ME T H O D S
IN
MO L E C U L A R BI O L O G Y
Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK
For other titles published in this series, go to www.springer.com/series/7651
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Vaccine Adjuvants Methods and Protocols
Edited by
Gwyn Davies St. George’s University of London, London, UK
Editor Gwyn Davies Department of Cardiac & Vascular Sciences St. George’s University of London Cranmer Terrace London Tooting United Kingdom SW17 0RE
[email protected]
ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-60761-584-2 e-ISBN 978-1-60761-585-9 DOI 10.1007/978-1-60761-585-9 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2009943283 © Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: Detection of antigen presention in vivo by immunofluorescence. 100 μg of EαGFP protein plus 1 μg LPS was injected into the neck scruff and 30 min or 24 hours later the skin injection site and draining brachial lymph nodes (BLNs) were collected, processed and stained. The cover illustration shows intrinsic EαGFP fluorescence (green) 30 min post injection at skin injection site. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)
Preface Over the last few years the field of research and development of vaccine adjuvants has become a very exciting and stimulating area to work in. Its scope is huge, from discoveries in fundamental immunology and mechanisms by which adjuvants are able to influence immune responses to antigens, to the safe application of products that modify immune responses, to optimization of adjuvant formulation with antigens, and last but not least, to the industrial and commercial considerations that are essential if an adjuvant is to be developed in a vaccine product. Its potential is also huge, as we understand more and more about how adjuvants may steer the immune system toward the responses required by unmet vaccination needs. Indeed, one of the complexities in putting a book like this together has been the decisions involving what to leave out rather than what to include. Where are the boundaries with pure immunology, and how do you avoid simply producing another vaccine protocol book? Also, much has recently been published about vaccine adjuvants, but relatively little specifically addressing the techniques and methods that are applied to understanding how adjuvants produce their effects in vaccines. This volume, then, aims to provide some guidance on how to go about assessing the activity of adjuvant products. Of course, the fact that there are very different mechanisms by which an adjuvant effect can be generated, reviewed in Chapter1, and different routes of administration of vaccines, means that many different aspects have to be addressed. The general philosophy of the volume has been not to describe individual adjuvants in development or in use in vaccines, although some types are described, but rather to provide information on measuring the responses produced by adjuvants. No preconception is made as the types of vaccine that could benefit from formulation with an adjuvant. Two chapters describe what might be considered as reference adjuvants. First, methods for the use of aluminium salts, that until recently were the only adjuvant products in licensed human vaccines, and then Freund’s adjuvants, are discussed. Use of the latter, particularly the complete Freund’s, is now restricted, but it has served as a reference for much adjuvant research, and so it is included here. There have inevitably been some overlaps with vaccine studies, although we have tried to keep the focus on the adjuvant throughout. There is also a small amount of overlap between some chapters, but it seemed preferable to maintain the integrity of each. Unfortunately, it has not been possible to include all relevant topics; commercial and other considerations have prevented some from being presented. Some topics are covered by reviews. The AS04 adjuvant has been described previously, but the approval of vaccines containing this adjuvant has opened up possibilities for novel adjuvants, and we considered a description of the methods used in its development as highly informative for this volume. The importance of adjuvant safety cannot be overestimated and a review has been dedicated to this. Also, there is a review of the use of cytokines as adjuvants. I sincerely hope that the methods presented here will have an enabling effect for those interested in working in vaccine adjuvant research and development. Also, I hope they
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will help to stimulate discussion on how we can best standardize adjuvant testing so that meaningful comparisons can be made, and above all, so that useful predictions can be made on which new products will most effectively and safely help to solve the current challenges in vaccination. Gwyn Davies
Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v ix
1.
Immunology of Vaccine Adjuvants . . . . . . . . . . . . . . . . . . . . . . . . Carla M.S. Ribeiro and Virgil E.J.C. Schijns
1
2.
Preclinical Development of AS04 . . . . . . . . . . . . . . . . . . . . . . . . . Nathalie Garçon
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Nonclinical Safety Assessment of Vaccines and Adjuvants . . . . . . . . . . . . . Jayanthi J. Wolf, Catherine V. Kaplanski, and Jose A. Lebron
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Aluminum Adjuvants: Preparation, Application, Dosage, and Formulation with Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . Erik B. Lindblad and Niels E. Schønberg
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Freund’s Complete and Incomplete Adjuvants, Preparation, and Quality Control Standards for Experimental Laboratory Animals Use . . . . . . . . . . . Duncan E.S. Stewart-Tull
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5.
6.
Liposomal Adjuvants: Preparation and Formulation with Antigens . . . . . . . . Jean Haensler
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Micro/Nanoparticle Adjuvants: Preparation and Formulation with Antigens . . . Padma Malyala and Manmohan Singh
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Adjuvant Activity on Human Cells In Vitro . . . . . . . . . . . . . . . . . . . . 103 Dominique De Wit and Michel Goldman
9.
Adjuvant Activity on Murine and Human Macrophages . . . . . . . . . . . . . 117 Valerie Quesniaux, Francois Erard, and Bernhard Ryffel
. . . . . . . . . . . . . . . . . . . . . 131
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In Vitro Effects of Adjuvants on B Cells Jörg Vollmer and Hanna Bellert
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NKT Cell Responses to Glycolipid Activation . . . . . . . . . . . . . . . . . . . 149 Josianne Nitcheu Tefit, Gwyn Davies, and Vincent Serra
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Tracking Dendritic Cells In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . 169 Catherine M. Rush and James M. Brewer
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Adjuvant Effects on Antibody Titre . . . . . . . . . . . . . . . . . . . . . . . . 187 Barry Walker and Ian Feavers
14.
Functional Antibody Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Ian Feavers and Barry Walker
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Determining Adjuvant Activity on T-Cell Function In Vivo: Th Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Thomas Lindenstrøm, Peter Andersen, and Else Marie Agger
16.
Quantitative Multiparameter Assays to Measure the Effect of Adjuvants on Human Antigen-Specific CD8 T-Cell Responses . . . . . . . . . . . . . . . 231 Laurent Derré, Camilla Jandus, Petra Baumgaertner, Vilmos Posevitz, Estelle Devêvre, Pedro Romero, and Daniel E. Speiser
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Large-Animal Model for Establishing E/T Ratio of Adjuvants . . . . . . . . . . 251 Luuk A. Th. Hilgers
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Determining the Activity of Mucosal Adjuvants . . . . . . . . . . . . . . . . . . 261 Barbara C. Baudner and Giuseppe Del Giudice
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Adjuvant Activity of Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Michael G. Tovey and Christophe Lallemand
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Contributors ELSE MARIE AGGER • Adjuvant Research, Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark PETER ANDERSEN • Adjuvant Research, Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark BARBARA C. BAUDNER • Novartis Vaccines and Diagnostics, Siena, Italy PETRA BAUMGAERTNER • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland HANNA BELLERT • Coley Pharmaceutical GmbH, A Pfizer Company, Düsseldorf, Germany JAMES M. BREWER • Centre for Biophotonics, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, UK GWYN DAVIES • European Adjuvant Advisory Committee, Cardiac and Vascular Sciences, St. George’s University of London, London, UK DOMINIQUE DE WIT • Institut d’Immunologie Médicale, Université Libre de Bruxelles, Charleroi, Belgium GIUSEPPE DEL GIUDICE • Novartis Vaccines and Diagnostics, Siena, Italy LAURENT DERRÉ • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland ESTELLE DEVÊVRE • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland FRANCOIS ERARD • Molecular Immunology and Embryology, University and CNRS, Orleans, France; IIDMM, University of Cape Town, Cape Town, South Africa IAN FEAVERS • National Institute for Biological Standards and Controls, Potters Bar, Hertfordshire, UK NATHALIE GARÇON • Global Adjuvant Center for Vaccine, GlaxoSmithKline Biologicals, Wavre, Belgium MICHEL GOLDMAN • Institut d’Immunologie Médicale, Université Libre de Bruxelles, Charleroi, Belgium JEAN HAENSLER • Sanofi Pasteur, Marcy l Etoile, France LUUK A. TH. HILGERS • Nobilon International BV, Boxmeer, the Netherlands CAMILLA JANDUS • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland CATHERINE V. KAPLANSKI • Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA CHRISTOPHE LALLEMAND • Laboratory of Viral Oncology, FRE2937 CNRS, Institut André Lwoff, Villejuif, France JOSE A. LEBRON • Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA
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ERIK B. LINDBLAD • Adjuvant Department, Brenntag Biosector, Frederikssund, Denmark THOMAS LINDENSTRØM • Adjuvant Research, Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark PADMA MALYALA • Novartis Vaccines and Diagnostics, Cambridge, MA, USA VILMOS POSEVITZ • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland VALERIE QUESNIAUX • Molecular Immunology and Embryology, University and CNRS, Orleans, France; IIDMM, University of Cape Town, Cape Town, South Africa CARLA M.S. RIBEIRO • Department of Cell Biology & Immunology, Wageningen University, Wageningen, The Netherlands PEDRO ROMERO • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland CATHERINE M. RUSH • Vascular Biology Unit, School of Medicine, James Cook University, Townsville, Queensland, Australia BERNHARD R YFFEL • Molecular Immunology and Embryology, University and CNRS, Orleans, France; IIDMM, University of Cape Town, Cape Town, South Africa VIRGIL E.J.C. SCHIJNS • Department of Cell Biology & Immunology, Wageningen University, Wageningen, The Netherlands NIELS E. SCHØNBERG • Adjuvant Department, Brenntag Biosector, Frederikssund, Denmark VINCENT SERRA • Wittycell SAS, Evry, France MANMOHAN SINGH • Novartis Vaccines and Diagnostics, Cambridge, MA, USA DANIEL E. SPEISER • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland DUNCAN E.S. STEWART-TULL • Division of Infection and Immunity, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, Scotland, UK JOSIANNE NITCHEU TEFIT • Wittycell SAS, Evry, France MICHAEL G. TOVEY • Laboratory of Viral Oncology, FRE2937 CNRS, Institut André Lwoff, Villejuif, France JÖRG VOLLMER • Coley Pharmaceutical GmbH, A Pfizer Company, Düsseldorf, Germany BARRY WALKER • National Institute for Biological Standards and Controls, Potters Bar, Hertfordshire, UK JAYANTHI J. WOLF • Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA
Chapter 1 Immunology of Vaccine Adjuvants Carla M.S. Ribeiro and Virgil E.J.C. Schijns Abstract In recent times vaccine adjuvants, or immunopotentiators, received abundant attention in the media as critical ingredients of current and future vaccines. Indeed, vaccine adjuvants are recognized to make the difference between competing vaccines based on identical antigens. Moreover, it is recognized that vaccines designed for certain indications require a matching combination of selected antigen(s) together with a critical immunopotentiator that selectively drives the required immune pathway with minimal adverse reactions. Recently, the mechanistic actions of some immunopotentiators have become clearer as a result of research focused on innate immunity receptors. These insights enable more rational adjuvant and vaccine design, which, ideally, is based on predictable immunophenotypes following vaccination. This chapter addresses immunopotentiators, classed according to their (presumed) mechanisms of action. They are categorized functionally in two major groups as facilitators of signal 1 and/or signal 2. The mode(s) of action of some well-known adjuvant prototypes is discussed in the context of this classification. Key words: Adjuvant, signal 1, signal 2, stranger, danger.
1. Introduction Vaccines have become one of most successful life-saving instruments in modern medicine due to their extremely efficient and cost-effective prevention of infectious disease (1). Traditionally, vaccines comprise either live-attenuated, replicating pathogens or non-replicating, inactivated pathogens or their subunits (2). Live vaccines, still used to immunize against measles or rubella, are safe for the majority of recipients. Although cost-effective and rather easy to manufacture, live vaccines may cause disease when given to a recipient with an unrecognized immunodeficiency (3). Inactivated vaccines consist of killed pathogens or G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_1, © Springer Science+Business Media, LLC 2010
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isolated non-replicating subunits. They are safe for immunocompromised individuals, but they often show limited immunogenicity. The vast majority of current vaccines act by inducing antibodies. However, many new vaccine targets require the induction of specific cell-mediated responses, in addition to antibodies (4). Special adjuvants, or immunopotentiators, are therefore required to elicit adequate immunity, for example, enhancement of T-cell responses, by targeting certain innate immune cells in most cases, with the additional benefits that less antigen doses and fewer administrations are necessary (5). Vaccine adjuvants come in many forms and are more or less effective at inducing the onset, magnitude, duration, and quality of an immune response against a co-formulated antigen. Hence, adjuvants (from Latin adjuvare meaning “to help”) can be defined as a group of structurally heterogeneous compounds able to enhance or modulate the intrinsic immunogenicity of an antigen (6). They can be classed according to their chemical nature or physical properties, yet related compounds frequently have divergent immunomodulating capacities. For example, saponin variants may differ in their capacity to stimulate Th1- or Th2-type immunity (7). Alternatively, adjuvants have been clustered according to the immunological events they induce (2, 8), although for many the exact mechanism of action is unknown. In 1989 Charles Janeway Jr. aptly termed vaccine adjuvants “the immunologist’s dirty little secret” (9). It reflected the general ignorance on the mechanisms of action of most known adjuvants at the time. Yet rational vaccine design involves a logical choice of immunopotentiator based on its mode of action and its expected effect on efficacy and safety of the vaccine (3). Despite the tremendous impact of the adjuvant choice, even today the key processes critical for immune induction in general, and those evoked by distinct adjuvants, in particular, are unknown and subject of debate among immunologists and vaccinologists (10).
2. Signal 1 and Signal 2 Facilitators
At present two major functional classes of vaccine adjuvants have been defined (8). The first category includes so-called facilitators of signal 1, influencing the fate of the vaccine antigen in time, place, and concentration, ultimately improving immunoavailability of the antigen. The second major group constitutes facilitators of signal 2, providing the correct co-stimulation signals for the antigen-specific adaptive immune cells during antigen recognition. Both classes of adjuvant are not mutually exclusive (Table 1.1). For a schematic illustration see Fig. 1.1.
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Table 1.1 Classification of adjuvants according to their stimulatory action Adjuvant category
Concept
Critical feature
Example
Signal 1
Improving immunoavailability
Time, place, dose of antigen
Alum-containing adjuvants, oil-based emulsions
Stranger (PAMP)
Lipopolysaccharide
Signal 2
Improving co-stimulation
Danger (DAMP)
Heat-shock proteins
Recombinant co-stimulus
Recombinant interferon
Release of natural immune system brakes
CTLA-4 inhibitory antibody
According to the two-signal model (11, 12) both the presentation of antigen (signal 1) and the additional secondary signals (signal 2) are required for activation of specific T and B lymphocytes, which form the adaptive arm of the immune system.
a
b
Fig. 1.1. The two-signal model. Recent advances in immunology have shown that the magnitude and specificity of the signals perceived by the innate immune cells following infection (and vaccination) can shape subsequent adaptive immune responses. Activation of (depicted) (a) T helper (Th) cells requires at least two different signals (b) from the antigen-presenting cell (APC) including signal 1 (antigen presentation) and signal 2 (co-stimulation).
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Secondary signals are delivered by co-stimulatory or co-inhibitory signals, and their overall balance and constellation determines the magnitude and quality of the ensuing adaptive immune reaction (Fig. 1.1). Stimulatory second signals (in most cases for adjuvants) inform the T cells that the presented antigens are proper subjects to initiate an immune response. Charles Janeway predicted that microbial pathogens are evolutionarily distant from their hosts and contain conserved molecular patterns, so-called pathogen-associated microbial patterns (PAMPs), which can be recognized by so-called pathogen recognition receptors (PRRs) (9). Today we know that PRRs exist. They include Toll-like receptors (TLRs), NOD-like receptors, dectin-1 or RIG-like helicases which are predominantly found on cells of the innate immune system. The rather recent discovery of PRRs and their capacity to detect molecular structures unique to pathogens (also referred to as signal 0) provided a mechanistic concept of the key upstream events leading to co-stimulation. This insight formed a breakthrough for rational vaccine design based on PRR ligands, comprising various PAMPs and later also endogenous TLR ligands, such as heat-shock proteins (Hsp) (5). Nowadays the innate immune system is studied extensively after the general appreciation by the scientific community that this system plays a critical role in PAMP sensing and instruction of adaptive immune reactions. It is considered critical in signal 2 induction and downstream activation of distinct T helper cell subsets. The category of signal 2 facilitating adjuvants has since been mechanistically defined at the molecular level (please see Section 4).
3. Signal 1 Facilitators In contrast to molecular events involved in the generation of signal 2, very little is known about the mechanisms governing the adjuvant effect of signal 1 facilitators. When soluble antigen is injected subcutaneously it is presented in two waves in the secondary draining lymph nodes (LN). Within 30 min free antigen enters the LN by afferent lymph vessels and is presented by resident dendritic cells (DC), while tissueresident DCs that acquire antigen at the injection site migrate to LN within 12–24 h and present Ag in MHC class II complexes. They sustain the activation of Ag-specific CD4+ cells initially activated by resident DC (13). For most vaccine adjuvants it is not known whether their activity is required at the site of injection or in the local
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draining lymph node. Naive T and B cells normally do not enter non-lymphoid areas, such as most injection sites, of the body efficiently. They rather circulate between secondary lymphoid organs, including the spleen and lymph nodes, via the blood and efferent lymphatics. Only memory and effector lymphocytes are able to penetrate non-lymphoid tissues during an inflammation (14, 15). Hence, the antigen has to reach the secondary lymphoid tissues in order to be recognized by adaptive immune cells. The geographical concept of immune reactivity (16) proposed that time, place, and dose of the antigen are critical factors for induction of adaptive T- and B-cell responses. It is supported by experimental observations of reduced immune responses early after surgical removal of the injection site (17) and in lymph node-deficient hosts (18). No immune response develops when the antigen is not able to reach the lymph node due to interruption of the afferent lymphatics (19–22). Also the fact that augmented immune responses were observed after repeated injection of minute amounts of poorly immunogenic antigens support this concept (23, 24). Obviously, motility and migration have a key role in many biological processes. However, despite the paramount importance of this class of vaccine adjuvants and their extensive application in existing vaccines, the mechanisms underlying the activity of presumed signal 1 facilitators remain a mystery. For prototype examples such as oil-based emulsions and aluminum-based adjuvants some aspects of its alchemy are described in the next section. 3.1. Oil-Based Emulsions
Already in 1968 Herbert attempted to mimic slow release of antigen by daily injections of tiny doses of ovalbumin in mice and noted an antibody production profile similar to a single full dose of ovalbumin formulated in a W/O emulsion (24). Interestingly, the antibody level in the circulation dropped soon after the daily injections were stopped. Freund studied the role of the depot function in rabbits by measuring antibody formation after surgical removal of the vaccine from the site of injection. Excisions performed between 30 min and 4 h after injection resulted in a decrease of the immune response when compared to the response in animals in which the injected areas were not removed. Remarkably, excisions performed after one or more days did not seem to affect the antibody response (17, 25). Hence, as yet there is no conclusive evidence for slow (i.e., more than 1 day) release as explanation for immune stimulating features of Freund’s adjuvants. Despite such investigations, little is known about the mechanisms responsible for adjuvant activity of the W/O emulsion in general. The structural requirements as well as the cellular and molecular immunological mechanisms within the host remain poorly
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understood. Indeed, this extremely powerful immune activating formulation has been shrouded in obscurity and is often referred as an example of “the immunologist’s dirty little secret.” As local reactions and residues represent major safety concerns for vaccines based on W/O emulsions, there is a clear demand for alternatives. Understanding the mechanism underlying W/O emulsion activity may help to further improve their formulation, thereby diminishing or eliminating potential hazards. Alternatively, such insights will be highly useful in developing novel vehicles in general. Dupuis and coworkers (26) showed that DCs internalize vaccine antigen and labeled adjuvant MF-59, an oil-in-water emulsion, after intramuscular injection. How DCs know where to go is likely based on chemokine-based attraction, preceded by recruitment of granulocytes and monocytes/macrophages as shown recently (27). In general, these cells are rapidly recruited into sites of tissue injury in response to inoculation with live or inactivated microorganisms, probably as a result of locally produced chemotactic factors. The transient influx of neutrophils (PMNs) or other innate immune cells (27) likely affects DC function. Upon arrival in the lymph nodes DC’s antigen capture and processing capacity declines, while their immunostimulatory function is up-regulated. 3.2. Aluminum-Based Adjuvants
Aluminum-containing adjuvants such as aluminum hydroxide and aluminum phosphate adjuvants (often referred to as alum) are generally assumed to adsorb the Ag on the alum particle which forms an Ag depot at the injection site. This is believed to prolong availability of Ag (signal 1) for Ag-presenting cells (APCs). However, this concept has been challenged in recent times. Various mechanisms have been proposed to be responsible for its adjuvant effect. Alum has been shown to fix complement (28), to cause granulomas (29), to recruit eosinophils and neutrophils (30), and induce the appearance in the spleen of IL-4secreting cells (31). Yet, how alum achieves these effects remains unknown. Interestingly, alum does not promote classical direct dendritic cell (DC) maturation (32), which questions whether its activity is mediated by pattern recognition receptors. Kool and coworkers showed that intraperitoneally injected alum triggers local inflammatory CD11b+ Ly6G– Ly6C+ F4/80int monocyte-type cells to differentiate into inflammatory dendritic cells, which is associated with the secretion of uric acid (33). Treatment with uricase reduced recruitment of antigen (OVA)-labeled monocytes in alum-immunized mice. However, effects on alum-driven antigenspecific antibody production were not measured. This concept is
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strongly challenged by earlier data showing that uric acid crystals augment CTL responses to co-injected antigen, which, by contrast, was not observed for aluminum hydroxide in the same animal experiments (34). Hence, uric acid polarizes the immune response toward a type-1 response-associated CTL reaction, while a Th2-type response is the hallmark of alum adjuvanted vaccines. The lack of proper CTL priming by alum represents its major deficiency as vaccine adjuvant. Both (35) and very recently (30) showed that alum is able to activate the NALP3 inflammasome in human peripheral blood mononuclear cells (PBMCs), and primary peritoneal macrophages of mice, respectively. However, such activation is dependent on priming of the cells with lipopolysaccharide (LPS), questioning whether inflammasome activation is involved during vaccination in the absence of a co-stimulatory LPS signal. By contrast, Sokoloska and coworkers showed in 2007 that alum-containing adjuvants directly stimulate the release of IL-1β and IL-18 via caspase-1 activation from mouse dendritic cells. Remarkably, in this study responses were noted without priming by LPS (36). In NALP3-deficient mice a partial (3- to 5fold) reduction of alum-induced antigen-specific antibody formation (IgE and IgG1 isotypes) was observed using OVA or human serum albumin (HAS) as antigens (30, 35). On the contrary, Franchi and Nunez showed that NALP3-deficient mice showed normal antibody responses when immunized in the context of alum adjuvant (37). However, of particular importance is a recent study (38) demonstrating that mice lacking MyD88, and therefore unable to respond to TLR signals as well as IL-1β and IL-18 (39), are still capable to generate normal antibody responses when augmented by repository vaccine adjuvants including aluminum hydroxide, Freund’s incomplete and complete adjuvant. Of importance is also the study of Pollock et al., who showed that endogenous IL-18 facilitates alum-induced IL-4 production, but effects on humoral immune responses such as OVA-specific immunoglobulin G1 (IgG1) and IgE production, the hallmark of alum’s adjuvant effects, remained unaffected in IL-18-deficient mice (40). Moreover, in an allergic asthma model, IL-1R1-deficient mice sensitized by OVA formulated in alum developed typical pulmonary Th2 responses, eosinophilic inflammation, antibody responses and CD4(+) T-cell priming in lymph nodes similar to normal mice (41). Together, these data do not support the recently proposed NALP3 pathway (30), nor the uric acid concept (33) for alum adjuvant activity. In addition, there is no evidence for IL-18 or IL-1β dependence.
