved in pathogenicity illustrates the enormous complications of selecting candidate mutations. For some diseases, including cholera, where a single product such as the cholera enterotoxin plays a central role in the pathogenic process, the target genes speak for themselves. However, attenuation in itself is not sufficient, because any attenuated strain must retain immunogenicity and the capacity to be formulated into a practical vaccine. Hence, mutations that impair protective immunity or affect the integrity of the pathogen so as to make them unsuitable for storage of formulation (e. g., freeze drying) cannot be utilized. Few studies have been made to carefully compare the impact of different or combinations of attenuating mutations on the protective capacity of candidate vaccine strains. However, some useful examples exist that can be used to illustrate the con-

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siderations involved. Hosieth and Stocker [18] used the murine model to show that aroA mutations of S. Typhimurium could be used as a basis on which to develop single-dose oral vaccines against salmonellosis. This work has subsequently been confirmed by many other workers [see 19]. aroA encodes an enzyme, 5-enolpyruvyl-shikimate-3-phosphate synthase, that is critical for biosynthesis of the aromatic ring by many bacteria. Since the availability of aromatic compounds in mammalian tissues is carefully regulated, S. enterica aroA mutants are starved of such compounds in vivo and fail to grow substantially in immunocompetent hosts. The construction of rationally attenuated Salmonella strains for practical use should ideally involve the generation of S. enterica derivatives harboring more than one attenuating mutation. Indeed, this is true for any live vaccine, in order to minimize the potential for reversion to virulence. Hence, although S. enterica aroA strains are useful experimental vaccines, it is important to identify compatible mutations to use in combination with aroA. Early efforts focused on mutations involving genes of the purine biosynthetic pathway, such as purA and purE [20, 21]. It was believed that targeting two metabolic pathways would be better than targeting a single (aro) pathway. Consequently, a candidate live oral typhoid vaccine was generated based on S. Typhi Ty2 harboring mutations in both aroA and purA. In volunteer studies (described later) this strain was not particularly immunogenic as a live oral vaccine. Subsequent studies in the mouse model showed that live oral vaccines based on S. Typhimurium aroA purA derivatives where significantly less immunogenic than similar vaccines based on isogenic S. Typhimurium aroA [22, 23]. Indeed, although S. Typhimurium aroA purA vaccines could induce the production of antibodies, they were unable to protect mice against virulent S. Typhimurium challenge, in contrast to vaccines based on S. Typhimurium aroA. Interestingly, vaccines based on S. Typhimurium purA were also unable to protect mice against virulent S. Typhimurium. Hence, purA appears to over-attenuate S. enterica in comparison to aroA. purE mutations, which act further down the purine biosynthetic pathway, only partially attenuate murine virulent S. Typhimurium, and are consequently totally unsuitable for live vaccine development [24]. However, combinations of different aro mutations, such as aroA and aroC or aroA and aroD, induce an indistinguishable level of attenuation compared to aroA alone, making feasible the generation of immunogenic S. enterica derivatives harboring multiple attenuating mutations [25]. Although these experiments provide a clear example of the impact of different combinations of mutations on virulence, attenuation, and immunogenicity, other factors, such as bacterial strain background and host genetics, may also have a significant impact on vaccine performance. Again, this points to the critical role of clinical studies for human vaccine development. The lessons taken from the example of the behavior of candidate S. enterica vaccines can be equally applied to the development of rationally attenuated live vaccines for other diseases.