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4. Signal 2 Facilitators 4.1. Signal Zero, PAMPs, and Stranger Motifs
The innate immune system of vertebrates uses germlineencoded pattern recognition receptors (PRRs) to sense invading pathogens. These PRRs are able to recognize so-called pathogenassociated molecular patterns (PAMPs), for example, peptidoglycan or unmethylated CpG DNA. PAMPs are unique and conserved molecular structures of a given microbial class (bacteria, viruses, fungi, and protozoa). Depending on their location these PRRs can be secreted receptors (e.g., pentraxins) found in blood and lymph associated with complement or opsonization, membrane receptors (e.g., C-type-like receptors, Toll-like receptors) on APC associated with endocytosis or induction of nuclear factorkB (NF-kB)- and mitogen-activated protein kinases (MAPKs)dependent signaling pathways or cytosolic receptors on APCs associated with induction of NF-KB and MAPK signaling pathways (42) (Fig. 1.2).
Fig. 1.2. Overview of major classes of pattern recognition receptors. MBL, mannose binding lectin; CRP, C-reactive protein; SR, scavenger receptor; CR, complement receptor; MR, mannose receptor; TREM, triggering receptor expressed on myeloid cells; TLR, Toll-like receptor; NOD, nucleotide oligomerization domain; NALP, Nacht, LRR and PYRIN domaincontaining proteins; RIG, retinoic acid-inducible gene; LDL, low-density protein, LPS, lipopolysaccharide, LTA, lipoteichoic acid, MDP, muramyl dipeptide, ss, single stranded. NBS- Nucleotide binding site, CARD - Caspax recruitment domain
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Among the PRRs, Toll-like receptors (TLRs) are the most well-studied and best-described class of receptors. They have been shown to play an essential role in the initial response of the innate immune system to infection. TLRs are type I transmembrane proteins with an extracellular domain of interspersed leucine-rich repeat (LRR) motifs that are involved in recognition of PAMPs. The cytoplasmic domain is characterized by a Toll/IL-1 receptor (TIR) motif which is involved in signal transduction (43). TLRs are either expressed on the plasma or endosomal membrane of APCs and they respond to specific bacterial, viral, fungal, and protozoan PAMPs. Recognition of PAMPs by TLRs results in recruitment of a set of TIR domain-containing adaptor proteins such as MyD88. These interactions trigger intracellular downstream cascades eventually leading to activation of nuclear factor-kB (NF-kB) and mitogen-activated protein kinases (MAPKs), which results in induction of inflammatory cytokines (e.g., interleukin-1β, tumor necrosis factor-α) and co-stimulatory ligands (B7-1 and B7-2). Importantly, TLRs not only trigger signal 2. They may also play a role in antigen presentation (signal 1). TLRs may control the generation of T-cell receptor (TCR) ligands from the phagosome guaranteeing the presentation of both microbial components and antigen to activated APCs (44). TLR engagement can initiate and also shape the adaptive immune response, for instance, by skewing the immune system toward a Th1 or Th2 response (45) or by activating or inhibiting Treg cells (46). Moreover, vaccine adjuvants that contain TLR ligands can induce higher avidity T-cell responses (47). This suggest the need for antigen and TLR agonist to be co-delivered, in order to target the same phagosome cargo of one APC and thereby induce optimal antigen presentation and subsequent stimulation of Ag-specific T-cell responses (6). Indeed, TLRs ability to link innate and adaptive immunity represents a promising mechanism to be explored in the design of new vaccines (5). However, antigen targeting and engulfment can also be mediated by other receptors present in the surface of APC, such as C-type lectin receptors (CLRs) and triggering receptors expressed on myeloid cells (TREMs). CLRs, including mannose receptor and DC-SIGN, recognize sugar moieties (e.g., N-acetylglucosamine, mannose) at the surface of the pathogens enabling binding to an array of bacteria, virus, and fungi. Intracellular cytosolic receptors such as NODs (nucleotide-binding oligomerization domain proteins), NOD-like receptors (NLRs), and retinoic acid-inducible gene I-like helicases (RLHs) recognize structures from intracellular bacteria or viruses, but possibly also aluminum-containing adjuvants (30) as mentioned earlier. These receptors may form new targets for vaccine adjuvant development.
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4.2. Danger Signals, Alarm Signals, or Damage-Associated Molecular Patterns (DAMPs)
According to the so-called danger theory, originally proposed by (48), the immune system evolved to focus primarily on danger rather than on microbial non-self signals described above (48–50). Accordingly, antigens can be divided into two major groups: those associated with danger and antigens positioned in a harmless situation. Danger signals can be released by stressed or damaged tissue, as organelles of cells undergoing necrotic (but not apoptotic) death. Although such organelles or molecules from dead cells are not well defined at the molecular level, likely candidates responsible for observed immune activation include mitochondrial and nuclear fractions of necrotic cells as well as heat-shock proteins (HSPs), cellular DNA, or uric acid (30). These may be referred to as danger-associated molecular patterns (DAMPs) which lead directly or indirectly to up-regulation of co-stimulatory signals for APCs. Together with antigens released from dying cells these danger signals are recognized by APCs which may trigger an immune response if the host is not tolerant for these antigens. Also endogenous cytokines, such as type I IFN produced by infected cells, can be considered a DAMP. Indeed, several adjuvants are known to cause local damage at the injection site and may act by induction of danger signals. Examples include aluminum hydroxide (30), saponins and possibly also oil-based emulsions (51). Theoretically, the danger model may explain, in part, some of the mystery of signal 1 facilitating adjuvants described earlier.
4.3. Recombinant Cytokines or Co-stimulatory Molecules Mimicking Endogenous Immune Amplifiers
Protection conferred by some vaccines, such as influenza subunit vaccine, is mainly achieved by induction of a humoral response. A humoral response is mediated by antibodies and their effector functions, which include neutralization and opsonization of pathogen and activation of the complement cascade. Nevertheless, to ensure that the pathogen is effectively eradicated, it is not only important to attain an adequate amount of antibody but also to generate a specific immunoglobulin isotype. Antibody isotypes differ in their ability to activate the complement cascade or the binding to receptors on phagocytes. Therefore, it is of great importance to use adjuvants which promote the synthesis of specific antibody isotypes which confer more protective activity (52). Synthesis of particular antibody isotypes is strongly influenced by the combination of locally produced cytokines. For example, interleukin-5 (IL-5) or transforming growth factor β can augment IgA antibody formation, while interleukin-4 (IL-4) and interleukin-13 (IL-13) are able to induce switching toward the IgE isotype, and interferon-γ (IFN-γ) increases the synthesis of IgG2a antibody (53). A number of cytokines represent the hallmarks of a polarized immune reaction. For example, IFN-γ and IL-12 are associated with Th1-type immune reactions, while
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IL-4, IL-5, and IL-13 are related to Th2-type immune pathways. Use of such molecules in recombinant form may skew responses in the desired direction. Therefore, recombinant cytokines constitute serious adjuvant candidates that are especially suitable for subunit vaccines, which are poorly immunogenic when compared to whole killed or live-attenuated pathogens (54). In addition, ligands and receptors of co-stimulatory pathways represent an attractive target for the development of adjuvants. For instance, type I IFNs promote DC maturation by increasing expression of co-stimulatory molecules including CD40, CD80, and CD86 and major histocompatibility complex (MHC) antigen. Recombinant IFNs co-delivered with influenza vaccine have been shown to enhance protection against virus challenge (55). Also CD40 stimulation by agonistic antibodies exerts adjuvant effects (56). As a result, various co-stimulatory agonists are currently considered as an important new class of adjuvants (57). 4.4. Release of the Brakes
As mentioned earlier TLR agonists can constitute potent adjuvants for infectious diseases. However, it has been demonstrated that certain TLR agonists were able to promote the induction of IL-10-secreting Treg cells (58). A recent study revealed that TLR agonists can induce IL-12 and IL-10 production concomitantly and therefore promote the development of Th1 and importantly also Treg cells (59). So during a physiological immune response stimulating signals are accompanied by natural inhibitory signals. It is now recognized that CTLA-4 blockade (60), or depletion of Treg cell development, enhances the efficacy of therapeutic vaccination against tumors (61). Also, selective inhibitors of MAPKp38 in dendritic cell vaccines suppressed IL-10 and enhanced IL-12 production, thereby augmenting Th1 response and suppressing Treg cells (59). Hence, attenuation of regulatory Tcell induction by proper TLR agonist, or inhibition of natural immune response attenuating signals, may improve the efficacy of some vaccines and represents a variant of signal 2 facilitation (59).
5. Outlook Despite accumulated knowledge on the adjuvant mechanisms of signal 2 facilitators described above, we know very little about the classical adjuvants, which are presumed to facilitate signal 1. In particular we lack knowledge on the most upstream events, the earliest interactions between adjuvant and the tissue at the injection site. Future studies will certainly unravel these events and contribute to rational vaccine design.
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References 1. Van Duin, D., Medzhitov, R., Shaw, A. C. (2006) Triggering TLR signalling in vaccination. Trends Immunol 27, 49–55. 2. Schijns, V. E. J. C. (2000) Immunological concepts of vaccine adjuvant activity. Curr Opin Immunol 12, 456–463. 3. Gershon, A. A. (2003) Varicella vaccine: rare serious problems-but the benefits still outweight the risks. J Infec Dis 7, 945–947. 4. Rappuoli, R. (2007) Bridging the knowledge gaps in vaccine design. Nat Biotechnol 25, 1361–1366. 5. Schijns, V. E. J. C., Degen, W. G. J. (2007) Vaccine immunopotentiators of the future. Clin Pharm Therap 82, 750–755. 6 Guy, B. (2007) The perfect mix: recent progress in adjuvant research. Nat Microbiol 5, 505–517. 7. Marciani, D. J. (2003) Vaccine adjuvants: role and mechanism of action in vaccine immunogenicity. Drugs Disc Today 8, 934–943. 8. Schijns, V. E. J. C. (2001) Induction and direction of immune responses by vaccine adjuvants. Crit Rev Immunol 21, 75–85. 9. Janeway, C. A., Jr. (1989) Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harbor Symp Quant Biol 54, 1–13. 10. Schijns, V. E. J. C., O’Hagan, D. (2006) Immunopotentiators in Modern Vaccines, Elsevier/Academic press, London. 11. Bretscher, P., Cohn, M. (1970) A theory of self-nonself discrimination. Science 169, 1042–1049. 12. Lafferty, K. J., Cunningham, A. J. (1975) A new analysis of allogenic interactions. Aust J Exp Biol Med Sci 53, 27–42. 13. Itano, A. A., McSorley, S. J., Reinhardt, R. L., Ehst, B. D., Ingulli, E., Rudensky, A. Y., Jenkins, M. K. (2003) Distinct dendritic cell populations sequentially present antigen to Cd4 T cells and stimulate different aspects of cell-mediated immunity. Immunity 19, 47–57. 14. Mackay, C. R. (1993) Homining of naïve memory and effector lymphocytes. Curr Opin Immunol 5, 423–427. 15. Butcher, E. C., Picker, L. J. (1996) Lymphocyte homing and homeostasis. Science 272, 60–66. 16. Zinkernagel, R. M., Ehl, S., Aichele, P., Oehen, S., Kundig, T., Hengartner, H. (1997) Antigen localisation regulates immune responses in a dose- and timedependent fashion: a geographical view
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cans through induction of IL10 and regulatory T cells. J Immunol 172, 3712–3718. 59. Jarnicki, A. G., Conroy, H., Brereton, C., Donnelly, G., Toomey, D., Walsh, K., Sweeney, C., Leavy, O., Fletcher, J., Lavelle, E. C., Dunne, P., Mills, K. H. (2008) Attenuating regulatory T cell induction by TLR agonists through inhibition of p38 MAPK signaling in dendritic cells enhances their efficacy as vaccine adjuvants and cancer immunotherapeutics. J Immunol 180, 3797–3806. 60. Pedersen, A. E., Ronchese, F. (2007) CTLA4 blockade during dendritic cell based booster vaccination influences dendritic cell survival and CTL expansion. J Immune Based Ther Vaccines 5, 9–15. 61. Sutmuller, R. P., van Duivenvoorde, L. M., van Elsas, A., Schumacher, T. N., Wildenberg, M. E., Allison, J. P., Toes, R. E., Offringa, R., Melief, C. J. (2001) Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. J Exp Med 194, 823–832.
Chapter 2 Preclinical Development of AS04 Nathalie Garçon Abstract Recent knowledge on vaccine-induced immunity led to the development of vaccine Adjuvant Systems specially designed and adapted to vaccine needs. AS04 is such a tailored Adjuvant System developed by GlaxoSmithKline Biologicals. This chapter focuses on the methods that were used during the preclinical evaluation of AS04. AS04 consists of the combination of aluminum salts and 3 -O-deacylated monophosphoryl lipid A (MPL), a detoxified lipid A derivative with retained immunostimulatory capacity. MPL also induces considerably less pro-inflammatory cytokines, as compared to the parent LPS molecule. Preclinical evaluation of AS04 allowed the determination of the optimal size of MPL particles. The added value of MPL in AS04-based formulations was evidenced by higher vaccine-elicited antibody responses, as well as the induction of higher levels of memory B cells, as compared to aluminum alone formulations. Preclinical evaluation demonstrated the relevance of using AS04 in situations where high and long-lasting antibody levels are needed. This represents the basis for the successful application of AS04 in vaccines against hepatitis B virus and human papillomavirus. Key words: AS04, Adjuvant System, MPL, formulation, immune response, TLR-4, vaccine.
1. Introduction Over the last century, vaccines have demonstrated their potential in reducing mortality and morbidity due to infectious diseases, being at the same time one of the safest and most cost-saving products developed for health care. The subtle molecular mechanisms that are involved in the initiation of the body’s response to a vaccine are now better understood. Indeed, in the last 20 years, progress has been huge in understanding the immune response mechanisms, particularly the intricate relationship between innate and adaptive immunity. At G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_2, © Springer Science+Business Media, LLC 2010
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the center of the immune system, the antigen-presenting cells (APCs) represent the cell population that is able to recognize the type of pathogen and to distribute the work appropriately to the different immune effectors according to the pathogen involved. APCs stimulate the other immune factors after being activate through innate receptors on their membrane that specifically recognize pathogen molecules called pathogen-associated molecular patterns (PAMPs). The most studied of these innate receptors is the family of Toll-like receptors (TLRs) in which up to 10 members have been identified so far (1, 2). The impact of this growing knowledge on vaccinology is remarkable. Indeed, mostly to increase the safety profile, the last generation of vaccines is composed of protein subunits and recombinant antigens instead of whole pathogens. This evolution toward less toxicity, unfortunately, brought a decrease in immunogenicity for some antigens, as the stimulatory signals provided by several PAMPs present in the whole pathogen had been lost, and with them the ability to adequately stimulate APCs. To compensate such loss, it has become evident that an appropriate adjuvantation was required (3). The use of adjuvants is not new. They were first recognized in the 1920–1930s, when it was observed that increased antibody responses were induced by adding aluminum salts to a given antigen (4, 5). Such aluminum salts have been for 70 years the only adjuvant approved for human use, but their action might be too restricted for the new challenges that have appeared with the subunit vaccines and to respond to the still unmet medical needs. The recently acquired knowledge in immunology makes possible the use of TLRdependent or TLR-independent molecule combinations that are able to act at the APC level to specifically activate the desired arms of the immune system. With such a versatile approach, it is feasible to adapt the adjuvantation to the specific immunological needs required for a given pathogen and/or a given target population. GlaxoSmithKline Biologicals (GSK Bio) has been developing such tailor-made Adjuvant Systems (AS), destined to be combined with the right antigen(s) and intended to promote a fast, strong, and sustained immune response, supported by cellmediated immunity. One of these proprietary Adjuvant Systems, AS04, is now present in two registered vaccines, FENDrixTM , the hepatitis B vaccine destined to pre- and hemodialysis patients, and CervarixTM , the human papillomavirus (HPV-16/18) cervical cancer vaccine, the first vaccine with a new adjuvant to be registered in the US, and approved in 100 additional countries including Japan. The chapter below discusses the methods that were used during the preclinical evaluation of AS04, which allowed successful development of the adjuvant in vaccines.
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2. The AS04 Adjuvant System AS04 originates from the need to ensure better vaccination outcomes in subjects with challenging immunological conditions or challenging pathogens. AS04 is composed of an aluminum salt (aluminum hydroxide for CervarixTM or aluminum phosphate for FENDrixTM ) combined with a TLR-4-dependent immunostimulant, namely the 3 -O-deacylated monophosphoryl lipid A (MPL). The combination of both molecules results in an AS able to promote a strong and sustained protection through the induction of high and persistent antibody titers, concomitant with effective B- and T-cell memory responses (6, 7). 2.1. Aluminum Salts
As already mentioned, aluminum salts are the oldest and also most widely used vaccine adjuvants. They are principally recognized to enhance antibody responses and their mechanism of action, though not fully understood, has been partially unveiled (for review, see (3)). It had been long accepted that adsorption of the antigen onto aluminum and its slow release at the site of injection (depot effect) were key to the adjuvant effect until it was demonstrated that adsorption, and thus the depot effect, might not be the crucial mechanism for the induction of the immune response. Results in mice rather suggested that aluminum salts impact on the APC function, inducing a Th2-biased immune response characterized by the promotion of antibody production via IL-4 signaling.