15.3

15.3 Clinical Studies Involving the Development of Live Recombinant Vaccines 15.3.1 Live Recombinant Salmonella Vaccines

Theoretically it should be possible to develop a live recombinant vaccine against any of the numerous S. enterica serovars that differ in terms of their host range and pathogenicity. However, there is little evidence, except for a short period of nonspecific immunity detectable 2–4 weeks after live vaccination, that inter-serovar protection is particularly extensive [23]. Hence, S. Typhimurium vaccines are unlikely to protect against S. Typhi, for example. Few attempts have been made to develop human vaccines for any S. enterica serovars other than S. Typhi. Most S. enterica serovars cause localized gastroenteritis in humans, associated with diarrhea in the absence of significant systemic spread. The mouse is not a good model for this disease syndrome, but is believed to more closely mimic human typhoid. Because S. Typhi is already highly attenuated in the mouse, murine S. Typhi infections cannot be used to model levels of attenuation, and results are on the whole modeled from murine S. Typhimurium infections. However, as stated above, there are inherent limitations in this approach, which are perfectly illustrated in clinical studies on S. Typhi galE mutations. The first live oral typhoid vaccine to achieve general commercial success was the S. Typhi Ty2 galE derivative Ty21a. Ty21was developed by using chemical mutagenesis of S. Typhi Ty2, and consequently, the nature of the attenuating mutations in Ty21 a were not defined. Nevertheless, Ty21 a has proved to be a very safe vaccine in extensive use throughout the world [26, 27]. S. Typhi Ty21 a was designed to harbor mutations in the gene galE encoding UDP-glucose-4-epimerase, because early experiments had indicated that galE mutants of S. Typhimurium were significantly attenuated in the murine model [28]. The experience obtained with Ty21 a encouraged the development of a rationally attenuated S. Typhi Ty2 harboring a defined galE deletion mutation [29]. Once, such a S. Typhi gale-negative Vi-negative strain, EX462, was generated by using site-directed mutagenesis [30]. Interestingly, two of four volunteers who ingested 7 × 108 viable EX462 went on to develop typhoid-like disease within a few days, with viable EX462 organisms being isolated from their blood. Each of the individuals who ingested EX462 exhibited a rise in local and systemic antibodies to S. Typhi antigens. Clearly, the presence of galE and via mutations in combination in this strain was insufficient to completely attenuate S. Typhi. Interestingly, some of the early studies using galE mutations in S. Typhimurium had indicated that additional mutations to galE (linked to effects on bacterial cell susceptibility to lysis in the presence of galactose) might be required for full attenuation. However, these experiments illustrate the need for extreme caution when moving from mouse to man in such experiments. The experience with S. Typhi galE mutant strains instilled a note of caution into future S. Typhi live oral vaccine studies. Encouraged by the exciting data produced by studies on S. Typhimurium aroAbased vaccines, efforts were stepped up to develop an oral typhoid vaccine based on

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aroA mutants. However, in these first experiments, instead of a single aroA mutation, the S. Typhi strains were constructed with double aroA purA mutations [31]. In volunteer studies, two S. Typhi Ty2 derivatives, 541 Ty aroA purA and 543 Ty aroA purA via were fed to volunteers in increasing doses (Table 15.1). Although no serious adverse reactions were observed in these volunteers, the humoral and local antibody responses to these vaccines were minimal, and they were deemed too poorly immunogenic to justify further volunteer studies. Retrospective assessment of this study and comparison with subsequent data obtained in the mouse suggests that purA mutations, particularly in combination with aro mutants, are potentially over-attenuating for S. Typhi [22]. Thus, follow-up experiments involved S. Typhi derivatives harboring double aro mutations (aroC aroD), since similar mutant combinations had performed well in the murine salmonellosis model. For the follow-up phase of volunteer studies, aroC and aroD deletion mutations were introduced into two different S. Typhi backgrounds. CVD 906 harbored aroC aroD in a Chilean S. Typhi isolate, whereas CVD 908 harbored the same mutations on a conventional Ty2 background [32]. CVD 906 was found to be quite immunogenic in terms of seroconversion to LPS and local antibody production, even when administered at a dose of 5 × 107 viable organisms. However, this vaccine was quite reactogenic in several volunteers (increased temperatures), and the vaccine strain was detected in the bloodstream of some of them. More encouraging results were obtained with CVD 908 in a series of volunteer studies. The strain proved consistently immunogenic, with high levels of seroconversion and appearance of IgA antiSalmonella antibody-secreting cells in the blood stream. Further, significant T cell responses were detected. However, although the strain was less reactogenic than CVD 906, significant numbers of volunteers harbored bacteria in their blood streams, socalled silent vaccinemia. These observations encouraged further modification of the CVD 906 and CVD 908 strains. HtrA is a gene encoding a serine protease involved in the Salmonella stress response and in clearance of misfolded proteins by a degradative pathway [33]. The HtrA protein is required for the efficient survival of Salmonella inside macrophages and facilitates the ability of these bacteria to resist killing by oxygen radicals. HtrA mutants of murine-virulent S. Typhimurium are attenuated, and S. Typhimurium aro htrA derivatives are excellent single-dose live oral vaccines in the mouse [34, 35]. CVD 906-htrA and CVD 908-htrA were developed and administered to volunteers as live oral vaccines [36]. Early studies using wet-harvested bacteria were encouraging, and volunteers tolerated well doses of between 107 and 109 bacteria. These strains retained some immunogenicity and, more significantly, bacteria were not detected in the bloodstream of volunteers, indicating that htrA had indeed further attenuated these strains. Work with CVD 908-htrA was recently extended to a phase II study incorporating the use of lyophilized preparations of vaccine [37]. Again, both safety and immunogenicity data were encouraging in terms of circulating IgA-producing cells and, to a lesser extent, seroconversion. Further human studies have indicated that CVD 908 and CVD 908-htrA can induce significant T cell proliferation to Salmonella antigens including flagellin, and circulating anti-Salmonella cytotoxic T cells have been detected [36–39]. Further aro htrA S. Typhi derivatives have been

15.3

generated and evaluated in volunteers. An S. Typhi aroA aroD htrA strain was constructed on a wild-type S. Typhi background CDC10–80 [32]. Interestingly, this derivative was apparently significantly more reactogenic in volunteers than CVD908, and some vaccinemias were detected. These data illustrate the influence of the S. Typhi strain background, as well as that of the combination of mutations, on the outcome of clinical studies. Another combination of mutations that received early attention for incorporation into live oral typhoid vaccines was cya crp – both involved in the gene regulation pathway involving cAMP generation in bacteria. S. Typhimurium cya crp mutants were effective live oral vaccines in the murine salmonellosis model, encouraging volunteer studies. Initial studies involved an S. Typhi Ty2 crp cya derivative 3927 [41]. This strain was highly immunogenic but quite reactogenic in volunteers, delaying subsequent volunteer studies. Subsequently, S. Typhi Ty2 cya crp cdt derivatives were further evaluated [42]. S. Typhimurium strains harboring mutations in the phoP phoQ two-component regulatory system are attenuated and good oral vaccines in the murine salmonellosis model [43]. An S. Typhi strain, Ty800 phoP phoQ, was constructed and evaluated in volunteers. This derivative performed well in a dose-escalation study in terms of immunogenicity, lack of adverse reactions, and lack of vaccinemias [44, 45]. In a recent study, the attenuation and immunogenicity of two novel Salmonella vaccine strains, ZH9 S. Typhi Ty2 aroC ssaV and WT05 S. Typhimurium TML aroC ssaV, were evaluated after oral administration to volunteers as single escalating doses of 107, 108, or 109 viable bacteria. The ssaV gene encodes a component of the SPI-2 type III secretion apparatus, and bacteria harboring mutations in ssaV are defective in their ability to replicate inside macrophages [46, 47]. This study is particularly interesting because it involved a direct side-by-side comparison of S. Typhi and S. Typhimurium derivatives harboring similar attenuating mutations. S. Typhimurium TML is a clinical isolate associated with gastroenteritis and diarrhea. S. Typhi ZH9 was well tolerated by the volunteers and was not detected in blood as a vaccinemia. In addition, this strain was only excreted in the stool for a short time after oral ingestion. Six out of nine volunteers elicited anti-S. Typhi LPS IgA antibody-secreting cell responses, and two out of three receiving 109 viable ZH9 bacteria elicited high-titer LPS-specific serum IgG. S. Typhimurium WT05 was also well tolerated by the volunteers, with no diarrhea, but interestingly, administration of 108 and 109 viable bacteria lead to shedding in stools for up to 23 days. Only volunteers immunized with 109 WT05 mounted detectable anti-S. Typhimurium LPS-specific antibody-secreting cell responses, and serum antibody responses were variable. Taken together, these data indicate that mutations in ssaV and type III secretion systems in general may provide a route to the development of live vaccines in humans. The study also highlights significant differences in the properties of S. Typhimurium- and S. Typhibased live vaccines.

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15.3.2 Live Cholera Vaccines