2.2. Monophosphoryl Lipid A (MPL)
MPL is a purified, detoxified derivative of the lipopolysaccharide (LPS) that is found on the outer membrane of the R595 strain of Salmonella minnesota. Detoxification is carried out through successive acid and base treatments. After purification through a succession of chromatography steps, it yields a molecule with the same immunomodulatory properties but with considerably less toxicity than the parent LPS molecule (8, 9). LPS is a group of structurally related complex molecules composed of three covalently linked regions (1): the innermost region is lipid A, containing glucosamine disaccharide units that carry long chains of fatty acids (2), the central region is the core oligosaccharide, and (3) the outer region is represented by the Ospecific polysaccharide chains, containing repeated oligosaccharide units. MPL displays the same basic structure as the lipid A region of LPS. It is noteworthy that there is biosynthetic variability in the assembly of the lipid A moiety and loss of fatty acids from the lipid A backbone during the hydrolytic steps of the manufacturing process. The resulting MPL is a mixture of
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HO
HO
O
O
P HO
O
O O
O NH
A
OH
HO HO NH
B C Number of Fatty Acids 6 5
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A C14(OC14) C14(OC14) C14OH ΔC14 C14OH Δ-C 14 H H
Position B C14 (OC12) C14 (OC12) C14 (OC12) C14 (OC12) C14 (OC12) C14 (OC12) C14 (OC12) C14 (OC12)
C C14(OC16) C14OH C14(OC16) C14(OC16) C14OH C14OH C14(OC16) C14OH
C14(OC16) = 3-(R)-hexadecanoyloxytetradecanoyl C14(OC14) = 3-(R)-tetradecanoyloxytetradecanoyl C14(OC12) = 3-(R)-dodecanoyloxytetradecanoyl C14OH = 3-(R)-hydroxytetradecanoyl Δ-C14 = tetradecanoyl
Fig. 2.1. Major 3-O-deacyl monophosphoryl lipid A congeners in MPL. Congener species all contain the same backbone consisting of a β -1 ,6-linked disaccharide of 2-deoxy-2aminoglucose, phosphorylated at the 4 position, but contain variable numbers and types of fatty acyl groups at the 2, 2 , and 3 positions. The 1, 3, 4, and 6 positions of the backbone are unsubstituted in all monophosphoryl lipid A species present in MPL. The 2, 2 , and 3 positions may be substituted with tetradecanoic, 3-(R)-hydroxytetradecanoic, or 3-(R)-acyloxytetradecanoic acids, depending on the position, so that the total number of fatty acyl groups varies from three to six (previously published in (6)).
closely related 3 -O-deacylated monophosphoryl lipid A species, called congeners (Fig. 2.1), and is the one referred to in the GSK Bio Adjuvant Systems. The composition of the mixture is always the same in reproducible manufacturing conditions and may vary when a different process is used. The resulting monophosphoryl lipid A then may display different properties. The Gram-negative bacterial LPS are PAMPs, specifically recognized by TLR-4 (10, 11), a receptor at the surface of the tissueresident macrophages and of immature APCs. Activation by LPS elicits a cascade of signals ultimately resulting in an immune response that is appropriate to combat the infection. MPL also acts through TLR-4 binding, leading to an activation pattern which is similar to that induced by LPS. 2.3. Formulation
Due to its hydrophobic nature, MPL is present in aqueous solution as a particulate structure and can be characterized by its particle size. It is available as a lyophilized triethylamine salt (TEA MPL) and the lyophilized powder is resuspended in water before it is processed to reach a particle size that allows sterile filtration,
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as measured by photon correlation light scattering. MPL bulk preparations are stable over time in terms of both chemical composition and physical properties. For vaccine preparation, MPL is adsorbed on preformed aluminum salt. MPL can be characterized by chromatographic analysis when present in aqueous solution or after desorption from the aluminum salts. If desorption is not possible because of the high binding affinity, a destructive method, such as gas chromatography–mass spectrometry, must be used to quantify MPL. As for any substance to be injected in humans, sterility and pyrogenicity need to be tested. Due to its nature, pyrogenicity of MPL cannot be evaluated with the classical limulus amebocyte lysate (LAL) test (12, 13) but only with the rabbit pyrogenicity assay (14).
3. The Preclinical Development of AS04 3.1. Immunological Evaluation of MPL
As previously mentioned, MPL exists as particles of different size in aqueous solutions. Therefore, besides studies on the optimal concentration, the impact of MPL particle size on the immune response was evaluated. Groups of Balb/c mice (n = 10) were immunized at days 0 and 15 by the intra-footpad route with 70 μL of the vaccine formulation (1/7 of the human dose), equivalent to 10 μL of aluminum hydroxide, 0.4 μg of hepatitis B surface antigen (HBsAg), and different amounts of MPL (Fig. 2.2). At day 7 after the second immunization, blood samples were taken and serum anti-HBsAg IgG2a antibody levels were measured by ELISA. These experiments in mice established that both parameters have an impact on the humoral response (Fig. 2.2). In the conditions of the experiment, small size MPL (100 nm) showed a typical dose–response curve, while larger MPL particles (500 nm) induced a bell-shaped humoral response curve. The results obtained with small MPL particles were confirmed in two open, randomized clinical trials in healthy adults that compared the immunogenicity of four different formulations. In these trials, hepatitis B vaccines, containing different concentrations of MPL (200 nm in size), 500 μg of aluminum salts, and 20 μg of HBsAg were administered to healthy adults (18–40 years), according to a 0 and 6 month immunization schedule. As negative control, the same formulation without MPL was used in a three-dose regimen (0, 1, and 6 months). One month after the last immunization, specific antibody levels were measured in serum by ELISA. The effect observed was dose related and a plateau was reached at 50 μg of MPL per human
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4000 100 nm MPL 500 nm MPL
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Fig. 2.2. Effect of MPL particle size and concentration on mouse IgG2a antibody response to hepatitis B surface antigen (HBsAg). Mice (n = 10 per group) were vaccinated with combinations of HBsAg/aluminum with MPL particles of two different sizes (100 or 500 nm) at concentrations ranging from 0 to 50 μg per vaccine dose. At day 7 after the second injection, serum samples were assayed for anti-HBsAg IgG2a antibodies by ELISA. Results are expressed as ELISA Units/mL (previously published in (6)).
dose (Fig. 2.3). Based on these preclinical results, manufacturability, and dose ranging in humans, the AS04 formulation selected for vaccination of adults is 50 μg per vaccine dose of MPL particles with a size allowing sterile filtration. The beneficial effects of AS04 on the immune responses, and particularly the added value of MPL (in AS04) as compared with aluminum alone, were established in animal models. Groups of mice were immunized at days 0 and 21 with the combination of both HPV-16 and HPV-18 L1 virus-like particles adjuvanted with aluminum salt alone or with aluminum salt supplemented with MPL (AS04). At days 14 and 37 after the second immunization, blood samples were taken and anti-HPV-16 and anti-HPV18 antibody levels were determined by ELISA. These experiments showed that the presence of MPL in the formulation greatly enhances the antibody levels elicited by vaccination (Fig. 2.4). 3.2. Safety Studies
Safety assessment is an integrated part of adjuvant development. To date, guidelines have been issued by the European authorities (15) and WHO (16) to regulate testing of vaccine or new adjuvant safety. Different safety aspects have been evaluated in several animal species. Altogether, the various preclinical safety evaluations did not indicate any adverse effect or systemic toxicity of MPL and/or AS04. Only transient inflammatory markers were observed locally, coherent with the use of an immunomodulator.
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Fig. 2.3. Effect of MPL concentration on human antibody response to hepatitis B surface antigen (HBsAg). Healthy human adults (18–40 years) were vaccinated with combinations of HBsAg/aluminum with MPL particles of 200 nm at concentrations ranging from 12.5 to 100 μg per vaccine dose. At day 30 after the second injection, serum samples were assayed for anti-HBsAg antibodies by ELISA. Results are expressed as ELISA Units/mL (previously published in (6)).
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14
anti-VLP16
× 105
anti-VLP18
GMT antibody titers (EU/mL)
GMT antibody titers (EU/mL)
12 8
6
4
2
10 8 6 4 2
0
0 Al(OH)3
AS04
Al(OH)3 14d post II
AS04
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Fig. 2.4. Antibody response in mice after immunization with adjuvanted HPV-16/18 L1 VLP vaccine. Mice (n = 12 per group) were vaccinated with the combination of HPV-16 and HPV-18 L1 VLPs adjuvanted with MPL adsorbed to aluminum salt (AS04) or with aluminum salt alone. After two intra-muscular injections (0 and 21 days), serum samples collected 14and 37-day post II were assayed for anti-HPV-16 or HPV-18 L1 VLP antibody response by ELISA. Results are expressed as ELISA Units/mL (GMT ± 95% CI) (previously published in (7)).
Garçon
The mode of action of an adjuvant needs to be established to the best of the current knowledge. Together with preclinical safety evaluation, insights into the mode of action allow the design of clinical trials and the safety follow-up during subsequent vaccine development. Despite detoxification, MPL has been shown to retain the capacity to act as an immunostimulant, like the original LPS molecule. The reduced endotoxicity of MPL has been related to its lower aptitude to induce pro-inflammatory cytokines, such as TNF-α, and to its favorable effect on IL-10 synthesis. TNF-α expression in response to MPL was assessed on human CD14+ cell population (Fig. 2.5a). Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were cultured for 6 h in the presence of MPL (10 μg/mL) or LPS (0.1 μg/mL). The frequency of TNF-α-producing CD14+ cells was measured by the combination of intracellular staining and flow cytometry analysis. CD14 is a surface marker associated with TLR-4, essentially found on macrophages, and TNF-α is a pro-inflammatory cytokine. Fixed and permeabilized PBMCs were incubated with anti-CD14 and anti-TNF-α antibodies. This allowed the determination of the percentage of TNF-α-producing CD14+ cells, which is representative of the pro-inflammatory potency of the immunomodulator. Alternatively, TNF-α production was also measured in the U937 bioassay (Fig. 2.5b). For that, a human 50
16000
A
B
14000 40
cytokine production (pg/mL)
3.3. Functional Properties
cytokine producing cells (%)
22
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20
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12000 10000 8000 6000 4000 2000
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control LPS
MPL
control LPS
MPL
Fig. 2.5. Comparative analysis of the production of the pro-inflammatory cytokine TNF-α upon stimulation by LPS or MPL. (A) PBMCs were purified from healthy human adults and cultured in the presence of LPS or MPL. Flow cytometry associated with intracellular staining was used to determine the frequency (%) of TNF-α-producing CD14+ cells (macrophages). (B) Human U937 cells, differentiated into macrophage-like cells, were cultured in the presence of LPS or MPL. TNF-α levels in culture supernatants were measured by ELISA and expressed in picograms per milliliter (previously published in (6)).
Preclinical Development of AS04
23
U937 monocytic cell line (ATCC, CRL-1597.2) was cultured in 10% FCS–RPMI medium (5 × 105 cells/mL) in 24-well culture plates and was differentiated into macrophage-like cells in the presence of phorbol myristate acetate (PMA, 30 ng/mL) for 72 h. After differentiation, the cells were washed with medium, resuspended in the presence of MPL (10 μg/mL) or LPS (0.1 μg/mL), and incubated for 4 h. Quantitation of TNF-α was carried out with ELISA. The intensity of the pro-inflammatory response was approximately 15 times lower with MPL than with the parent molecule. Since MPL is adsorbed onto aluminum salts in the adjuvant formulation, it was necessary to explore whether aluminumadsorbed MPL maintains its ability to act as a TLR-4 agonist, like free MPL. U937 bioassay was used, in conjunction with ELISA. U937 cells were cultured in 10% FCS–RPMI medium (5 × 105 cells /mL) in 24-well culture plates and were differentiated into macrophage-like cells in the presence of PMA (30 ng/mL) for 72 h. After differentiation, the cells were washed with medium and resuspended in the presence of various doses of aluminum hydroxide, MPL, or both (AS04) for 6 h (Fig. 2.6). After incubation, supernatants of the culture plate wells were collected and the concentration of TNF-α determined by ELISA. The results were expressed as relative potency, using an internal 6
Relative potency
5
4
3
2
1
0 10 _
30 _ 100 _
_
_
_
1
3
10
10 1
30 100 Al(OH)3 3 10 MPL
Fig. 2.6. Aluminum-coupled MPL displays the same potency for inducing TNF-α as free MPL. U937 cells were incubated and differentiated into macrophage-like cells in the presence of various doses of aluminum alone, MPL alone, or AS04 (MPL coupled onto aluminum). TNF-α in culture supernatants was measured by ELISA, and results were expressed as relative potency compared to an internal MPL reference (previously published in (6)).
24
Garçon
MPL reference (10 μg/mL) as comparator. It was observed that aluminum-adsorbed MPL displays the same potency for inducing TNF-α than free MPL, which indicates that adsorption of MPL onto aluminum does not impact on its immunological properties.
4. The Added Value of AS04 in the Clinic
The selection of AS04 as adjuvant for the HPV-16/18 cervical cancer vaccine was based on clinical studies in which a higher antibody response was consistently observed against the vaccine antigens formulated with AS04, compared with aluminum hydroxide or non-adjuvanted formulations. The induction of higher antibody levels by AS04 reflected the agonistic TLR-4 activity of MPL, in addition to the antibody-inducing effect of aluminum, highlighting the benefit of using combination of molecules in an Adjuvant System. In these clinical studies, volunteers received three intramuscular administration of HPV-16 and HPV-18 L1 VLP antigens formulated with either AS04 (aluminum hydroxide + MPL) or aluminum hydroxide alone. Blood samples were taken at different time points and the levels of anti-HPV-16 and antiHPV-18 antibodies were measured by ELISA. Briefly, microwell titer plates were coated with either purified recombinant L1 VLP-16 (210 ng/100 μL) or L1 VLP-18 (270 ng/100 μL) overnight at 4◦ C. After blocking of the active sites, serial dilutions of the human sera were added to the wells, followed, after washing, by peroxidase-conjugated goat anti-human IgG. Bound IgG were revealed by addition of tetramethylbenzidine, a peroxidase substrate, which causes a colorimetric reaction; this reaction was stopped with sulfuric acid and quantified by optical density measurements at 450 and 620 nm (Fig. 2.7). Vaccination must not only elicit immediate high titers of antibody but also afford protection over the long term. Persistent serum antibody levels after vaccination reflect the generation of antigen-specific memory B cells. In this respect, the added value of MPL in eliciting memory B cells was investigated on the same panel of volunteers as above. The quantification of antigenspecific memory B cells was performed with a memory B-cell ELISPOT, adapted from the assay developed by the Lanzavecchia laboratory (17). Briefly, PBMCs were purified from blood samples and plated into culture plates in the presence of CpG (3 μg/mL), which induces the pool of memory B cells to differentiate into antibody-producing plasma cells. Among the pool, HPV-16- and HPV-18-specific plasma cells were detected by plating the cultures onto HPV-16 or HPV-18 pre-coated 96-well filter plates (ELISPOT plates). In wells containing anti-HPV-16
Preclinical Development of AS04
GMT antibody titers (EU/ml)
10000
6000
anti-HPV16
*
anti-HPV18 *
5000
8000 Al(OH)3 AS04
6000
25
4000
Al(OH)3 AS04
*
3000 4000
*
*
2000 *
2000
*
0
8
*
*
*
*
*
0
*
1000 0
16
24
32
Time (months)
40
48
0
8
16
24
32
40
48
Time (months)
Fig. 2.7. AS04 Adjuvant System induces a higher and longer lasting antibody response to HPV-16/18 L1 VLP antigens in humans. In two separate clinical trials, human subjects were vaccinated with HPV-16 and HPV-18 L1 VLPs adjuvanted with AS04 or with aluminum salt alone. Antibody responses against HPV-16 or HPV-18 L1 VLPs were evaluated by ELISA at several time points and expressed as geometric mean titers (GMT) in ELISA Units/mL. Significant differences (p < 0.05) between the antibody titers of the AS04 and the aluminum salt group are indicated by asterisks (n = 9–19 subjects for the aluminum salt group, n = 21–37 subjects for the AS04 group). Arrows indicate vaccination time points (previously published in (7)).
or anti-HPV-18-producing plasma cells, the antibodies directly bound the coated antigens. After washing the cells, bound antibodies are locally detected with a biotinylated mouse anti-human IgG, followed by peroxidase-labeled avidin and the addition of a peroxidase substrate to obtain a colored spot. Quantification of the number of spots occurred in an automated ELISPOT plate reader. Results are expressed as the frequency or percentage of HPV-16- or HPV-18-specific antibody-producing plasma cells (reflecting the specific memory B cells) among the total IgGproducing plasma cell population in PBMC, as determined in parallel with a control ELISPOT assay for total IgG. The results (Fig. 2.8) show that AS04-adjuvanted antigens elicit much higher levels of memory B cells than when adjuvanted with aluminum hydroxide alone. This can be attributed to the direct effect of MPL on the APCs, which induces an adequate cascade stimulation to induce long-term immune responses.
5. Conclusions Over the last decades, GSK Bio has accumulated a unique experience in combining immunologically active molecules to develop the family of Adjuvant Systems, each member being specifically designed for a given antigen and a given population. AS04 is one of the Adjuvant Systems developed by GSK Bio. Preclinical investigations showed that the presence of AS04 in a vaccine greatly enhances the production of antibodies against the vaccine antigen, with no safety issue. AS04 also induces
Frequency of HPV16/18-specific memory B cells
26
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HPV16
16000
HPV18
3000 (13)*
(14) Q3
12000 2000 8000 (11)
1000 4000 (21) (6)
0
(8)
pre
Median
(22) (12)
(11)
(13)
(5)
Q1
(9)
0 day 60
day 210
pre
Al(OH)3
day 60
day 210
AS04
Fig. 2.8. Frequency of HPV-16- and HPV-18-specific memory B cells in humans. Subjects from two separate clinical trials were vaccinated with HPV-16 and HVP-18 L1 VLPs adjuvanted with AS04 or aluminum salt alone. Memory B-cell responses directed against HPV-16 or HPV-18 L1 VLPs were quantified by ELISPOT at two time points post vaccination. Results are represented as the frequency of HPV-16- or HPV-18-specific memory B cells per 106 PBMCs. The number of subjects is given in parenthesis. Asterisk represents significant difference between the aluminum and the AS04 group (p < 0.05) (previously published in (7)).
high levels of memory cells, which accounts for sustained production of specific antibodies. These characteristics are found particularly in CervarixTM , the GSK Bio HPV-16 and HPV18 AS04-adjuvanted cervical cancer vaccine, that has demonstrated long-term efficacy against HPV-16 and HPV-18 infection and associated cervical lesions (18, 19), but also in FENDrixTM , an AS04-adjuvanted hepatitis B vaccine destined to pre- and hemodialysis patients, able to induce high and persistent-specific antibody responses (20, 21). The use of AS04 in other candidate vaccines is currently under investigation.
Acknowledgments The author is thankful to Ulrike Krause and Pascal Cadot for their assistance in the preparation of the chapter. FENDrix and Cervarix are trade marks of the GlaxoSmithKline group of companies. References 1. Takeda, K., Akira, S. (2005) Toll-like receptors in innate immunity. Int Immunol 17(1), 1–14. 2. Beutler, B., Jiang, Z., Georgel, P., et al. (2006) Genetic analysis of host resistance:
Toll-like receptor signaling and immunity at large. Annu Rev Immunol 24, 353–389. 3. McKee, A. S., Munks, M. W., Marrack, P. (2007) How do adjuvants work? Impor-
Preclinical Development of AS04
4.
5. 6.
7.
8.
9.
10.
11.
12.
13.
tant considerations for new generation adjuvants. Immunity 27(5), 687–690. Glenny, A. T., Buttlem, G. A. H., Stevens, M. F. (1931) Rate of disappearance of diphtheria toxoid injected into rabbits and guinea pigs: toxoid precipitated with alum. J Pathol 34, 267–275. Ramon, G. (1926) Procédés pour accroître la production des antitoxines. Ann Inst Pasteur 40, 1–10. Garçon, N., Van Mechelen, M., Wettendorff, M. (2006) Development and evaluation of AS04, a novel and improved immunological adjuvant system containing MPL and aluminium salt, in (Schijns V., O’Hagan D., eds.) Immunopotentiators in Modern Vaccines. Elsevier Academic Press, London, pp 161–177. Giannini, S. L., Hanon, E., Moris, P., et al. (2006) Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only. Vaccine 24(33–34), 5937–5949. Myers, K. R., Truchot, A. T., Word, J., Hudson, Y., Ulrich, J. T. (1990) A critical determinant of lipid A endotoxic activity, in (Nowotny A., Spitzer J. J., Ziegler E. J., eds.) Cellular and Molecular Aspects of Endotoxin Reactions. Elsevier Science Publishing Co., New York, pp 145–156. Johnson, D. A., Keegan, D. S., Sowell, C. G., et al. (1999) 3-O-Deacyl monophosphoryl lipid A derivatives: synthesis and immunostimulant activities. J Med Chem 42(22), 4640–4649. Hirschfeld, M., Ma, Y., Weis, J. H., Vogel, S. N., Weis, J. J. (2000) Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine Tolllike receptor 2. J Immunol 165(2), 618–622. Tapping, R. I., Akashi, S., Miyake, K., Godowski, P. J., Tobias, P. S. (2000) Toll-like receptor 4, but not Tolllike receptor 2, is a signaling receptor for Escherichia and Salmonella lipopolysaccharides. J Immunol 165(10), 5780–5787. Iwanaga, S., Morita, T., Harada, T., et al. (1978) Chromogenic substrates for horseshoe crab clotting enzyme. Its application for the assay of bacterial endotoxins. Haemostasis 7(2–3), 183–188. Nakamura, S., Morita, T., Iwanaga, S., Niwa, M., Takahashi, K. (1977) A sensi-
14.
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18.
19.
20.
21.
22.
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tive substrate for the clotting enzyme in horseshoe crab hemocytes. J Biochem 81(5), 1567–1569. European Pharmacopea. (1971). Test for pyrogens, Vol. 11. Published under the direction of the Council of Europe, Maisonneuve SA, Sainte-Ruffine, France, pp 58–60. The European Medicines Agency (EMEA). (2005) Committee for medicinal products for human use (CHMP). Guideline on adjuvants in vaccines for human use. (EMEA/CHMP/VEG/134716/2004) (http://www.emea.europa.eu/pdfs/human/ vwp/13471604en.pdf). World Health Organization. (2005) Guidelines on nonclinical evaluation of vaccines. WHO Technical Report Series No. 927, Annex 1, pp 31–63 (http://www.who.int/ biologicals/publications/trs/areas/vaccines/ nonclinical_evaluation/en/). Bernasconi, N. L., Traggiai, E., Lanzavecchia, A. (2002) Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298(5601), 2199–2202. Harper, D. M., Franco, E. L., Wheeler, C., et al. (2004) Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet 364(9447), 1757–1765. Harper, D., Gall, S., Naud, P., et al. (2008) Sustained immunogenicity and high efficacy against HPV-16/18 related cervical neoplasia: long-term follow up through 6.4 years in women vaccinated with CervarixTM (GSK’s HPV 16/18 AS04 candidate vaccine). Gynecol Oncol 109(1), 158–159. Kong, N. C. T., Beran, J., Kee, S. A., et al. (2008) A new adjuvant improves the immune response to hepatitis B vaccine in hemodialysis patients. Kidney Int 73(7), 856–862. Beran, J. (2008) Safety and immunogenicity of a new hepatitis B vaccine for the protection of patients with renal insufficiency including pre-haemodialysis and haemodialysis patients. Expert Opin Biol Ther 8(2), 235–247. Kool, M., Pétrilli, V., De Smedt, T., Rolaz, A., Hammad, H., van Nimwegen, M., Bergen, I. M., Castillo, R., Lambrecht, B. N., Tschopp, J. (2008) Cutting edge: Alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome. J Immunol 181(6), 3755–3759, Sep 15.
Chapter 3 Nonclinical Safety Assessment of Vaccines and Adjuvants Jayanthi J. Wolf, Catherine V. Kaplanski, and Jose A. Lebron Abstract To ensure the safe administration of vaccines to humans, vaccines (just like any new chemical entity) are evaluated in a series of nonclinical safety assessment studies that aim at identifying the potential toxicities associated with their administration. The nonclinical safety assessment of vaccines, however, is only part of a testing battery performed prior to human administration, which includes (1) the evaluation of the vaccine in efficacy and immunogenicity studies in animal models, (2) a quality control testing program, and (3) toxicology (nonclinical safety assessment) testing in relevant animal models. Although each of these evaluations plays a critical role in ensuring vaccine safety, the nonclinical safety assessment is the most relevant to the evaluation in human clinical trials, as it allows the identification of potential toxicities to be monitored in human trials, and in some cases, eliminates candidates that have unacceptable risks for human testing. This review summarizes the requirements for the nonclinical testing of vaccines and adjuvants needed in support of all phases of human clinical trials. Key words: Vaccine, safety, adjuvant, nonclinical safety assessment.
1. Introduction There is probably no other type of medicinal product that is responsible for saving lives to the same extent as vaccines, which have been credited for saving hundreds of millions of lives over the past 100 years. Despite all the therapeutic advances in medicine, vaccines remain an integral part of the diseasefighting arsenal by preventing and/or ameliorating the effects of a gamut of infectious diseases. Given the success of vaccines, many companies and government institutes are working on developing new vaccines. Unlike small molecule drugs, vaccines are one of the most diverse type of medicinal products including purified and recombinant proteins, polysaccharide preparations, G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_3, © Springer Science+Business Media, LLC 2010
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plasmid DNA, recombinant viruses, virus-like particles, living irradiated cells, synthetic peptides, and attenuated or live organisms—viruses, bacteria, or parasites. Vaccines are often formulated with adjuvants, which are substances that enhance the immune response induced by the vaccine antigens. Like vaccines, adjuvants also encompass a range of products including inorganic salts (e.g., aluminum-based adjuvants), oligonucleotides (e.g., CpG DNA sequences), oil emulsions (e.g., MF59), and R saponin-based mixtures (e.g., QS-21 and ISCOMATRIX ). More recently, vaccine administration is being evaluated using novel delivery devices, which include needleless injectors, electroporation, microneedles, and nanoparticles. However, regardless of the considerable variety in vaccine formulation and delivery methods, the nonclinical safety assessment of vaccines follows a common approach aimed at evaluating their potential for local and systemic toxicity. This review focuses on summarizing the regulatory expectations and types of toxicology studies available for the nonclinical safety assessment of vaccines and adjuvants, concluding with a discussion on how to interpret the toxicology data and determine its implications for human safety.
2. Regulatory Considerations Several regulatory guidelines have been published (Table 3.1) which provide information about the nonclinical safety assessment studies that need to be performed for new vaccines and adjuvants. Nonclinical safety assessment programs are usually designed on a case-by-case basis, since some vaccine types under development require special considerations. The addition of a novel adjuvant in vaccine formulations needs to be justified in preclinical studies in order to demonstrate that inclusion of the adjuvant will provide a benefit to immunogenicity and/or efficacy. Dose–response studies in animals are performed to select the vaccine antigen and adjuvant concentrations to be tested in subsequent nonclinical and clinical studies. Toxicology studies for vaccines with adjuvants are designed to evaluate the safety profile of the adjuvant and adjuvant/vaccine combination. The World Health Organization (WHO) (1) and European Medicines Agency (EMEA) (2) have published guidelines on the overall nonclinical safety evaluation of vaccines. The EMEA has published a comprehensive guideline for vaccine adjuvants which covers aspects of nonclinical and clinical testing for new adjuvants (3). In addition to these and other regulatory guidelines for vaccines (Table 3.1), nonclinical safety assessment studies need to be performed in compliance with Good Laboratory Practices (GLP) regulations as detailed in 21 CFR Part 58 (4).
Guideline on Adjuvants in Vaccines for Human Use (3) Note for Guidance on Pharmaceutical and Biological Aspects of Combined Vaccines (7) Note for Guidance on the Quality, Preclinical and Clinical Aspects of Gene Transfer Medicinal Products (8) Points to Consider on the Manufacture and Quality Control of Human Somatic Cell Therapy Medicinal Products (9)
Adjuvanted vaccines Combination vaccines Viral vector and DNA vaccines Cell-based vaccines
(continued)
Note for Guidance on Preclinical Pharmacological and Toxicological Testing of Vaccines (2)
All vaccines
European Medicines Agency (EMEA)
ICH S6: Preclinical Safety Evaluation of BiotechnologyDerived Pharmaceuticals (6)
Recombinant vaccines (although it is more relevant for other biologics)
Guidelines for Assuring the Quality and Nonclinical Safety Evaluation of DNA Vaccines (5)
DNA vaccines
International Conference on Harmonization (ICH)
WHO Guidelines on Nonclinical Evaluation of Vaccines (1)
All vaccines
World Health Organization (WHO)
Guideline
Vaccine class
Regulatory agency
Table 3.1 Guidelines for the nonclinical safety assessment of vaccines Nonclinical Safety Assessment of Vaccines and Adjuvants 31
Guidance for Industry. Considerations for Developmental Toxicity Studies for Preventative and Therapeutic Vaccines for Infectious Disease Indications (11) Guidance for Industry: Considerations for Plasmid DNA Vaccines for Infectious Disease Indications (12) Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy (13) Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology (14)
Vaccines for women of childbearing potential DNA vaccines Viral vector and cell-based vaccines Recombinant protein/peptide vaccines
United States Food and Drug Administration (FDA)
21 CFR Part 610: General Biological Product Standards (10)
All biological products
United States Code of Federal Regulations (US CFR)
Guideline
Vaccine class
Regulatory agency
Table 3.1 (continued)
32 Wolf, Kaplanski, and Lebron
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33
3. Toxicology Studies Performed for Vaccines 3.1. Selection of Animal Models
Unlike toxicology programs for new chemical entities which require two species, one rodent and one nonrodent, the toxicology program for vaccines typically use a single species which must be demonstrated to be a relevant animal model based on the immunogenicity or efficacy of the vaccine in the selected species. In general, vaccine toxicology programs use either rodents (rats or mice) or non-rodents (rabbits). Non-human primates are only used if there is no other option; for example, for a therapeutic vaccine, non-human primates might be the only species that has homology with the human antigen. The size of the treatment group in toxicology studies depends on the species that is chosen. Generally, 10 animals/gender/group/necropsy time points are used for studies with rodents, whereas fewer animals per group are used in studies with larger animals.
3.2. Immunogenicity Evaluation
The immunogenicity of the vaccine candidate is typically evaluated within the toxicology study, as recommended in the WHO guideline (1). The measure of the expected immune response allows a demonstration of the exposure to the vaccine and confirms the relevance of the animal model for evaluating the potential toxicity of the vaccine. In addition, this measure might help the toxicologist with data interpretation, particularly in correlating any observed toxic effects with the degree of the specific immune response to the vaccine. The evaluation of the immune response to the vaccine relies on immunoassays that are developed in order to measure the most relevant endpoint, i.e., antibody response or cellular immune response. For the measure of specific antibodies, standard ELISA formats are now frequently replaced by multiplex assays for multiple antigens vaccine candidates. The simultaneous evaluation of multiple antigen-specific antibody responses in a single serum R sample is achieved using technologies such as Luminex or elecR trochemiluminescence detection (ECLA, e.g., MSD ) (15). When the candidate vaccine targets the cellular arm of the immune response, assays measuring cytokine-secreting antigenspecific T lymphocytes (such as γ-interferon ELIspot (16)) allow the evaluation of the most relevant endpoint. However, these assays are quite resource intensive, since peripheral blood mononuclear cells collected from animals administered the candidate vaccine need to be stimulated ex vivo with the corresponding peptide antigen(s) and the number of cytokine-secreting T lymphocytes is evaluated.
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3.3. Toxicology Studies
Vaccine toxicology studies evaluate the inherent toxicity of the vaccine formulation, which includes any adjuvants and excipients, in addition to evaluating any toxicity that might be due to the vaccine immune response. The types of toxicology studies that could be performed are listed below; however, since vaccine safety assessment programs are designed on a case-by-case basis not all of these studies will need to be performed. The vaccine formulation used in toxicology studies should be representative of the proposed clinical formulation; therefore, for adjuvanted vaccines the vaccine and adjuvant are tested together. The adjuvant alone can be included as a control group in these studies, but is usually not tested in separate studies unless it is a new synthetic adjuvant. In the latter case, the new adjuvant is regarded as a new chemical entity and requires single- and repeat-dose toxicity studies in two species (one rodent and one nonrodent), assessment of genotoxicity, and possibly other tests (3). More details on considerations for new synthetic adjuvants are provided in Section 3.4.
3.3.1. Single-Dose Toxicity Studies
The purpose of single-dose toxicity studies is to determine the acute effects of a vaccine by evaluating parameters such as mortality, clinical signs, body weight, and food consumption. Since single-dose evaluations can be included within a repeat-dose toxicity study, separate single-dose toxicity studies are typically not performed for vaccines intended for clinical use in a repeat-dose schedule.
3.3.2. Repeat-Dose Toxicity Studies
Repeat-dose toxicity studies evaluate the effects of repeated administrations of the vaccine in animals. The same route of administration as the clinical route is used in the animal study, and usually one more dose is administered in animals when compared with the number of doses in the clinical regimen (this is sometimes referred to as the “N+1” rule (17)). The full human dose of the vaccine needs to be tested, or when not possible due to formulation constraints, then the maximum feasible dose should be administered into the selected animal species. An adjuvant-alone control group or saline control group could be included in the study as a concurrent control. Antemortem parameters evaluated include mortality, physical signs, body weights, food consumption, ophthalmic examinations, urinalyses, hematology, serum biochemistry, coagulation, and immunogenicity assessments as described in Section 3.2. Necropsies are usually performed at two time points, a few days (e.g., 2–7 days) after the last vaccine dose (to determine the early effects after vaccine dosing) and 2–4 weeks after the last vaccine dose (to detect any delayed toxicity and determine whether any detected effects have resolved over time). Postmortem evaluations include gross examination of all major organs, organ weights for selected organs, and histopathology
Nonclinical Safety Assessment of Vaccines and Adjuvants
35
on a complete list of tissues (following the WHO vaccine guidance) (1). Histopathological examination is usually focused on the pivotal organs (brain, kidneys, liver, reproductive organs), immune organs (spleen, thymus, draining lymph nodes), and the site of vaccine administration (e.g., quadriceps at the injection site and skin over the quadriceps, if the vaccine is administered intramuscularly) (1). In cases where there are specific theoretical safety concerns, additional parameters may be included in the study. For example, potential pathogenic autoimmune responses against a particular tissue could be evaluated by more detailed immunohistochemistry. When using an oligonucleotide-based adjuvant, the development of anti-DNA or anti-RNA antibodies could be evaluated. Potential systemic inflammatory responses could be evaluated by examining inflammatory biomarkers such as serum IL-6. Treatment-related effects that are typically observed in vaccine repeat-dose toxicity studies include inflammation at the site of injection, enlargement and hyperplasia of lymph nodes draining the injection sites, increase in spleen weight, and hematological and serum biochemical changes (such as increases in white blood cells, increases in serum globulin, and decreases in serum albumin). These observations are regarded as the intended immunological and inflammatory responses to the vaccine and are not considered adverse effects unless unusually severe. For vaccines, in most cases the highest dose level tested (full human dose) is the No-Observed-Adverse-Effects-Level (NOAEL). However, with the addition of novel adjuvants that might cause severe reactogenicity, more than one vaccine/adjuvant dose levels could be tested in the toxicology study such that a NOAEL can be determined. 3.3.3. Local Tolerance Studies
The purpose of local tolerance studies is to evaluate potential irritation at the injection site both macroscopically and histopathologically. In the interest of reducing animal use, an assessment of local tolerance can be performed within the repeat-dose toxicity study.
3.3.4. Safety Pharmacology Studies
The purpose of safety pharmacology studies is to evaluate the potential for undesirable effects of a substance on physiological functions, particularly on the cardiovascular system, respiratory system, and central nervous system (18). Separate safety pharmacology studies are generally not performed for vaccines (17). Safety pharmacology evaluations, such as body temperature, electrocardiogram, and central nervous system evaluations, could be incorporated into the repeat-dose toxicity study (1), if needed.
3.3.5. Developmental and Reproductive Toxicity Studies
Developmental and reproductive toxicity studies are needed for vaccines that will be administered to women of childbearing potential, since these studies provide information on potential
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effects of the vaccine on fertility, fetal development, and postnatal development of the offspring (11). According to the US FDA guideline on vaccine developmental toxicity studies (11), for vaccines indicated for women of childbearing potential, subjects may be included in clinical trials without developmental toxicity studies, provided appropriate precautions are taken to avoid vaccination during pregnancy. Data from the developmental toxicity studies can then be supplied with the Biologics License Application submission. In the developmental toxicity study, female animals are immunized a few weeks before mating in order to ensure peak immune responses during the critical phases of pregnancy (e.g., organogenesis). Vaccine booster doses are then administered during gestation (embryo-fetal period) and lactation (postnatal period) to evaluate potential direct embryotoxic effects of the components of the vaccine formulation and to maintain an immune response throughout the remainder of gestation. If an adjuvant is included in the vaccine, an adjuvant-alone control group could also be included. 3.3.6. Genotoxicity Studies
According to the “WHO Guidelines on Nonclinical Evaluation of Vaccines” (1) and the EMEA “Note for Guidance on Preclinical Pharmacological and Toxicological Testing of Vaccines” (2), genotoxicity studies are generally not required for vaccines. For adjuvants of biological origin, genotoxicity studies might not be regarded as relevant (6). However, for synthetic adjuvants, which are considered to be new chemical entities, the standard tests that are used to assess the potential for gene mutation, chromosome aberrations, and primary DNA damage are needed (19).
3.3.7. Carcinogenicity Studies
According to the “WHO Guidelines on Nonclinical Evaluation of Vaccines” (1) and the EMEA “Note for Guidance on Preclinical Pharmacological and Toxicological Testing of Vaccines” (2), carcinogenicity studies are generally not required for vaccines. Carcinogenicity studies are also not needed for adjuvants. According to the EMEA “Guideline on Adjuvants in Vaccines for Human Use” (3), adjuvants are intended to be used only a few times with low dosages such that the risk of induction of tumors by these compounds is very small.
3.3.8. Other Toxicity Studies
Specialized toxicity studies are needed for certain types of vaccines. For example, virulence and neurovirulence studies may be needed for new live attenuated virus vaccines that have either a theoretical or an established potential for reversion of attenuation (20) or neurotropic activity (1). Note that the current vaccine strains of measles, mumps, and varicella viruses that have a good safety record should not require re-evaluation in the neurovirulence test(s) when there are minimal changes to seed lots or to manufacture (20). Biodistribution studies, and in some cases integration studies, are performed for nucleic acid and viral
Nonclinical Safety Assessment of Vaccines and Adjuvants
37
vector-based vaccines to determine the tissue distribution following administration and the potential for the vector to integrate into the host genome (5, 12). 3.4. Toxicology Studies Required for Adjuvants Alone
4. Evaluation of Toxicology Data and Risk Assessment
New chemically synthesized adjuvants are regarded as new chemical entities and require a separate toxicological testing program, in addition to their evaluation as part of the vaccine formulation in the tests described in Section 3.3. Separate tests required for novel chemical adjuvants include single- and/or repeat-dose toxicity studies in two species, one rodent and one nonrodent, unless a specific scientific rationale can be provided for testing in a single species (3). Since adjuvants may stimulate the immune system, tests for the induction of systemic hypersensitivity in appropriate models might be needed, although no test is really considered predictive. For synthetic chemical-based adjuvants, an assessment of genotoxicity is needed using the standard battery of tests (e.g., potential for gene mutation, chromosome aberrations, and primary DNA damage) (19). Pharmacokinetic studies (e.g., determining serum concentrations of antigens) are not required for vaccines (1, 2); however, these studies might help in further understanding the mechanism of action of the adjuvant. Additionally, an in vivo test for pyrogenicity might be needed, with the possibility of using alternative in vitro tests for fever-inducing substances if such tests are validated (3). The results from toxicology studies of the adjuvant alone could be included in a separate Drug Master File that is referenced in the Investigational New Drug (IND) application, which contains toxicology data for vaccine adjuvant–antigen combinations.
The goals of the nonclinical safety assessment program are to identify (1) a safe starting dose in the human clinical trials (2), potential toxicities and their reversibility, and (3) target organs of toxicity. The next step after the toxicological studies are completed is to interpret the results with these goals in mind and taking into consideration the intended indication, the target population (e.g., infants, adults), the margin-of-safety (MOS), and the limitations, if any, in extrapolating the results in animals to human safety. The intended indication plays a critical role in the risk/benefit evaluation and can be classified into two major categories— prophylactic and therapeutic. Prophylactic vaccines, for example, influenza, rotavirus, and hepatitis B vaccines, are designed to prevent disease. These types of vaccines are generally administered to healthy individuals, most of which are infants and
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children. In contrast, therapeutic vaccines, such as vaccines for cancer and Alzheimer’s disease, are designed to take advantage of the patient’s immune system to treat a disease already manifested in the patient. Thus, given that the targeted population of prophylactic and therapeutic vaccines are different (healthy vs. disease), and that tolerance for risk is directly proportional to the disease severity, the expectation is that prophylactic vaccines have minimal adverse effects and higher MOS, while some adverse effects might be tolerated with therapeutic vaccines, with smaller MOS. Generally, the safe starting human dose for vaccines corresponds to the NOAEL identified in the repeat-dose toxicity study. For vaccines, the MOS is usually expressed as the fold excess by body weight between humans and the animal species since vaccines are generally given to animals at a full human dose regardless of the animal body weight. For example, if a full human dose of a vaccine is administered into a 0.25 kg rat, and the vaccine is intended for adult individuals (assume an average body weight of 70 kg), then the MOS based on body weight (assuming the human dose evaluated in rats is the NOAEL) will be calculated as the ratio between the human weight and the rat weight since both will be receiving the full human dose; thus, 70 kg/0.25 kg = 280-fold MOS based on body weight. Another consideration when extrapolating toxicology results to potential human risk is the animal model used for the toxicology studies. For example, when vaccine adjuvants that are tolllike receptor 9 (TLR-9) agonists are evaluated in rodents, the adjuvant may cause toxicities resulting from an overstimulation of the immune system (exaggerated pharmacology). These toxicities may be more pronounced in rodents than in non-human primates or humans, which have a more limited distribution of TLR-9. Thus, the animal model used for the toxicology study needs to be taken into account when developing the overall vaccine risk assessment. Finally, note that the assignment of the risk/benefit ratio of the vaccine needs to be continuously refined as new data from the clinic and additional nonclinical studies becomes available.
5. Conclusion In this review we highlighted the regulatory expectations and types of toxicology studies available for the nonclinical safety assessment of vaccines and provided a discussion on the interpretation of the toxicology data and its implications for human safety. We also discussed how the nonclinical safety assessment strategy
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is impacted by the type of indication, and whether the vaccine uses novel adjuvants. Although use of these novel approaches promises to make available vaccines currently not available to prevent serious diseases such as HIV and malaria, increasing safety concerns on the use of these novel technologies have resulted in increased regulatory requirements for both the nonclinical and the clinical development aspects of vaccines. The ultimate goal of vaccine developers should be to develop safe and efficacious vaccines. Although the nonclinical safety assessment is an integral part toward achieving this goal, it is important to remember that ensuring the safety of vaccines is a multi-component approach that includes (1) the evaluation of vaccines in animal models of efficacy and immunogenicity, (2) a quality control testing program, (3) nonclinical safety assessment studies, and (4) clinical studies. This multi-component approach is what ultimately will support the marketing application and eventual licensure of the vaccine.