Live oral vaccines against cholera have several attractions. Cholera, caused by the bacterial pathogen Vibrio cholerae, is a mucosal infection of the intestine and not an invasive disease. Consequently, local mucosal immunity could play an important role in protection. The best correlate of protection against cholera is the presence of vibriocidal antibodies in the serum of the host, and the critical target antigen appears to be somatically located on the bacteria rather than being antitoxic. Hence, although cholera enterotoxin plays a central role in cholera pathogenesis, antitoxic immunity does not appear to be essential for the induction of a protective immune response. The application of recombinant DNA technology to live oral cholera vaccine development has focused on inactivation of the cholera toxin gene encoding the A subunit, the ctxA gene. Several groups have created Vibrio cholerae O1 mutants harboring deletions within ctxA that completely obliterate the production of toxic enterotoxin [48–50]. Early studies utilized wild-type El Tor strains such as N16961 and classical strains such as 395 as hosts for ctxA deletion mutations. ctxA-negative derivatives of both these strains were significantly attenuated in volunteers, and one strain, JBK70 based on N16961, was able to induce protective immunity against virulent challenge with a dose as low as 106 viable bacteria. Unfortunately, JK70 and other early prototype candidate vaccines were also reactogenic, inducing some limited diarrhea and some nausea [51]. Further attenuated derivatives were generated by introducing mutations into either tcpA, encoding the subunit of the toxin coregulated pilus, or toxR, involved in expression of both the pilus and cholera enterotoxin. However, both these strains were markedly reduced in immunogenicity and were deemed unsuitable for oral vaccine development [52]. These early investigations were followed up by a series of volunteer studies designed to identify an immunogenic but nonreactogenic live oral cholera vaccine candidate. Extensive efforts led to the development of CVD 103-HgR [53]. This Vibrio cholerae 01 classical inaba strain 569B derivative harbors a deletion mutation in ctxA and has a mercury-resistance determinant inserted into the hemolysin hlyA gene, which acts as a genetic marker. CVD 103-HgR moved successfully through extensive volunteer studies with an excellent safety and immunogenicity profile. The strain was formulated as a live oral cholera vaccine for commercial development and was tested in several parts of the world for safety, immunogenicity, and efficacy. In volunteer studies using people from the USA or Europe, CVD 103-HgR consistently raised the detectable levels of vibriocidal antibodies in the serum of volunteers at least four fold after a dose of 5 × 105 viable organisms by mouth [54, 55]. When CVD 103-HgR was evaluated in volunteers from developing countries such as Thailand, it performed less well. In Thai volunteers, a dose of 5 × 109 viable organisms was required to get significant increases in vibriocidal antibodies. This lack of ‘take’ in individuals from developing countries was attributed to factors such as differences in the normal flora or to the presence of priming to cross-reactive antigens [56–58].

15.3

Eventually, a randomized, double-blind, placebo-controlled efficacy trial of the CVD 103-HgR live oral cholera vaccine was performed in Indonesia. Over 60 000 individuals were immunized in the study. The vaccine was extremely well tolerated overall, with vibriocidal blood responses detected in about 70 % of vaccinees. Significantly, the incidence of confirmed cholera cases was lower than expected throughout the duration of the trial, and little to no protective efficacy was observed [59]. Recent follow-up studies indicated that factors such as worm burden, immunosuppression, and small-bowel overgrowth in vaccinees can effect the immunogenicity of CVD 103-HgR, and this study highlighted the problems of moving live vaccines from Western volunteers out into the field in developing countries [60–62]. Other V. cholerae live vaccine candidates have been developed and are being evaluated [63, 64]. In efforts to complement the field studies, a randomized, double-blind, placebocontrolled, multicenter study of CVD 103-HgR vaccine efficacy was designed to test longer-term protection using 108 viable CVD 103-HgR against moderate and severe El Tor cholera challenge. American volunteers were challenged three months after immunization with wild-type V. cholerae O1 El Tor Inaba strain N16961. Over 90 % of the vaccinees had a greater than four-fold rise in serum vibriocidal antibodies after vaccination. The vaccine exhibited a protective efficacy of approximately 80 %, an indication that CVD 103-HgR could be used to prevent cholera in travelers [65]. Efforts continue to improve the formulation and potentially the efficacy of live oral cholera vaccines. One option is to expand the microbial target of vaccines to cover the novel V. cholerae O139 derivatives that have appeared in Bangladesh [66]. Much more needs to be learned about the impact of diet, normal flora, and other pathogens on the immunogenicity of live oral vaccines, and CVD 103-HgR is proving to be an excellent and safe model vaccine for this purpose. 15.3.3 Live Shigella Vaccines

Shigella species are the cause of dysentery in humans. Dysentery is a mucosal infection associated with invasion of the gut mucosal surface, inflammation, and cellular damage, leading to the generation of bloody diarrhea. Several antigenically distinct Shigella differ in the type of dysenteric syndrome they induce, with Shigella dysenteriae being associated with the most severe disease. A consequence of this antigenic variation is that Shigella vaccines are likely to protect only against the homologous serotype. Many attempts have been made to generate live oral vaccines against Shigella, but in the West we still do not have an acceptable live vaccine, although this type of work has progressed further in China. The key problem with developing live Shigella vaccines is that it has so far proved impossible to separate immunogenicity from reactogenicity. In other words, vaccines with acceptable levels of immunogenicity are usually over-reactogenic in volunteers. This again illustrates the problem of using surrogate models, even primates, for vaccine development. It is extremely difficult to predict reactogenicity in man from such models. Like S. Typhi, Shigella are host adapted and are often poorly pathogenic in many model systems, limiting their potential.