Acknowledgments The authors would like to thank Dr. Brian Ledwith and Dr. Tom Monticello for reviewing this chapter. References 1. WHO Guidelines on Nonclinical Evaluation of Vaccines. (2003) WHO/BS/03.1969. WHO, Geneva, Switzerland. 2. Note for Guidance on Preclinical Pharmacological and Toxicological Testing of Vaccines. (1997) EMEA. CPMP/SWP/465/95. 3. Guideline on Adjuvants in Vaccines for Human Use. (2005) EMEA. EMEA/CHMP/VEG/134716/2004. 4. Good Laboratory Practice Regulations. (2009) Code of Federal Regulations, Title 21, Part 58 (21 CFR 58). 5. Guidelines for Assuring the Quality and Nonclinical Safety Evaluation of DNA Vaccines (2005) WHO, Geneva, Switzerland. 6. ICH S6: Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals. (1997) International Conference on Harmonization. 7. Note for Guidance on Pharmaceutical and Biological Aspects of Combined Vaccines. (1998) EMEA. CPMP/BWP/477/98. 8. Note for Guidance on the Quality, Preclinical and Clinical Aspects of Gene Trans-
9.
10. 11.
12. 13. 14.
fer Medicinal Products. (2001) EMEA. CPMP/BWP/3088/99. Points to Consider on the Manufacture and Quality Control of Human Somatic Cell Therapy Medicinal Products. (2001) EMEA. CPMP/BWP/41450/98. General Biological Products Standards. (2009) Code of Federal Regulations, Title 21, Part 610 (21 CFR 610). Guidance for Industry. Considerations for Developmental Toxicity Studies for Preventative and Therapeutic Vaccines for Infectious Disease Indications. (2006) CBER, FDA. Guidance for Industry: Considerations for Plasmid DNA Vaccines for Infectious Disease Indications. (2007) CBER, FDA. Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy. (1998) CBER, FDA. Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology. (1985) CBER, FDA.
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15. Opalka, D., Lachman, C. E., MacMullen, S. A., et al. (2003) Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexed luminex assay. Clin Diagn Lab Immunol 10, 108–115. 16. Casimiro, D. R., Tang, A., Perry, H. C., et al. (2002) Vaccine-induced immune responses in rodents and nonhuman primates by use of a humanized human immunodeficiency virus type 1 pol gene. J Virol 76, 185–194. 17. Gruber, M. F. (2003) Non-clinical safety assessment of vaccines, in CBER Counter Terrorism Workshop, Bethesda, MD.
18. ICH. (2000) Safety Pharmacology Studies for Human Pharmaceuticals. Topic S7A, Step 5, ICH Harmonized Tripartite Guideline. International Conference on Harmonization, Geneva, Switzerland. 19. ICH. (1997) S2B Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. International Conference on Harmonization, Geneva, Switzerland. 20. The Organisers, IABs. (2006) IABs scientific workshop on neurovirulence tests for live virus vaccines. Biologicals 34, 233–236.
Chapter 4 Aluminum Adjuvants: Preparation, Application, Dosage, and Formulation with Antigen Erik B. Lindblad and Niels E. Schønberg Abstract Important new knowledge about the effect of aluminum adjuvants on the immune response in terms of their impact on cytokine profiles, uptake by antigen-presenting cells (APC), and surface marker expression has been published in recent years. However, although the knowledge about these adjuvants is thus more comprehensive now than ever before, the user is often still confined to a more empirical approach when confronted with practical issues when it comes to the handling and use of these adjuvants. In this chapter we have given focus to the user’s perspective, discussing practicalities like dosage, temperature stability, relevant monographs, and preparation with antigen. Key words: Aluminum hydroxide gel, aluminum phosphate gel, monographs, dosage, autoclaving, freezing, adsorption.
1. Introduction Aluminum compounds have the longest history and by far the most comprehensive record of use as adjuvants in practical vaccination of both animals and humans. Both aluminum hydroxide (AlhydrogelTM ) and aluminum phosphate (Adju-PhosTM ) adjuvants are generally regarded as safe to use in human vaccines when used in accordance with current vaccination schedules (1–3). Vaccine preparations based on adsorption of the antigen onto a preformed aluminum hydroxide or aluminum phosphate adjuvant are referred to as aluminum-adsorbed vaccines (see Note 1). In practical vaccination the adsorption of antigen onto preformed aluminum hydroxide and aluminum phosphate gels has G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_4, © Springer Science+Business Media, LLC 2010
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facilitated standardization of vaccine production and has now almost completely substituted the original old technique where potassium alum was made to co-precipitate with the antigen by exposing the potassium alum + antigen mixture to alkaline conditions (4). The historical and more detailed theoretical background of the aluminum adjuvants have been left out, primarily as they are beyond the scope of this chapter which is meant to provide a more practical approach, but also since they have been reviewed extensively elsewhere (5–9). 1.1. Preparation of Aluminum Adjuvants
Aluminum hydroxide and aluminum phosphate adjuvants are generally prepared by exposing aqueous solutions of aluminum ions to alkaline conditions in a well-defined and controlled chemical environment. Various soluble aluminum salts can be used for the production of aluminum hydroxide, typically aluminum sulfate or chloride is used. However, the experimental conditions— temperature, concentration, and even the rate of addition of reagents—strongly influence the results (10, 11). Anions present at time of preparation may co-precipitate and change the characteristics away from those of “pure” aluminum hydroxide. One very important example of an anion having such influence is phosphate. Aluminum phosphate gel can be seen as an example of such a preparation where the soluble aluminum salts are exposed to alkaline conditions in the presence of sufficient amounts of phosphate ions.
1.2. Application of Aluminum Adjuvants
Aluminum adjuvants have been used in both experimental immunology (12, 13) and to raise murine monoclonals of the IgG1 isotype as well as polyclonal antisera for analytical purposes, immunoprecipitation assays, etc. In veterinary medicine aluminum adjuvants have been used in a large number of vaccine formulations against viral (14–18) and bacterial (19–22) diseases, as well as in attempts to make antiparasite vaccines (23–26). In human vaccination aluminum adjuvants have been primarily used in tetanus, diphtheria, pertussis, and poliomyelitis vaccines as part of standard child vaccination programs for more than 50 years in many countries, including combination vaccines. Later aluminum adjuvants were also introduced in hepatitis A, hepatitis B virus vaccines, and in the new vaccines against human papillomavirus (HPV)-induced cervical cancer. Other aluminumadsorbed vaccines, against, e.g., anthrax and botulinus toxin, are available for special risk groups. Aluminum phosphate has a potential of being applied also in DNA vaccines with promising results, whereas aluminum hydroxide has been shown to inhibit the transcription of the nucleotide in DNA vaccines. The content of phosphate in the DNA molecule
Aluminum Adjuvants
43
apparently gives a high binding affinity of the nucleotide to the aluminum hydroxide, which in turn prevents the host RNA from getting access to and translating the nucleotides into protein (27). One obvious limitation for the application of aluminum adjuvants lies in the clear Th2 profile of these adjuvants with adsorbed protein vaccines. A Th2-biased immune response is not likely to protect against diseases for which Th1 immunity and MHC class I-restricted CTLs are essential for protection, such as with, e.g., intracellular parasites or tuberculosis (28). Another limitation lies in the fact that traditional aluminum-adsorbed vaccines are frost sensitive and therefore not lyophilizable. 1.3. Dosing Aluminum Adjuvants
Although there are no generally accepted limits for the dosages of aluminum adjuvants to be used in experimental immunology, many countries have established local animal ethics committees that have to approve the individual experimental protocol before commencing the experiments. For the use of aluminum adjuvants (and other adjuvants as well) to raise polyclonal antibodies for, e.g., analytical and diagnostic purposes ECVAM (a generally recognized European body engaged in animal ethics) established a set of guidelines in 1999 (29). In the preclinical phase of vaccine development the optimum dose of adjuvant is normally determined empirically in a pilot trial, but helpful guidelines are available in the literature. In veterinary vaccines there is no defined maximum limit for the allowed content of aluminum adjuvants. Here the dose is normally set from a balance between efficacy and local reactogenicity. In vaccines for humans the allowed amount of aluminum in adsorbed vaccines is subject to limitations. These limits are 1.25 mg aluminum per dose in Europe (30) and in USA 0.85 mg aluminum per dose if determined by assay, 1.14 mg if determined by calculation, and 1.25 mg if safety and efficacy data justifies it (31). These limit values refer to aluminum calculated as metallic aluminum and not as the salt.
1.4. Temperature Stability of Aluminum Adjuvants
In practical terms there are two major considerations to be observed here. The first is that aluminum adjuvants are sensitive to freezing. Aluminum hydroxide and phosphate adjuvants are suspensions of hydrated colloid particles (aluminum oxyhydroxide and aluminum hydroxyphosphate, respectively) with a slow sedimentation in water. The slow sedimentation is partly due to the oriented water molecules that are an intrinsic constituent of the adjuvants (giving buoyancy), and partly because the adjuvant particles have a charge that gives electrical repulsion among the particles in the suspension and to some extent prevents packing. If the adjuvant is subjected to freezing then the free supernatant water as well as the oriented water molecules will eventually
Lindblad and Schønberg
freeze, and upon thawing, the water molecules formerly oriented in the gel structure itself will not take up their former position and as a result the gel structure is irreversibly damaged (Fig. 4.1). Aluminum adjuvants thus damaged by freezing lose their protein adsorption capacity (Fig. 4.2). At the other end of the temperature scale autoclaving is commonly applied to achieve sterility of the adjuvant preparations. It is a well-established observation that autoclaving leads to a slight
Fig. 4.1. Two vials of aluminum hydroxide gel adjuvant. The vial to the left has not been exposed to freezing. The vial to the right has been frozen and subsequently thawed. The gel structure in this vial is collapsed and the colloid water released into the supernatant.
30,00 25,00
mg HSA/mL
44
20,00 15,00 10,00 5,00 0,00 0% destruction
25% destruction
75% 50% destruction destruction
100% destruction
Fig. 4.2. Reduction of protein adsorption capacity (HSA onto aluminum hydroxide gel) after controlled partial and complete freezing of the adjuvant prior to incubation with antigen.
Aluminum Adjuvants
45
reduction of protein adsorption as well as a minor reduction of pH in non-buffered adjuvant preparations. Burrell and coworkers investigated the effects of autoclaving on aluminum hydroxide and aluminum phosphate. They found that autoclaving aluminum hydroxide increased the degree of crystallinity as measured by the width at half-length of the major band in the X-ray diffractogram. It was confirmed that the pH decreased during autoclaving, suggesting that deprotonation and dehydration reactions resulted in a reduced surface area as both the protein adsorption and the viscosity decreased following autoclaving. With aluminum phosphate adjuvant the amorphous structure remained after autoclaving; however, also in this case deprotonation and dehydration reactions occurred as evidenced by a decrease in pH. In addition the protein adsorption capacity, rate of acid neutralization at pH 2.5, and PZC also decreased, indicating that the reactions resulted in a decreased surface area (32). 1.5. Relevant Monographs
A number of pharmacopeias and handbooks contain monographs on aluminum hydroxide and aluminum phosphate. However, these may apply to preparations intended for oral intake as antacids, and not for parenteral use as adjuvants. Examples of such monographs are the USP monograph on aluminum hydroxide gel and the Ph. Eur. monographs on hydrated aluminum phosphate no. 1598 and 2166. Such monographs have focus on acid neutralizing capacity and may encompass the option of adding flavors like peppermint oil or eucalyptus to make the taste better. In addition, there is little focus on the sterility of the preparation. Handbook of Pharmaceutical Excipients contain monographs on both aluminum hydroxide and aluminum phosphate for use in vaccines (33, 34). A monograph on aluminum hydroxide specifically for use in adsorbed vaccines was launched by the European Pharmacopoeia in July 2004 (35).
1.6. Formulation with Antigen
As a general guideline for many protein antigens adsorption is best accomplished in the pH interval between the isoelectric point (IEP) of the protein antigen and the point of zero charge (PZC) of the adjuvant, which is the equivalence of the IEP, but for the adjuvant (Fig. 4.3). This applies for both aluminum hydroxide and aluminum phosphate adjuvants. In this interval the adjuvant and the antigen will have opposite electrical charges, facilitating electrostatic attraction and adsorption (36). In the formulation with antigen a pH close to the physiological pH is preferred, i.e., pH 6–8, to reduce vaccination discomfort. Applying this principle aluminum hydroxide is the preferred adjuvant for adsorption of antigens with an acidic IEP and aluminum phosphate is the preferred adjuvant for adsorption of antigens with an alkaline IEP (see Note 2).
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Fig. 4.3. In the pH range between the isoelectric point (IEP) of the antigen and the point of zero charge (PZC) of the mineral adjuvant there is a basis for electrostatic attraction, due to opposite charge. The alkaline PZC for Al(OH)3 makes it suitable for adsorption of acidic IEP proteins as exemplified by albumin, whereas the acidic PZC of AlPO4 makes it suitable for adsorption of alkaline IEP proteins, as exemplified by hen egg lysozyme.
Antigen preparations that contain fragments of phospholipid membranes or phosphorylated proteins may in addition adsorb to aluminum hydroxide in particular by an alternative mechanism, known as ligand exchange (37). However, the same mechanism may reduce adsorption by electrostatic attraction if anions with affinity for ligand exchange with hydroxyl on the surface of aluminum hydroxide, in particular phosphate ions, are used in the formulation (e.g., in buffers) (38). In general incubation for 1–2 h at room temperature or over night at 4◦ C for both aluminum hydroxide and aluminum phosphate adjuvant is recommended to allow adsorption to take place.
2. Materials 2.1. Production of Aluminum Adjuvants 2.1.1. Aluminum Hydroxide ad modus Gupta and Rost
1. 5× aluminum chloride/sodium acetate solution: 0.257 M aluminum chloride and 0.05 M sodium acetate (62.05 g aluminum chloride and 6.8 g of sodium acetate per liter of distilled water). Sterilize by autoclaving or by filtration (0.2 μm) (see Note 3). The pH of the 5× concentrate is between 3 and 4. Store at room temperature (20–30◦ C) (39). 2. 5× sodium hydroxide solution: 0.257 M sodium hydroxide (10.25 g of sodium hydroxide per liter of distilled water). Sterilize by autoclaving or filter sterilization (see Note 3).
Aluminum Adjuvants
47
The pH of the 5× concentrate is between 12 and 14. Store at room temperature (20–30◦ C). 3. Acetic acid (sterilized). 2.1.2. Method for Preparation of Aluminum Phosphate Adjuvant ad modus WHO (40)
1. Sterile distilled water. 2. K1 filter sheets. 3. Aluminum chloride solution: dissolve 3 kg of pure aluminum chloride (AlCl3 ·6H2 O) in distilled water to a final volume of 30 L. The solution is then filtered through a soft (K1) filter sheet. 4. Trisodium phosphate: dissolve 4.73 kg of pure trisodium phosphate dodecahydrate (Na3 PO4 ·12H2 O) in distilled water to a final volume of 30 L. The solution is then filtered through K1 sheets. 5. 0.36% sodium chloride (NaCl) in water (100 L).
2.2. Testing of Aluminum Adjuvants
1. Bunsen burner.
2.2.1. Determination of Aluminum
3. pH meter with combined pH electrode.
2. Precision balance, d = 1 mg. 4. Titriplex solution, pH 6.0: 37.224 g Titriplex III (ethylenediaminetetraacetic acid, analysis quality GR) in H2 O ad 1,000 mL (0.100 M final). Adjust to pH 6.0 using sodium acetate powder. 5. Xylenol orange potassium nitrate mixture: grind together 1 g xylenol orange tetrasodium salt and 99 g KNO3 in a mortar until a fine powder with a homogenous color is obtained. (KNO3 is added to improve the stability.) 7. Zinc sulfate solution (0.100 M): 28.754 g zinc sulfate heptahydrate (ZnSO4 ·7H2 O) analysis quality GR in H2 O ad 1 L. 8. Saturated sodium acetate solution: sodium acetate trihydrate (CH3 COONa·3H2 O) analysis quality GR in water. 9. 32% w/v sodium hydroxide (NaOH) solution: 320 g NaOH in Aqua purificata ad 1,000 mL. 10. Concentrated hydrochloric acid (HCl 37%). 11. Aluminum titrisol standard solution: dilute the contents of one vial of standard liquid [aluminum chloride (AlCl3 ) in H2 O, 1.000 g Al ± 0.002 g (Merck) which contains 1.000 g Al] to 100 mL with Aqua purificata. This solution then contains 10 mg Al per milliliter and has a density of 1.039 g/mL.
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2.2.2. Determination of the Protein Adsorption Capacity
1. Test-tubes: Immuno Tube MiniSorp 12 mL (Biotech Line) 2. Orbit rotator 3. Desiccator with silica gel 4. Bench centrifuge 5. Double beam UV–VIS spectrophotometer 6. Imidazole buffer, pH 6.5: 3.4 g imidazole, 2.2 g NaCl, and 18 mL 1 M HCl in water ad 1,000 mL. Filter through a 0.45 μm filter (Sartorius) and store in a refrigerator 7. HSA standard solution: 200 mg HSA (ICN or Sigma) in 100 mL imidazole buffer 8. BCA Protein Assay Reagent A, 250 mL (Pierce) 9. BCA Protein Assay Reagent B, 25 mL (Pierce) 10. HSA stock solution: 2.0 g HSA (ICN or Sigma) in 200 mL imidazole buffer
3. Methods 3.1. Production of Aluminum Adjuvants
3.1.1. Aluminum Hydroxide ad modus Gupta and Rost
Whereas the production details of the commercially available aluminum adjuvants are subject to confidentiality, a recipe for the production of aluminum hydroxide adjuvant in laboratory scale has been published by Gupta and Rost (39). A method for preparing aluminum phosphate adjuvant has previously been published by WHO (40). 1. Stir the contents continuously during the procedure at 40–60 rpm (39). 2. First add 0.257 M NaOH solution (20% of final volume) to a mixing vessel. 3. Add sterile distilled water (50–55% of final volume). 4. Add 0.257 M aluminum chloride/sodium acetate (AlCl3 /CH3 COONa) solution (20% of final volume) to the mixture at a rate of 1–2 L/min. During the addition of this solution, monitor pH and maintain it between 5.5 and 6.5 (optimal pH for tetanus and diphtheria toxoids) or any other range suitable for a particular antigen. 5. Adjust to the final volume with sterile distilled water. Mix the suspension for 2 h. 6. Adjust the final pH to 5.9–6.1 (for tetanus and diphtheria toxoids) or to the optimal pH with 5 N NaOH or 5 N acetic acid.
Aluminum Adjuvants
3.1.2. Method for Preparation of Aluminum Phosphate Adjuvant ad modus WHO (40)
49
1. Filter 150 L of distilled water through K1 sheets into a container of 300 L capacity. Slowly add the 30 L of AlCl3 solution and mix well. 2. Add the Na3 PO4 solution with good stirring until a pH of 5.0 is reached (about 27–29 L are necessary). 3. Finally, add 30 L of distilled water, mix the suspension well, and leave standing for 7 days. 4. After 7 days siphon off the clear supernatant and add the same volume of sterile distilled water. Mix the suspension well and again leave standing for 7 days. 5. After 7 days again siphon off the clear supernatant, add distilled water to bring the volume to 150 L, and then add 100 L of 0.36% NaCl. The total volume is now 250 L. 6. Distribute the resulting suspension of AlPO4 with continuous mixing into 6 L volumes in 10 L bottles steamed for 30 min and autoclaved for 40 min at 121◦ C. 7. After 2 or 3 days test the sterility of the phosphate suspension. 8. This procedure yields bottles each containing 6 L of AlPO4 suspension with 6 mg of aluminum phosphate per milliliter or 1.32 mg of aluminum per milliliter (see Note 4).
3.2. Testing of Aluminum Adjuvants
This section presents two of the most important tests that are recommended prior to using preformed aluminum adjuvants in vaccine production. Commercial producers of aluminum adjuvants supply a Certificate of Analysis (CoA) with each batch supplied. It is highly recommended that the end user verifies the analytical data of the CoA in their own incoming material control. In addition, the end user should undertake testing for additional critical parameters that may apply to their specific, intended use of the adjuvant, i.e., vaccine-specific requirements (see Note 5). Below we describe the necessary tests for (1) determination of the aluminum concentration in the product (important for the dosing into the vaccine) and (2) a method for quantitative determination of the protein adsorption capacity (in the context of standardization it may be preferable over the pass–not pass limit test of the Ph. Eur. monograph 1664). In the example presented, the test is based on the adsorption of human serum albumin onto aluminum hydroxide, but the principle can be utilized with other proteins as well, including purified toxoids.
3.2.1. Determination of Aluminum
The aluminum content in aluminum hydroxide gel can be determined by titration of Titriplex–EDTA. The test is based on the principles given in USP and European Pharmacopoeia test 2.5.11 (complexometric titrations). The test is carried out in duplicates.
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The result of the test will be given as the mean of the two duplicates. 1. Determination of volume B by measuring precisely 50 mL 0.100 M Titriplex solution, pH 6.0, and adding a small amount of xylenol orange potassium nitrate mixture to produce a clear yellowish brown color. Titrate with 0.100 M ZnSO4 until the yellowish brown changes to pink. The amount (mL) of 0.100 M ZnSO4 used is B. 2. Prepare quantity A by shaking the product bottle well and take a sample of 6 g of the product if you analyze aluminum hydroxide adjuvant (Alhydrogel) or 10 g of the product if you analyze aluminum phosphate adjuvant (Adju-Phos). The exact amount is noted. This amount is hereafter called A. 3. Determine quantity b by transferring the 6 g if analyzing Alhydrogel or 10 g if analyzing Adju-Phos (quantity A) of the product from step 2 above into a 250 mL beaker. As a reference use 6 g of the Titrisol standard solution. Add 50 mL H2 O and 5 mL 32% w/v NaOH solution. In case there should be any Al-containing product on the sides of the beaker, rinse with as little water as necessary. The Adju-Phos should now be a clear solution. Heat Alhydrogel using a Bunsen burner until the solution is clear. Allow the solution to cool down to room temperature and then neutralize the solution by drop-wise addition of concentrated HCl. During this neutralization a precipitate of aluminum oxide forms. Re-dissolve this precipitate by further dropwise addition of concentrated HCl until pH = 1.0. Now add precisely 50 mL of 0.100 M Titriplex solution, pH 6.0, heat until boiling, and then keep warm using a pilot light for approximately 10 min. Allow the solution to cool down to room temperature and adjust the pH to 6.0 using saturated sodium acetate solution. Add a little amount of xylenol orange-KNO3 (to produce a clear yellowish brown color, very little is required). Titrate with 0.100 M ZnSO4 until the yellowish brown color changes to pink. The amount (mL) of 0.1 M ZnSO4 used is b. 4. Calculate mg/mL Al = “C” using the following formula: C = (B − b) × d × M × 27 A where B and b are defined above in the text d = density = approx. 1.03 g/mL for Alhydrogel 2.0%, approx. 1.01 g/mL for Adju-Phos 1.039 g/mL for the aluminum standard solution. M = the molarity of ZnSO4
Aluminum Adjuvants
51
27 = the molecular weight of Al A is defined above in the text. 3.2.2. Determination of the Protein Adsorption Capacity (see Notes 6–8)
Determination of the protein adsorption capacity of the adjuvant is highly recommended and can be measured by a variety of analytical methods (see Notes 7 and 8). It is normally done by comparing the protein content in the aqueous phase of the antigen solution before and after adsorption onto the adjuvant. We use a test based on a modified BCA (bicinchoninic acid) assay. The BCA method was developed in 1985 by Smith et al. in USA (41) and seems in some respect to be superior to other methods for protein determination with respect to sensitivity as well as user friendliness. It is based on the principle that copper ions will, through interaction with peptide bonds, pass from the Cu++ state to the Cu+ state and that Cu+ has a high binding affinity to BCA. The complex will then have a light absorption maximum at 562 nm, i.e., this complex may be used for a photometric protein determination. This test principle has been utilized in the present method, adapted by Lindblad for determining the protein adsorption capacity of Alhydrogel. When the test is carried out, the protein solution should be at room temperature, just as all other applied reagents. All reagents have to be used within 2 days. Prepare a standard curve for every lot of HSA. 1. Plot a standard curve for HSA by preparing the samples of different protein concentration as shown in Table 4.1. For each HSA concentration 0.2 mL is transferred into a test tube. Prepare two tubes with “blanks,” referred to as Sample 0.
Table 4.1 Preparation of the standard row of HSA in imidazol buffer Name
Concentration (mg/mL)
HSA standard solution (mL)
Imidazole buffer (mL)
0 (blank)
0.0
0
10
1
0.2
1
9
2
0.4
2
8
3
0.6
3
7
4
0.8
4
6
5
1.0
5
5
6
1.2
6
4
7
1.4
7
3
8
1.6
8
2
9
1.8
9
1
10
2.0
10
0
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2. Prepare a working solution (W.S.) by mixing 50 mL of Reagent A with 1 mL of Reagent B. Mix thoroughly. Add 4.0 mL W.S. to each tube and mix thoroughly. Incubate samples for 2 h at room temperature. 3. Measure the absorbance at 562 nm for the standards against a blank sample and plot absorbance against HSA concentration. 4. Using the HSA stock solution (2.0 g HSA in 200 mL imidazole buffer) prepare nine samples in 10 mL test tubes [0 (i.e., blank), 1, 2, . . . 8] as shown in Table 4.2.
Table 4.2 For Alhydrogel 2% Preparation of Alhydrogel with HSA prior to adsorption HSA stock solution (mL)
Imidazole buffer (mL)
0
0.00
7.00
1
26
2.60
4.40
2
30
3.00
4.00
3
34
3.40
3.60
4
38
3.80
3.20
5
42
4.20
2.80
6
46
4.60
2.40
7
50
5.00
2.00
8
54
5.40
1.60
Name 0 (blank)
Amount of HSA added (mg)
5. Shake the Alhydrogel flask thoroughly and add 2.0 mL of the Alhydrogel to each of the test tubes and attach them to the wheel of the blood rotator. Let the Alhydrogel adsorb the HSA for at least 1 h by rotating at room temperature. 6. After adsorption, transfer the tubes to the bench centrifuge and spin down by centrifugation for 10 min at 2,550 × g. For each sample 0.2 mL is transferred to a test tube. Avoid transferring any sediment (i.e., fine fractions of Alhydrogel), as this may influence the readings (see Note 9). 7. Prepare a working solution (W.S.) by mixing thoroughly four parts of reagent B with 200 parts of reagent A. For the testing of each batch of Alhydrogel use approximately 40 mL of W.S. 8. To each sample tube, add 4.0 mL W.S. and mix thoroughly. Incubate samples for 2 h at room temperature. 9. Measure the absorbance of the samples at 562 nm. As reference use the imidazole buffer.
Aluminum Adjuvants
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Fig. 4.4. Determining the protein adsorption capacity of aluminum adjuvant (exemplified by adsorption of HSA onto aluminum hydroxide gel). The crossing point of the graph through the measurements with free HSA in the supernatant with the X-axis gives the adsorption capacity for the 2 mL sample taken into analysis. The result is divided by 2 to state mg HSA adsorbed per milliliter Alhydrogel.
10. Calculate the binding capacity of that particular batch (mg HSA adsorbed per milliliter Alhydrogel). The following is an example calculation of the case where the standard curve shows that a sample after adsorption, centrifuging, and filtering has a HSA concentration of 0.225 mg HSA per milliliter. The total volume was 9 mL as the tube from which the sample was taken contained 7 mL sample and 2 mL Alhydrogel. The total amount of free protein after adsorption was thus 0.225 mg × 9 = 2.03 mg. The total amount of protein before adsorption in the same test tube was 38.00 mg. The difference, equivalent to the amount
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adsorbed, is thus (38.00 – 2.03) mg = 36 mg protein, and since it was adsorbed on 2.0 mL Alhydrogel, it corresponds in this single test to an adsorption capacity of 36 divided by 2 (equivalent to 18 mg HSA per milliliter Alhydrogel). This calculation should be made for each of the adsorption samples (see Note 10). The calculated values for free HSA in the supernatant (Y-axis) is plotted against the amount of HSA added to the sample (X-axis) (Fig. 4.4). The adsorption capacity for the 2 mL Alhydrogel added is given by the intercept between a line through the calculated values for free HSA in the supernatant and the X-axis.
4. Notes 1. In the literature the word “alum” is sometimes used to describe both aluminum hydroxide and aluminum phosphate gels, but that is incorrect use of terminology. Potassium alum, KAl(SO4 )2 ·12H2 O, is in accordance with the chemical definition of an alum, whereas neither aluminum hydroxide nor aluminum phosphate is. Although both aluminum hydroxide and aluminum phosphate adjuvants are chemically referred to as Al(OH)3 and AlPO4 , respectively, neither of these formulas should be seen as stoichiometrically true. As a consequence any calculation that takes basis in the formulas mentioned will only be an approximation. 2. Antigens with a distinct polarity in terms of one part of the molecule having a clearly acidic IEP and a distant part of the same molecule having a clearly alkaline IEP may bind well to both, e.g., Al(OH)3 and AlPO4 . But in such cases there may be a difference in the orientation of the adsorbed molecule (42). 3. If filtration is used, test the compatibility of filter membranes with the solution. 4. Cleaning of equipment: aluminum adjuvants may leave a white deposit on stainless steel and aluminum surfaces in tanks and holding vessels upon prolonged exposure or drying. Such deposits are not easy to rinse away with water alone, but can be removed exposing the surfaces to 4–5% NaOH at 80◦ C. 5. The Ph. Eur. monograph 1664 contains a requirement for a Limulus Amoebocyte Lysate (LAL) testing of the aluminum hydroxide adjuvant. The LAL test can be performed either as a clot assay or as a chromogenic assay.
Aluminum Adjuvants
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Although these assays have a long history of use for the purpose neither of the two procedures are particularly well suited for the testing of aluminum hydroxide, due to some intrinsic characteristics of the aluminum adjuvant. The binding affinity of LPS to aluminum hydroxide is well established and was much higher than to aluminum phosphate (43). With the clot assay, the problem is that aluminum hydroxide acts as an adsorbant, meaning that the clotting of reagents, which, in applications where there is no adsorbant present, is specific for the LAL–endotoxin interaction, cannot be deemed as a specific reaction in this particular application. With the chromogenic assay, problems also arise due to the adsorption of endotoxin by the aluminum hydroxide. This adsorption has been measured to be in the magnitude of 283 μg/mL Al (43). Lipid A is typically composed of a glucosamine (GlcN) disaccharide backbone [β-D-glucosaminyl-(1-6)-α-D-glucosamine disaccharide] which carries two ester-bound phosphate groups at positions 1 and 4 , and amide or ester-linked long fatty acids at positions 2, 2 , 3, and 3 (44, 45). The adsorption of endotoxin by aluminum hydroxide gel is due to two mechanisms, which are a direct consequence of the structure of the endotoxin molecule: At physiological pH aluminum hydroxide adjuvant is positively charged, whereas endotoxin typically is negatively charged at pH conditions higher than 2. As a result there is attraction between the positively charged aluminum hydroxide and the negatively charged endotoxin. As described above, the LPS contains phosphate residue that can displace surface hydroxyl from the aluminum hydroxide gel. This process is known as ligand exchange, and the affinity is very high indeed. The impact is profound when, for example, preparing a standard curve by making a dilution series of LPS from, e.g., E. coli. If aluminum hydroxide is present LPS will be taken up by the adjuvant and as a consequence will not be freely accessible in the supernatant, i.e., before the adjuvant is saturated. If aluminum hydroxide is excluded from the LPS dilutions used for the standard curve, good standard curves can be prepared, but cannot be correlated with aluminum hydroxide-containing samples. LPS cannot be determined by “spiking” a sample of aluminum hydroxide with a controlled amount of LPS and then correlating it to the standard curve from the LAL assay. 6. It has been observed that acute toxicity is reduced in adsorbed vaccines as compared to the non-adsorbed antigen preparation. It is conceivable that the acute toxicity is reduced simply by a delayed release of toxic vaccine
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constituents, like pertussis toxin, peptidoglycans from Gram-negative cell walls, or LPS (lipopolysaccharides) from the injection site. 7. ELISA methods have been designed (46) in which aluminum-adsorbed antigens could be used directly as antigens in ELISA assays. ELISA methods were also applied for in vitro assessment of various viral antigens, i.e., pseudorabies, porcine parvovirus, and infectious bovine rhinotracheitis vaccines adsorbed onto aluminum hydroxide adjuvant (47). 8. In a complex mixture of antigens some individual components may adsorb to the adjuvant to a larger extent than others, meaning that what is actually adsorbed is not reflecting the composition of the original complex solution quantitatively. The reason for this could be either the presence of phosphorylated amino acids or differences in the isoelectric point of some of the components. An HPLC chromatogram can be run on the complex antigen mixture prior to adsorption and compared with the bands of a HPLC run on the supernatant of the same mixture after adsorption. Unadsorbed components will retain their peak pattern, whereas missing peaks or reduced height of peaks are indicative of complete or partial protein adsorption. Similarly, if an antiserum raised against the complex antigen mixture is available, a crossed, two-dimensional immunoelectrophoresis before and after adsorption may reveal if single components from the complex solution of proteins remain unadsorbed. In this case it is based on the precipitation band pattern from the electrophoresis (48). 9. The fine fraction of particles of aluminum adjuvants may give lead to light scatter that interferes with photometrical readings. For that reason it is recommended to pass the supernatants through a 0.22 μm nitrocellulose sterility filter after harvest and before further processing to retain such particles. 10. This way of calculating gives an approximate value. The reason for this is that Alhydrogel is a suspension of hydrated particles in water. After centrifugation the 2 mL of Alhydrogel is packed at the bottom of the test tube together with the adsorbed protein. Hence, it is only an approximation to state that concentration of HSA measured in the free supernatant relates to the 9 mL.
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57
References 1. Immunological Adjuvants. (1976) Technical Report Series 595 World Health Organization, Geneva. 2. Edelman, R. (1980) Vaccine adjuvants. Rev Infect Dis 2(3), 370–383. 3. Goldenthal, K. L., Cavagnaro, J. A., Alving, C., Vogel, F. R. (1993) NCVDG working groups: safety evaluation of vaccine adjuvants. AIDS Res Hum Retrovirus 9(suppl. 1), s47–s51. 4. Volk, V. K., Bunney, W. E. (1942) Diphtheria immunization with fluid toxoid and alumprecipitated toxoid. Am J Pub Health 32, 690–699. 5. Hem, S. L., White, J. L. (1984) Characterization of aluminum hydroxide for use as an adjuvant in parenteral vaccines. J Parent Sci Tech 38(1), 2–11. 6. Hem, S. L., HogenEsch, H. (2007) Aluminum-containing adjuvants: properties, formulation and use, in (Singh, M., ed.), Vaccine Adjuvants and Delivery Systems. John Wiley & Sons, Hoboken, NJ, pp 81–114. 7. Lindblad, E. B. (2004) Aluminum compounds for use in vaccines. Immunol Cell Biol 82, 497–505. 8. Lindblad, E. B. (2004) Aluminum adjuvants – in retrospect and prospect. Vaccine 22, 3658–3668. 9. Lindblad, E. B. (2006) Mineral adjuvants, in (Schijns, V. and O’Hagan, D. eds.), Immunopotentiators in Modern Vaccines. Elsevier Science Publishers, Burlington, MA, pp 217–233. 10. Willstätter, R., Kraut, H. (1923) Über ein Tonerde-Gel von der Formel Al(OH)3 II: mitteilung über Hydrate und Hydrogele. Ber Dtsch Chem Ges 56, 1117–1121. 11. Willstätter, R., Kraut, H. (1924) Über die Hydroxide und ihre Hydrate in den verschiedenen Tonerde-Gelen. V: mitteilung über Hydrate und Hydrogele. Ber Dtsch Chem Ges 57, 1082–1091. 12. Uede, T., Huff, T. F., Ishizaka, K. (1982) Formation of IgE binding factors by rat T lymphocytes. V. Effect of adjuvant for the priming immunization on the nature of IgE binding factors formed by antigenic stimulation. J Immunol 129(4), 1384–1390. 13. Uede, T., Ishizaka, K. (1982) Formation of IgE binding factors by rat T lymphocytes. VI. Cellular mechanisms for the formation of IgE-potentiating factor and IgEsuppressive factor by antigenic stimulation of antigen primed spleen cells. J Immunol 129(4), 1391–1397.
14. McDougall, J. S. (1969) Avian infectious bronchitis: the protection afforded by an inactivated virus vaccine. Vet Rec 85, 378–380. 15. Wilson, J. H. G., Hermann-Dekkers, W. M., Leemans-Dessy, S., de Meijer, J. W. (1977) Experiments with an inactivated hepatitis leptospirosis vaccine in vaccination programmes for dogs. Vet Rec 100, 552–554. 16. Sellers, R. F., Herniman, K. A. J. (1974) Early protection of pigs against foot-andmouth disease. Br Vet J 130, 440–445. 17. Hyslop, N. S. G., Morrow, A. W. (1969) The influence of aluminium hydroxide content, dose volume and the inclusion of saponin on the efficacy of inactivated foot-and-mouth disease vaccines. Res Vet Sci 10(2), 109–120. 18. Pini, A., Danskin, D., Coackley, W. (1965) Comparative evaluation of the potency of beta-propiolactone inactivated newcastle disease vaccines prepared from a lentogenic and a velogenic strain. Vet Rec 77(5), 127–129. 19. Thorley, C. M., Egerton, J. R. (1981) Comparison of alum-adsorbed or non-alumadsorbed oil emulsion vaccines containing, either pilate or non-pilate bacteroides nodosus cells in inducing and maintaining resistance of sheep to experimental foot rot. Res Vet Sci 30, 32–37. 20. McCandlish, I. A. P., Thompson, H., Wright, N. G. (1978) Vaccination against canine bordetellosis using an aluminium hydroxide adjuvant vaccine. Res Vet Sci 25, 51–57. 21. Nagy, L. K., Penn, C. W. (1974) Protective antigens in bovine pasteurellosis. Dev Biol Stand 26, 65–76. 22. Ris, D. R., Hamel, K. L. (1979) Leptospira interrogans serovar pomona vaccines with different adjuvants in cattle. NZ Vet J 27, 169–171. 23. Leland, S. E., Sofield, W. L., Minocha, H. C. (1988) Immunogenic effects of culturederived exoantigens of Cooperia punctata on calves before and after challenge exposure with infective larvae. Am J Vet Res 49(3), 366–379. 24. Monroy, F. G., Adams, J. H., Dobson, C., Bast, I. J. (1989) Nematospiroides dubius: influence of adjuvants on immunity in mice vaccinated with antigens isolated by affinity chromatography from adult worms. Exp Parasitol 68(1), 67–73. 25. Carlow, C. K. S., Bianco, A. E. (1987) Resistance of Onchocerca lienalis microfilariae in mice conferred by egg antigens of homologous and heterologous onchocerca species. Parasitology 94(3), 485–496.
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26. Gamble, H. R., Murrell, K. D., Marti, H. P. (1986) Inoculation of pigs against Trichinella spiralis using larval excretorysecretory antigens. Am J Vet Res 47(11), 2396–2399. 27. Kwissa, M., Lindblad, E. B., Schirmbeck, R., Reimann, J. (2003) Co-delivery of a DNA vaccine and a protein vaccine with aluminum phosphate stimulates a potent and multivalent immune response. J Mol Med 81(8), 502–510. 28. Lindblad, E. B., Elhay, M. J., Silva, R., Appelberg, R., Andersen, P. (1997) Adjuvant modulation of immune responses to tuberculosis subunit vaccines. Infect Immun 65(2), 623– 629. 29. Leenaars, P. P. A. M., Hendriksen, C. F. M., de Leeuw, W. A., Carat, F., Delahaut, P., Fischer, R., Halder, M., Hanley, W. C., Hartinger, J., Hau, J., Lindblad, E. B., Nicklas, W., Outschoorn, I. M., Stewart-Tull, D. E. S. (1999) The production of polyclonal antibodies in laboratory animals: the report and recommendations of ECVAM/FELASA Workshop 35. ATLA 27, 70–102. 30. Ph. Eur. 6.ed. (2009) Vaccines for human use pp. 3971. 31. Code of Federal Regulations. (2009) 21, vol. 7: sec. 610.15 (Constituent Materials), Revised April 1. 32. Burrell, L. S., Lindblad, E. B., White, J. L., Hem, S. L. (1999) Stability of aluminumcontaining adjuvants to autoclaving. Vaccine 17, 2599–2603. 33. Hem, S. L., Klepak, P., Lindblad, E. B. (2009) Monograph: aluminum hydroxide adjuvant, in Handbook of Pharmaceutical Excipients. Pharmaceutical Press Ltd., London. 34. Hem, S. L., Klepak, P., Lindblad, E. B. (2009) Monograph: aluminum phosphate adjuvant, in Handbook of Pharmaceutical Excipients. Pharmaceutical Press Ltd., London. 35. European Pharmacopeia. (2004) Monograph 1664: Aluminium hydroxide, hydrated, for adsorption (Aluminii hydroxicum hydricum ad adsorptionem). 36. Seeber, S. J., White, J. L., Hem, S. L. (1991) Predicting the adsorption of proteins by aluminium-containing adjuvants. Vaccine 9, 201–203. 37. Morefield, G. L., Jiang, D., RomeroMendez, I. Z., Geahlen, R. L., HogenEsch, H., Hem, S. L. (2005) Effect of phosphorylation of ovalbumin on adsorption by aluminum-containing adjuvants and elution upon exposure to interstitial fluid. Vaccine 23(12), 1502–1506.