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Early efforts with nonrecombinant, genetically manipulated Shigella derivatives indicated that oral vaccines could be protective in humans [67], with serum and local antibody production being correlated with different degrees to protection. Early efforts to apply recombinant technology to Shigella vaccine development focused on aro mutants [68]. Several aroD derivatives of Shigella flexneri were constructed and shown to have significantly reduced virulence in infection models, including monkeys. In a series of volunteer studies, different aro auxotrophic S. flexneri derivatives, including SFL124 aroD, were evaluated in various parts of the world. Overall, the vaccines proved to be immunogenic in terms of their ability to induce anti-LPS, but some reactogenicity was detected at higher doses of these vaccines [69–71]. Nevertheless, the results were encouraging enough to consider progressing Shigella aro derivatives in combination with other attenuating mutations. An example of this approach is the generation of the S. flexneri 2 a derivative CVD 1203, which harbors mutations in aroA and virG/icsA on strain background 2457T [72, 73]. virG, also called icsA, encodes an 120-kDa outer membrane protein involved in activating actin polymerization at the poles of Shigella cells. VirG enhances the ability of Shigella to spread within and between eukaryotic cells. Again, CVD1203 proved to be immunogenic in volunteers, but reactogenicity was detected at an increasing level with higher doses (up to 109 bacteria) [74]. An additional Shigella flexneri 2 a derivative known as CVD1207 was subsequently constructed. CVD 1207 harbors specific deletion mutations in virG, sen, set, and guaBA. Sen and set encode enterotoxins that contribute to Shigella-associated diarrhea, and guaBA are genes encoding enzymes involved in guanidine biosynthesis, with guaBA mutant derivatives being auxotrophic [75]. In a dose-escalation study, up to 1010 viable CVD 1207 were administered orally to American volunteers, and the vaccine was well tolerated except for some significant reactogenicity at the higher doses. An immunoglobulin A antibody-secreting cell response specific to S. flexneri 2 a O was detected in the blood of many volunteers; this was associated with a Th1like response when cells from the vaccinees were stimulated with Shigella antigens, with production of interferon g without detectable IL-4 or IL-5. Thus, CVD 1207 represents a lead candidate for further development of a live oral Shigella vaccine. Other efforts have been made to develop live oral Shigella vaccines. Perhaps the most advanced are S. flexneri 2 a SC602 harboring mutations in iuc (involved in iron acquisition via siderophore production) and virG and S. dysenteriae 1 derivative strain SC599 harboring mutations in entF virG stxA fes [76]. fes and entF inactivate the iron chelator enterochelin complex and stxA inactivates the shiga toxin.

15.4 Expression of Heterologous Antigens in Live Bacterial Vectors

Live vaccines are attractive, because they can be used to induce immune responses that mimic those detected following real infection. They can also be delivered by the natural infection route, inducing local as well as systemic immunity. Thus, live vaccines can potentially be administered parenterally, or, for vectors based on enteric

15.4 Expression of Heterologous Antigens in Live Bacterial Vectors

bacteria, via the oral route. This combination of potent immunogenicity and mucosal administration has highlighted the potential of live enteric vaccines as vectors for delivering heterologous antigens to the mammalian immune system. This approach involves the expression of foreign heterologous antigens in particular live vaccine derivatives. Heterologous antigens can theoretically be derived from any source, including bacteria, viruses, parasites, and even mammals. The foreign gene must be adapted to facilitate expression in the particular live vector of choice. This can involve the addition of appropriate transcription and translation signals or even the generation of codon-optimized synthetic genes. There are now many examples in the literature involving an array of different live vectors and heterologous genes. Live vectors have been used as a basis to develop experimental vaccines against infectious agents, cancer, and allergens [77]. Expression vectors have been designed that exploit expression of the foreign antigen within the bacterial vector or even within the host, using eukaryotic transcription/translation signals [78–81]. The latter approach involves designing vectors that direct the expression of the foreign antigen within target eukaryotic host cells after delivery of the DNA (usually an extrachromosomal plasmid element) by the live vector. There have been many descriptions of the successful application of live vector-based gene delivery for vaccination, in which protection was induced against a particular infectious agent or cancer, but so far, these studies have been mainly restricted to experimental models and not clinical disease. The choice of vector has a significant influence on the type of immune response induced against a particular heterologous antigen. Pathogens vary greatly in their mode of pathogenicity and in the type of i