38. Rinella, J. V., White, J. L., Hem, S. L. (1995) Effect of anions on model aluminumadjuvant-containing vaccines. J Colloid Interface Sci 172, 121–130. 39. Gupta, R. K., Rost, B. E. (2000) Aluminum compounds as vaccine adjuvants, in (O’Hagan D. T., ed.), Vaccine Adjuvants, Preparation Methods and Research Protocols. Humana Press, Totowa, NJ, pp 65–89. 40. WHO (1977). World Health Organization Manual for the production and control of vaccines: Diphtheria toxoid; Appendix D.21: Preparation of aluminium phosphate suspension. BLG/UNDP/77.1. Rev. 1, pp 90–91 41. Smith, P. K., Krohn, R. I., Hermanson, G. T., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76–85. 42. Dagoussat, N., Robillard, V., Haeuw, J. F., Plotnicky-Gilquin, H., Power, U., Corvaia, N., Nguyen, T., Bonnefoy, J. Y., Beck, A. (2001) A novel bipolar mode of attachment to aluminium-containing adjuvants by BBG2Na, a recombinant subunit hRSV vaccine. Vaccine 19(30), 4143–4152. 43. Shi, Y., HogenEsch, H., Regnier, F. E., Hem, S. L. (2001) Detoxification of endotoxin by aluminium hydroxide adjuvant. Vaccine 19, 1747–1752. 44. Burton, A. J., Carter, H. E. (1964) Purification and characterization of lipid A component of the lipopolysaccharides from Escherichia coli. Biochemistry 3, 411–418. 45. Gmeiner, J., Lüderitz, O., Westphal, O. (1969) Biochemical studies on lipopolysaccharides of Salmonella R. mutans. Eur J Biochem 7, 370–379. 46. Thiele, G. M., Rogers, J., Collins, M., Yasuda, N., Smith, D., McDonald, T. L. (1990) An enzyme-linked immunosorbent assay for the detection of antitetanus toxoid antibody using aluminiumadsorbed coating antigen. J Clin Lab Anal 4, 126–129. 47. Katz, J. B., Hanson, S. K., Patterson, P. A., Stoll, I. R. (1989) In vitro assessment of viral antigen content in inactivated aluminium hydroxide adjuvanted vaccines. J Virol Methods 25, 101–108. 48. Weeke, B., Weeke, E., Løwenstein, H. (1975) The adsorption of serum proteins to aluminium hydroxide gel examined by means of quantitative immunoelectrophoresis, in (Axelsen, N. H., ed.), Quantitative Immunoelectrophoresis. New Developments and Applications. Universitetsforlaget, Denmark, pp 149–154.
Chapter 5 Freund’s Complete and Incomplete Adjuvants, Preparation, and Quality Control Standards for Experimental Laboratory Animals Use Duncan E.S. Stewart-Tull Abstract Quality control and quality assurance procedures are discussed for the agreed benchmark standard Freund’s complete adjuvant (FCA). In addition, the use of the incomplete adjuvant (FIA) in the preparation of antisera is discussed. A major problem is the use of a safe and suitable mineral oil in FCA and FIA; manufacturers should provide infra-red spectra and gas liquid chromatography analyses. A range of safety tests, toxicity, pyrogenicity and endotoxin assays and advice on practical procedures for the use of these adjuvants are described. Key words: Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), immunopotentiator, Mycobacterium tuberculosis, Montanide, immunization procedures, haemolytic activity, creatine kinase assay, rabbit pyrogenicity test, mouse weight gain test.
1. Introduction It is now more than 80 years since Lewis and Loomis (1) found that tuberculous guinea-pigs inoculated with sheep erythrocytes produced higher levels of sheep haemolysins than those in healthy animals. They injected rabbits with 50 μg of Mycobacterium bovis cells and obtained similar results (2). Their studies were continued by Dienes who injected guinea-pigs with 1–2 mg Mycobacterium tuberculosis O5 to induce a tuberculous nodule which was then inoculated with ovalbumin (3, 4). In 1993, EU and USA scientists and administrators discussed the harmonization of regulatory procedures for veterinary products. One opinion was G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_5, © Springer Science+Business Media, LLC 2010
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that “adjuvants are too reactive for inclusion in vaccines” (5). In terms of human vaccines Robbins was of the opinion that “we are prepared to accept no toxicity, any toxicity that we accept is a compromise” (6). This ignored the previous experience in the 1950s when some 18,000 American Service personnel were injected with an oil-adjuvanted influenza vaccine (7–10). In addition, 23,917 human beings were injected with an oil-adjuvanted poliomyelitis vaccine but only 93 individuals had an adverse reaction and only 14 developed nodules at the site of injection (11, 12). 1.1. Freund’s Complete Adjuvant
Subsequently, Freund used a water-in-mineral oil emulsion containing M. tuberculosis cells—this was termed Freund’s complete adjuvant, FCA (13). Adverse reactions to FCA reported from earlier studies included the formation of an epithelioid granuloma at the injection site (14–16). This effect was particularly noticeable in the New Guinea vaccination trial of 1964 during which pregnant Maprick tribal women were vaccinated with an adjuvanted tetanus vaccine in an attempt to prevent neonatal deaths. It was their normal practice to cover the umbilical stump on the newborn with mud and this sometimes caused a fatal disease. Unfortunately, in this trial many women developed similar granulomatous lesions at the site of injection, some of which had to be surgically removed (17). This was a setback for the use of an adjuvanted vaccine, but little attention was directed to the quality and composition of the mineral oil or the emulsifying agent. A detailed assessment of FCA is provided in a number of review articles (18, 19). It is important to qualify the constituents used in the preparation of experimental FCAs as they may vary considerably in their composition. The inclusion of dead M. tuberculosis is deemed to be unacceptable for use in human vaccines because of sensitization to other proteins, e.g. tuberculin. However, in 1990 the late Prof. Jonas Salk did confirm to me “If I thought that the inclusion of FCA would provide an effective vaccine in the fight against AIDS I should have no hesitation in recommending its use.” It should also be recognised that several existing commercial vaccines in routine use do contain microgram levels of an immunomodulator as shown in Table 5.1 (18). Conversely, the use of Freund’s incomplete adjuvant—FIA, without the mycobacterial component, did not cause the formation of such acute granulomatous adverse reactions in many experimental studies. Consequently, it was assumed that the mycobacterial cell component alone was responsible for the adverse reactions at the site of injection of FCA. Such conclusions, however, ignored the effects that the emulsifiers or mineral oils, used in the preparation of FCA, might have had on the formation of adverse histological reactions at the site of injection. Prior to the 1970s, the early studies used oils produced by the
Freund’s Complete and Incomplete Adjuvants
61
Table 5.1 Immunomodulator content of some vaccines Approximate weight (μg) of immunopotentiator injected during normal immunization schedules Vaccine
Peptidoglycan
Lipopolysaccharide
BCG
3.0–5.0
–
Whooping cough
6.5–50.0
6.0–35.0
Cholera
0.3–12.0
0.3–7.0
acid treatment or oleum method in the petroleum industry. At the beginning of the 1970s white mineral oils were produced by the single or double hydrogenation procedure and superior grades of oil were obtained which lacked the more toxic contaminants responsible for the adverse reactions. Therefore, it was not surprising that at the North Atlantic Treaty Organization meeting in Sounion, Greece, in 1989 it was agreed that Freund’s complete adjuvant (produced by the Statens Seruminstitut, Copenhagen, and available from Brenntag Biosector, Frederikssund, Denmark) consisting of 85% Marcol 52, 15% Arlacel A (mannide monooleate) and 500 μg heat-killed, dried M. tuberculosis 2.0 mg/mL and aluminium hydroxide adjuvant (Brenntag, Denmark) should be accepted as the “gold standard adjuvants” for experimental use (20). The FCA preparation was chosen as the standard adjuvant preparation because of its extensive use in the development of experimental vaccines in animals, its strong adjuvant effect and the considerable literature describing its activity. The mixture is used in a 1:1 ratio with the antigen-containing aqueous phase. The combination of mineral oil (Marcol 52) and dead mycobacterial organisms in FCA produces a specific cellular reaction in experimental animals since the mycobacteria stimulate the formation of epithelioid macrophage cells and also the maturation of plasmablasts to plasma cells. This effect was greater when the oil and mycobacterial fraction were combined. Numerous studies have shown that the oil emulsion was responsible for the retention of the antigen at the site of inoculation. This depot provides a slow and prolonged antigenic stimulus to antibody-forming cells. In addition, FCA stimulates both humoral and cell-mediated immune responses (see Note 1). In order to measure efficacy with a new adjuvant preparation it is important that its activity should be compared to these gold standards. Efficacy is dependent upon the vigorous mixing of the FCA and the antigen until a stable emulsion has formed, see below.
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There are increasing concerns about the general use of FCA for the routine production of antisera because Freund’s incomplete adjuvant (FIA) will, in most cases, achieve an acceptable result. From an ethical viewpoint there are few reasons to use FCA for routine antisera production and it is reasonable to use FIA. Some suggestions have been made that FCA should be limited to essential studies on the cell-mediated immune responses or where it is to be used as the standard adjuvant when a comparison is made with new adjuvanted vaccines. It should be emphasised that FIA or less reactive FCA products are more likely to be used. A non-ulcerative, commercial preparation of Non-Ulcerative Freund-type complete adjuvant—NUFA—has been introduced by Morris of Guildhay Ltd. (Guildford, Surrey, UK); this can be administered by intramuscular, subcutaneous and intradermal routes in small doses at multiple sites (see Note 2). The intramuscular site creates a longer depot stimulus and fewer adverse reactions than the other routes. 1.2. Freund’s Incomplete Adjuvant (FIA)
The killed M. tuberculosis component of FCA is not included in FIA. The latter is routinely used for boosting immunizations subsequent to a primary injection of FCA. It can also be used for the initial immunization, particularly when a strong antigen is used or moderate antibody levels are sufficient. As with FCA, the FIA is available from The Statens Seruminstitut, Copenhagen, and is composed of 85% Marcol and 15% Arlacel A. The search for less-reactogenic hydrocarbons and emulsifiers has continued. The Montanide series of oils (SEPPIC, France) based on anhydromannitol ether octadecanoate (oleic acid is a naturally occurring carboxylic acid in fats and oils—CH3 (CH2 )7 CH: CH (CH2 )7 COOH) is non-toxic in the Berlin test in a 50:50 (v/v) emulsion. The LD50 value in mice is equivalent to 25.0 g/kg bodyweight. Montanide ISA 720 is a ready-touse preparation, containing a highly refined emulsifier, a natural, metabolizable oil with a pharmaceutical grade mineral oil; Drakeol 6VR has been the choice for experimental studies in Glasgow and no adverse reactions have been recorded (see Notes 3 and 4).
1.3. The Mineral Oil
Many early studies employed the use of ill-defined mineral oils obtained from the petroleum industry. Indeed for my own early studies in the 1960s a free supply of Bayol F was obtained from the Esso Petroleum Company. An engineer walked to a outlet point in the Thames-side refinery where he suggested that “This cut-off should provide a supply of oil with the correct carbon chain length”. There may be concomitant adverse side-effects because of the reactogenicity of some types of mineral oil, particularly the formation of the epithelioid macrophage granuloma at the site of
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63
injection and local ulceration when the injection is given subcutaneously. My research in Japan with Shozo Kotani’s group was done with “a specially formulated mineral oil for adjuvant research”, but this caused severe disruption of cell membrane bilayers with consequent gross tissue destruction. Subsequently, gas liquid chromatography studies in Glasgow revealed that this mineral oil preparation contained short-chain hydrocarbons, nC6 H14 to nC10 –H22, and these were highly reactogenic and immunosuppressive. The most suitable mineral oils for experimental vaccine inclusion were shown to be fully saturated hydrocarbons, nC16 H34 –nC19 H40 (22) (see Note 4). The type of emulsion may affect the level of antibody stimulation; the injection of ovalbumin in a water-in-oil emulsion produced a stronger immune response in mice than in an oil-in-water emulsion. The hydrophilic and lipophilic groups should be in equilibrium in the adjuvant emulsion for the effective immunological activity of Freund’s adjuvants.
2. Materials 2.1. Preparation of Freund’s Complete Adjuvanted Experimental Vaccines
1. Heat-killed M. tuberculosis cells (the FCA available from Brenntag, or the Guildhay NUFA non-ulcerative Freund’s adjuvant (see above) is recommended) 2. Bacillus Calmette Guérin (BCG vaccine BP, BNF [id] John Bell and Croydon, London, UK) or from health organizations or direct from Movianto (SSI brand) 3. Mineral oil: examples are Bayol F (e.g. Esso is one source, but if a local source is available QA-QC information should be forthcoming), Marcol 52 or Drakeol 6VR (local source plus QA-QC record) or equivalent 4. Emulsifier such as Arlacel A (mannide mono-oleate) 5. Montanide 720 (Seppic)
2.2. Preparation of Freund’s Incomplete Adjuvanted Experimental Vaccines
1. Mineral oil: e.g. Bayol F, Marcol 52 or Drakeol 6VR.
2.3. Immunization Procedures
1. Experimental vaccine prepared as in Section 2.1.
2.4. Comparative Tests to Measure the Safety and Efficacy of Adjuvants
1. Cyanmethaemoglobin standard (Merck Ltd: Cyanmethaemoglobin standard for photometric determinations of haemoglobin or Sigma haemoglobin standard)
2. Emulsifier such as Arlacel A (mannide mono-oleate). 3. Montanide 720 (Seppic).
2. Experimental animal.
2. Drabkins reagent
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3. Creatine kinase diagnostic kit (Sigma) 4. Rabbits (adult New Zealand White, obtained from a reputable breeder) 5. Swiss white mice 6. Guinea-pigs
3. Methods 3.1. Preparation of Freund’s Complete Adjuvanted Experimental Vaccines
1. Dissolve the antigen, 10.0 mg in 0.2 mL sterile physiological saline (see Note 5). 2. Suspend 1.0 mg heat-killed, M. tuberculosis cells evenly in 0.6 mL sterile mineral oil by mild ultrasonication (see Note 6). 3. Add 0.2 mL of an emulsifier (see Note 7) to this oil phase and mix thoroughly. Alternatively, a suitable Montanide, e.g. Montanide 720, combined oil and emulsifier can be used. 4. Add the aqueous phase to the oil phase and mix thoroughly until a creamy white emulsion is produced. This can be achieved by either drawing up into a 1.0-mL glass Luer syringe fitted with a medium bore needle, or passing the mixture from syringe to syringe through a sterile adapter or by mild ultrasonication. 5. The nature of the resulting emulsion should be tested to ensure that a water-in-mineral oil emulsion has been prepared. Expel a drop of the emulsion onto the surface of water in a shallow dish: An oil-in-water emulsion will immediately disperse over the surface, whereas a water-in-oil emulsion will retain the integrity of the drop. 6. Store the final mixture at 4◦ C or at room temperature, depending on the nature of the antigen, but do not freeze because this will cause the emulsion to breakdown.
3.2. Preparation of Freund’s Incomplete Adjuvanted Experimental Vaccines
1. The procedure is as described in the Section 3.1, but the mycobacterial component or its equivalent is omitted from the mixture (see Note 8).
3.3. Immunization Procedures
1. The experimental vaccine should be warmed to 37◦ C before injection (see Note 9). This avoids shock to the small animal and helps the vaccine to flow from the syringe. 2. The size of the injection dose will depend on the animal and the route of injection. For example, 0.2 mL of a waterin-oil emulsion containing 2.0 mg of antigen and 200 μg
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M. tuberculosis im into the left hindlimb of a guinea-pig is suitable. On the other hand, 200 μg M. tuberculosis in the vaccine dose will completely suppress the humoral response in a mouse; in this case 25–50 g is an optimal dose. Different regimens may be required for larger animals; those for rabbits and sheep have been described where it may be advisable to use multiple injection sites (23). 3.4. Comparative Tests to Measure the Safety and Efficacy of Adjuvants
In 1988 at the First NATO Advanced Studies Institute conference on “Immunological Adjuvants and Vaccines” a unified approach to the assessment of immunological adjuvants was discussed (20, 25). This followed our previous studies on acceptable procedures to allow QA and QC in the use of mineral oils for use in adjuvant emulsions (21). In view of the expansion of the European Community it is important that these issues are widely discussed by researchers and industrialists so that each member country can accept agreed procedures acceptable to the EU and also to other regulatory bodies. By 1990, at the Second NATO conference on “Vaccines—Recent trends and progress” some recommendations were made and accepted; as mentioned previously it was agreed that FCA and aluminium hydroxide should be classed as the standard adjuvants against which new products would be compared (20, 23, 24). In addition, a number of QC and QA assays were discussed as shown below (and see Notes 11 and 12).
3.4.1. Haemolytic Activity of Adjuvants: Spectrophotometric Determination of Haemoglobin Release
The spectrophotometric determination of haemoglobin released from fresh whole rabbit blood by the accepted method gave the most reproducible and stable results. 1. Prepare reference solutions of cyanmethaemoglobin from 0.055 g/L to 0.85 g/L and measure the A540 nm values. 2. Add the test sample of fresh blood (20 μL), collected in a heparinized tube, to 4.0 mL of Drabkins reagent, mix and leave for 5 min after which the A540 nm values are recorded. 3. Prepare a standard curve with the reference standards and use to measure rabbit test results for haemoglobin concentration. If the percentage haemolysis in the negative control is >5.0% or in the positive control is <1.0 or >20% the test is invalid (25).
3.4.2. Haemolytic Activity of Adjuvants: Creatine Kinase Assay
There is a slight advantage with this method as a commercial kit is available from Sigma Chemical Company (Creatine kinase diagnostic kit Cat 520 or 520-C). 1. Three days after the injection of the adjuvanted vaccine a sample of blood is taken and the creatine kinase (CK) activity is measured according to the manufacturer’s instructions (see Note 10).
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3.4.3. Rabbit Pyrogenicity Assay
If the result of Test 1 is negative whether the adjuvant is required to stimulate a humoral or cell-mediated response a pyrogenicity test should be done (see Note 11). 1. All glassware, diluents and solutions to be used should be pyrogen-free. 2. The rabbits are required to be sham-tested 7 days prior to use. 3. Before the actual test, the rectal temperature of the rabbit is taken for every 30 min up to 90 min prior to the injection into a marginal ear vein of sterile physiological saline. If the temperature varies by either >0.2◦ C or if the range of readings exceeds 0.4◦ C the rabbit would be excluded. 4. Subsequently, the test sample, the immunomodulator in sterile physiological saline, is injected and the process is repeated; animals with a temperature rise of >0.6◦ C fail the test (26, 27).
3.4.4. Endotoxin Assays: Mouse Weight Gain Test
If the experimental vaccine fails the test described in 3.3.1, 3.3.2 or 3.3.3 above, it is unnecessary to proceed to further tests as the result indicates that the adjuvant is too reactive. This is a standard and reliable method to assess the presence of endotoxin as the mouse shows a decrease in total weight during the 24 h after inoculation. 1. If the animal shows a steady rise in body weight for 7 days after inoculation the inoculum is acceptable. 2. Conversely, if the animal continues to lose weight or even dies the inoculum fails the toxicity test (29).
3.4.5. Practical Procedures in the Preparation and Application of Adjuvant Mixtures
1. Standard control antigens: egg-white ovalbumin was originally chosen because it was a poor antigen but influenza haemagglutinin was also recommended (20). 2. The choice of test animals (see Note 13): It was pointed out that guinea-pigs and rabbits do not respond to influenza virus and outbred mice could introduce a considerable degree of laboratory to laboratory variation (20). Therefore, it was agreed to use guinea-pigs and mice. The mice should have (a) variable genetic background and similar H-2 haplotypes and (b) a constant genetic background and variable H-2. Dr Chella David (USA) recommended the following scheme: mouse strain and histocompatibility specificity 1, BALB/c H-2d ; 2, DRA/c H-2d ; 3, C57BL/10 H-2b ; 4, C3H H-2k ; 5 C3H.B10 H-2b . Tests should be done on either mouse groups 1, 2, 4 and 5 or with groups 3, 4 and 5. Other animals suggested for testing vaccines before field trials were goats and monkeys (see Note 14). It seems that an international agreement would be required in this matter.
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Table 5.2 Doses and sites of injection for various animals Injection sites
Species Mice or hamsters Guinea-pigs or rats
Maximum volume per injection site
Primary response
Secondary response
50 μL
Im
im
im into one hindlimb
oral im into one hindlimb
200 μL 200 μL 300 μL
3.4.6. Recommended Volumes of Injection Doses and Routes of Inoculation for Animals
oral
Rabbits
250 μL (if in multiple sites <25 μL/site)
im into one thigh muscle intradermal
into one thigh muscle id
Chicken
250 μL
im
im
Large animal
500 μL
For example sheep or donkey
(if in multiple sites ∼ 100 μL/site)
sc or im into one hindlimb id
sc or im into one hindlimb id
1. In general, as shown in Table 5.2 the doses and the site of the injection vary according to the size of the animal being inoculated for routine antisera production (see Notes 15–21).
4. Notes 1. Other contraindications for FCA have been recorded, e.g. pyrogenicity, stimulation of experimental autoimmune diseases and adjuvant arthritis (reviewed in detail by StewartTull (18, 20)). 2. The main difference between the Guildhay product and others relies on the use of BCG vaccine (Evans Medical) to provide the mycobacterial component for FCA. The BCG vaccine is formulated for intradermal use (BCG vaccine BP, BNF [id] supplied by John Bell and Croydon, London, UK). After reconstitution, 0.1 mL is added to 0.9 mL of the aqueous antigen solution and 2.0 mL of the FIA
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containing highly refined base oil that conforms to US and EU Pharmacopoeia requirements and complies with FDA regulations 21 CFR 172.878 and 178.3620(a). The dosage varies from one animal species to another but the total recommended dosage is between 1.0 and 3.0 mL. 3. Montanide ISA 720 and ISA 51 preparations should not be stored below 4◦ C after they have been included in a water-in-oil emulsion, otherwise there is separation into two phases. 4. The ratio of the oil phase to the aqueous phase may be altered depending on the particular product used. For example, the FIA from Statens Seruminstitut is used at a 1:1 ratio, whereas the Montanide ISA 720 (Seppic, Paris, France) is used in the ratio 70% Montanide and 30% aqueous phase containing the antigen to yield stable emulsions. It is not recommended to economize by reducing the amount of Montanide because if used at different ratios the emulsion may separate into two phases and reduce the storage time of the experimental adjuvant mixture. Montanide ISA 51 is a mixture of Montanide 80 in Drakeol 6VR which is a ready-to-use preparation in the ratio 1:1 with the aqueous phase containing the antigen (21). 5. Since the use of ill-defined oils in Freund-type mixtures may give rise to severe toxic reactions with accompanying ulceration it is recommended that manufacturers should provide evidence of QA and QC tests on the oil; for example, GLC spectrometer analyses and systemic toxicity and pyrogenicity test results for a production batch. GLC analyses are carried out with a 2.77 column packed with 3% OV-17 coated on Gas Chrom Q (Phase Separations Ltd.). Samples are dissolved in ether (ca. 5 mg/mL) and injected (1–2 μL) via a self-sealing septum into the analyzer. The analyses are done initially at 100◦ C for 16 min followed by temperature programming at 8◦ C/min to 275◦ C. Standard hydrocarbons nC11 H22 , nC18 H38 and nC20 H42 are used as reference compounds. The optimum hydrocarbon chain length was shown to be between C14 and C24 . Some commercial preparations are much less reactogenic, partly because of changes in the production of the mineral oil (21) and improved methods to measure the hydrocarbons in the oil (22). 6. If the M. bovis BCG mycobacterial cells are to be used these must be suspended in the saline component. 7. Examples of mineral oils that can be used are Bayol, Marcol 52, Drakeol 6VR or equivalent. 8. An example of an appropriate emulsifier is Arlacel A (mannide mono-oleate).
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9. For large-scale use in the field warming to 37◦ C may not always be possible but it is not recommended to use vaccine immediately from the refrigerator. 10. However, in our laboratory it was difficult to assay standard CK values for each animal species (20, 25). The normal range of CK activity must be obtained for different animals. 11. The Limulus amoebocyte lysate assay has been accepted as an alternative for the rabbit pyrogenicity test by the European Pharmacopoeia, although not by the US Food and Drug Administration. It has been suggested that both tests could be assayed during the development phase of a new adjuvanted vaccine in order to gain general approval of this method (28). 12. Induction of adjuvant arthritis in Lewis strain rats revealed positive reactions with mineral oils produced prior to 1970 (30), and it would seem unlikely that this should now be considered as a routine test if an experimental vaccine fails as described in 3.3.1, 3.3.2 or 3.3.3, but it could be relevant during the QA of a new immunomodulator. 13. “Too often the choice of the test animal is determined by availability rather than its suitability for the testing procedure. Some attention is given to the sex, age and species of animal, but little or no care is given to the selection of animals of uniform genetic composition with predictable test characteristics” (Manclark et al. (20)). 14. Jonas Salk (USA) did suggest, “If you can document safety, why not test efficacy in humans?” (24). 15. It is not recommended that these mineral oil adjuvants should be injected intradermally, with the possible exception of the Guildhay preparation, or subcutaneously because of the local reactivity and ulceration. 16. There may be species differences found after intraperitoneal injection; for example, the ip route caused ascites production in Balb/c mice, but booster injections in rabbits or sheep did not cause adverse effects. 17. If an experimental vaccine is under development for human use it should be remembered that the majority of such marketed vaccines are administered by an intramuscular injection (32). Therefore, the use of sc or id routes of injection may produce in vivo effects that are not representative of the human reaction. 18. Care must be exercised to ensure that the operator does not accidentally inject himself/herself or assisting person; a veterinary surgeon required amputation of part of a finger because of granuloma formation after accidental
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inoculation. If self-inoculation does occur it is wise to seek medical attention at the earliest opportunity (36). 19. As mentioned above manufacturers should provide details about the oils to be used in adjuvant mixtures. However, the vaccine candidate antigen may also have an effect on the inoculum, so it would be wise to check the immunogenic activity of the vaccine candidate antigen prior to its inclusion in the oil emulsion. 20. Some relevant information may be gleaned from the published information on adjuvants before the necessity for further animal experimentation. In the “Rules Governing Medicinal Products in the European Community—Volume 5” it states “it is recognized that for veterinary products a degree of toxicity and hazard for the animal are acceptable, provided that such toxicity has no consequences for man”. Such toxicity could include mineral oil residues remaining in meat products; the maximum hydrocarbon level for a pig is 25%/dose and 50% for cattle. It has been calculated that a 60 kg human has a daily intake of 50 g/day/kg. It has been calculated that a 100 kg pig receiving a 2.0 mL injection dose would have 3.0 mg oil/kg meat and a 500 kg cow would have 5.0 mg oil/kg meat. The amount of oil ingested by the 60 kg man would be 5 g/kg/day and 6 g/kg/day, respectively. These values should be calculated for other food products where an adjuvant in an oil emulsion is used for vaccination. 21. It is worth reiterating the statement of Davenport: “aside from the question whether the aluminium ion in adjuvant 65 is metabolizable, how curious it is that we accept in therapeutic medicine the principle that nonmetabolizable sutures, splints and prostheses are good but the employment of non-metabolizable vehicles in preventative medicine is evil” (10). It is to be hoped that under the auspices of the multi-national European Adjuvant Advisory Committee some agreements may be reached among academics and industrialists to answer Davenport’s criticisms. References 1. Lewis, P. A., Loomis, D. (1924) Allergic irritability. The formation of anti-sheep hemolytic amboceptor in the normal and tuberculous guinea-pig. J Exp Med 40, 503–515. 2. Lewis, P. A., Loomis, D. (1926) Allergic irritability. III. The influence of chronic infections and of trypan blue on the formation of specific antibodies. J Exp Med 43, 263–273.
3. Dienes, L., Schoenheit, E. W. (1927) Local hypersensitiveness I. Sensitization of tuberculous guinea-pigs with eggwhite and timothy pollen. J Immunol 14, 9–42. 4. Dienes, L. (1929) The technique of producing the tuberculin type of sensitisation with egg-white in tuberculous guinea-pigs. J Immunol 17, 531–538.
Freund’s Complete and Incomplete Adjuvants 5. Stewart-Tull, D. E. S., Brown, F. (1993) First steps towards an international harmonization of veterinary biologicals. Vaccine 11, 692–695. 6. Robbins, J. (1980) in (Mizrahi, A., et al., eds.) New Developments with Human and Veterinary Vaccines. AR Liss Inc., New York, pp 393–394. 7. Salk, J. E., Bailey, M. L., Laurent, A. (1952) Use of adjuvants in studies on influenza immunization, increased antibody formation in human subjects inoculated with influenza virus vaccine in water-in-oil emulsion. Am J Hyg 55, 439–456. 8. Davenport, F. M., Hennessy, A. V., Houser, H. B., Cryns, W. F. (1956) Evaluation of adjuvant influenza virus vaccine tested against influenza B, 1954–1955. Am J Hyg 64, 304–313. 9. Bell, J. A., Philip, R. N., Davis, D. J., Beem, M. O., Beigelman, P. M., Engler, J. I., Mellin, G. W., Johnson, J. H., Lerner, A. M. (1961) Epidemiologic studies on influenza in familial and general populations, 1951–1956: IV vaccine reactions. Am J Hyg 73, 148–163. 10. Davenport, F. M. (1968) Seventeen years experience with mineral oil adjuvant influenza virus vaccines. Ann Allergy 26, 288–292. 11. Stewart-Tull., D. E. S. (2003) Adjuvant formulations for experimental vaccines, in (Robinson, A., Hudson, M. J., Cranage, M. P., eds.) Vaccine Protocols, 2nd edn. Humana Press, Totowa, NJ. 12. Cutler, J. C., Lesesne, L., Vaughn, I. (1960) Use of poliomyelitis virus vaccine in light mineral oil adjuvant in a community immunization program and report of reactions encountered. J Allergy 33, 193–209. 13. Freund, J. (1951) The effect of paraffin oil and mycobacteria on antibody formation and sensitisation. A review. Am J Clin Path 21, 645–656. 14. White, R. G. (1959) The adjuvant effects of mycobacterial cells and fractions, in (Shaffer, J. H., Lo Grippo, G. A., Chase, M. W., eds.) Mechanisms of Hypersensitivity. Churchill, London, pp 637–645. 15. Suter, E., White, R. G. (1954) Response of reticulo-endothelial system to injection of ‘purified wax’ and lipopolysaccharide of tubercle bacilli; a histologic and immunogenic study. Am Rev Tuberc 70, 793–805. 16. Paraf, A. (1970) Mechanisme d’action des adjuvants de l’immunite. Annales des l’Institut Pasteur 118, 419–441. 17. MacLennan, R., Schofield, F. D., Pittman, M., Hardegree, M. C., Barile, M. F. (1965) Immunization against neonatal tetanus in
18.
19.
20. 21.
22.
23.
24.
25. 26.
27.
28.
29.
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New Guinea. Antitoxin response of pregnant women to adjuvant and plain toxoids. Bull World Health Org 32, 683. Stewart-Tull, D. E. S. (1983) Immunologically important constituents of mycobacteria: adjuvants, in (Ratledge, C., Stanford J., eds.) The Biology of the Mycobacteria. Academic Press, London, pp 3–84. White, R. G. (1959) The adjuvant effects of mycobacterial cells and fractions, in (Lo Grippo, G. A., Chase, M. W., eds.) Mechanisms of Hypersensitivity. Churchill, London, pp 637–645. Lederer, E. (1971) The mycobacterial cell wall. Pure Appl Chem 25, 135–165. Stewart-Tull, D. E. S. (1989) Recommendations for the assessment of adjuvants (immunopotentiators), in (Gregoriadis, G., Allison, A. C., Poste, G., eds.) Immunological Adjuvants and Vaccines. Plenum Press, London, pp 213–226. Stewart-Tull, D. E. S. (1985) Immunopotentiating activity of peptidoglycan and surface polymers, in (Stewart-Tull, D. E. S., Davies, M., eds.) Immunology of the Bacterial Cell Envelope. John Wiley & Sons Ltd., Sussex, pp 47–89. Stewart-Tull, D. E. S. (1995) Freund-type mineral oil adjuvant emulsions, in (StewartTull, D. E. S., ed.) The Theory and Practical Application of Adjuvants. John Wiley & Sons, Chichester, pp 1–19. Stewart-Tull, D. E. S., Shimono, T., Kotani, S., Knights, B. A. (1976) Immunosuppressive effect in mycobacterial adjuvant emulsions of mineral oils containing low molecular weight hydrocarbons. Int Arch Allergy Appl Immunol 52, 118–128. Stewart-Tull, D. E. S., Rowe, R. E. C. (1975) Procedures for large-scale antiserum production in sheep. J Immunol Methods 8, 37–46. Stewart-Tull, D. E. S. (1989) Recommendations for the assessment of adjuvants, in (Gregoriadis, G., Allison, A. C., Poste, G., eds.) Immunological Adjuvants and Vaccines. NATO ASI Series A: Life Sciences, Plenum, New York, vol 179, pp 213–226. Stewart-Tull, D. E. S. (1991) The assessment and use of adjuvants, in (Gregoriadis, G., Allison, A. C., Poste, G., eds.) Vaccines Recent Trends and Progress. NATO ASI Series A: Life Sciences, Plenum, New York, vol 215, pp 85–92. Gray, J. E., Weaver, R. N., Moran, J., Feenstra, E. S. (1974) The parental toxicity of clindamycin-2-phosphate in laboratory animals. Toxicol Appl Pharmacol 27, 308. British Standards Institution. (1990) Evaluation of medical devices for biological hazards.
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Part 11. Method of test for haemolysis. Br Stand 5736, 11. 30. British Standards Institution. (1990) Evaluation of medical devices for biological hazards. Part 5. Method of test for systemic toxicity; assessment of pyrogenicity in rabbits of extracts from medical devices. Br Stand 5736, 5. 31. Wardlaw, A. C., McCartney, A. C. (1985) Endotoxic activities of lipopolysaccharides, in (Stewart-Tull, D. E. S., Davies, M., eds.) Immunology of the Bacterial Cell Envelope. John Wiley & Sons, Chichester, pp 203–238. 32. Christodoulides, M., Sidey, F. M., Parton, R., Stewart-Tull, D. E. S. (1987) Acellular pertussis vaccine prepared by a simple extraction and toxoiding procedure. Vaccine 5, 199–207.
33. Pearson, C. M. (1956) Development of arthritis, periarthritis and periostitis in rats given adjuvant. Proc Soc Exp Biol Med 91, 95–101. 34. Kohashi, O., Tanaka, A., Kotani, S. (1980) Arthritis – inducing ability of a synthetic adjuvant, N-acetylmuramyl peptides, and bacterial disaccharide peptides related to different oil vehicles and their composition. Infect Immunol 29, 70–75. 35. Royal Pharmaceutical Society of Great Britain and British Medical Association. (2008) British National Formulary 14. Immunological Products and Vaccines, pp 651–674. 36. Stones, P. B. (1979) Self injection of veterinary oil-emulsion vaccines. Br Med J 1, 1627.
Chapter 6 Liposomal Adjuvants: Preparation and Formulation with Antigens Jean Haensler Abstract Many preclinical and clinical results indicate that liposomal systems can serve as effective adjuvants to subunit vaccines by enabling the formulation and delivery of vaccine antigens and immunopotentiators. The adjuvant effect of liposomes usually depends on both the composition of the lipid vesicles and their physical association with the vaccine antigen. This chapter describes methods for the preparation and characterization of sterile small, mostly unilamellar, lipid vesicles and for their association with vaccine antigens. It gives also some recommendations for the optimization of liposomal vaccines in preclinical testing. The most common immunopotentiators used in liposomal adjuvants are also described. Key words: Liposome, preparation, adjuvant, immunostimulant, immunopotentiator, vaccine, antigen, formulation.
1. Introduction Liposomes are small lipid vesicles containing an internal aqueous space. Depending on their lipid composition, liposomes have the ability to adsorb or encapsulate a wide variety of substances, including protein, peptide, and polysaccharide antigens. Lipophilic substances may be trapped within the lipid bilayers and hydrophilic substances may be encapsulated into the internal aqueous space, adsorbed or chemically linked onto the surface of the vesicles. As a consequence, liposomes provide a delivery
G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_6, © Springer Science+Business Media, LLC 2010
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system for antigens and immunopotentiators and an ideal tool for combining antigens and immunopotentiators within a single particulate carrier, i.e., a tool affording “the perfect mix” for modern adjuvant approaches (1, 2). In fact, liposomes have been used for over 20 years for the co-formulation of antigens and immunopotentiators (3, 4), and potent adjuvant effects have been obtained in a number of studies with antigens encapsulated, inserted, or adsorbed in liposomes containing immunostimulants. This chapter will provide simple methods for the manufacture of liposomes at lab scale (i.e., 2–100. mL) and for the association of antigens with these liposomes. These methods were selected because they are versatile (i.e., can adapt a wide range of lipids and lipoidal immunostimulants and a wide range of protein, peptide, or polysaccharide antigens) and they are readily scalable. I also wanted to focus on techniques for the manufacture of small liposomes (≤ 200 nm) that would be compatible with sterilization by filtration, which is an important feature for the manufacture of parenteral vaccines.
2. Materials 2.1. Starting Raw Materials and Equipment 2.1.1. Liposomal Lipids, Lipoidal Immunostimulants, and Selected Buffers
The most common ingredients of liposomal membranes are phospholipids (PL) and cholesterol (CHOL). However, apart from phosphatidylserine (PS), which is capable of interacting with a specific PS receptor on macrophages (5), and the lysophospholipids, which form upon phospholipid oxidation during the acute phase response to tissue trauma or infection (6), the natural lipids are not known to display specific immunological effects, and the adjuvant effect of standard PL/CHOL liposomes is thought to result essentially from their capacity to deliver/present antigens to APCs. To potentiate the adjuvant effects of liposomes, lipoidal immunostimulants have been incorporated into the liposomal membranes. These comprise QuilA, a saponin extracted from the bark of the South American tree Quillaja saponaria Molina and its purified subfraction QS21 (7–10), bacterial cell wall components such as lipopolysaccharide (LPS), monophosphoryl lipidA (MPLA) and its synthetic analogues (3, 9–11), muramyl dipeptide (MDP) and its lipophilic derivatives (3, 12), trehalose dimycolate (TDM) and its synthetic analogue trehalose dibehenate (TDB) (13, 14), a number of synthetic TLR2-binding lipopeptides (15), archeo lipids (16), and synthetic cationic lipids such as dioctadecyldimethylammonium
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bromide (DDAB), 3β-[N-(N , N’-dimethylaminoethane)carbamoyl]cholesterol Hydrochloride (DC-Chol), 1,2-dioleoyl3-trimethylammoniumpropane (DOTAP), and Ethylphosphatidylcholine (EPC) (14, 17, 18) (see Note 1). For the lipids to optimally organize into liposomes, the preparation of liposomes needs to be carried out at a temperature above the gel–liquid crystalline transition temperature (Tc ) of the lipid components. When liposomes are formed from a mixture of lipids, dissolution of theses lipids in organic solvent and removal of organic solvent to form a homogeneous dry lipid mix appears inevitable to ensure homogeneous distribution of the lipids over the bilayer after lipid resuspension in the selected liposome buffer. 1. Liposomal lipids can be obtained from different sources such as Avanti Polar Lipids (Alabaster, AL, USA), Lipoid (Ludwigshafen, Germany), Genzyme (Liestal, Switzerland), Sigma-Aldrich (St. Louis, MO, USA) (see Note 2). Store lipids under argon at ≤ –20◦ C. 2. Lipoidal immunostimulants such as some of those listed above can be obtained from Avanti Polar Lipids, EMC Microcollection (Tuebingen, Germany), Bachem (Bubendorf, Switzerland), InvivoGen (San Diego, CA, USA), Axxora (San Diego, CA, USA), and Sigma-Aldrich among others. Store lipoidal immunostimulants under argon at ≤ –20◦ C. 3. The Tc of lipids may be determined by using a differential scanning microcalorimeter such as supplied by MicroCal, Northampton, MA, USA, or equivalent. 4. Solvent removal from starting lipid mixture may be performed by using a rotary evaporator such as Buchi, rotavapor-RTM or equivalent. 5. Most commonly used buffers for the preparation of liposomes (“liposome buffers”) are phosphate, tris, and HEPES buffers at pH around 7.0–8.0 with or without 0.9% NaCl or with 5% sucrose if freeze-drying of the liposomes is planned (see Note 3). The different salts to prepare these buffers are available from Sigma-Aldrich. Buffers are sterilized by filtration. 2.1.2. Equipment for the Homogenization and Filtration of Liposomes
1. Extrusion: liposomes may be calibrated by extrusion using a LipexTM extruder (Northern Lipids Inc, Vancouver, Canada). The extruder is operated according to the operating and safety instructions provided by the supplier. 2. High-pressure homogenizers: liposome suspensions may be homogenized by using an M-110Y microfluidizer (Microfluidics Corporation, Newton, MA, USA). Alternatively, the
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EmulsiFlexTM C3 high-pressure homogenizer (Avestin, Ottawa, Canada) or other high-pressure homogenizers may be used (see Note 4). The high-pressure homogenizers are operated according to the operating and safety instructions provided by the supplier. 3. Filters for sterile filtration: commonly used filters for the sterilization of liposomes are made of polyvinylidene fluoride (PVDF) or polyethersulfone (PES) and are available from different suppliers such as Millipore, Pall, and Sartorius (see Note 5). Use syringe-driven filter units with PVC housing, filter pore size of 0.2 μm. The 13 mm units are recommended for filtering volumes from 1 to 10 mL. The 25 mm units are recommended for filtering volumes from 10 to 100 mL. 2.1.3. Ethanol Injection
1. Lipids are dissolved in absolute ethanol (Sigma-Aldrich). 2. Injection is performed with standard gastight Hamilton syringe with Teflon plunger (Sigma-Aldrich). 3. Temperature is controlled by using a circulating water bath.
2.1.4. Detergent Dialysis
1. Lipids are solubilized in non-ionic detergent n-octyl-β-Dglucopyranoside (Calbiochem, San Diego). 2. Detergent is removed by using Spectra/PorTM 7 dialysis tubing (Spectrum Laboratories, Breda, The Netherlands).
2.2. Purification of Liposomal Antigen 2.2.1. Sucrose Gradient
1. Liposome buffer. Heat sterilize. Store at 4◦ C. 2. Sixty percent sucrose solution prepared by dissolving 60 g sucrose in liposome buffer and completing the volume to 100 mL. Heat sterilize. Store at 4◦ C. 3. Thirty percent sucrose solution prepared by vol/vol dilution of the 60% sucrose with liposome buffer. Heat sterilize. Store at 4◦ C.
2.2.2. Ultracentrifugation 2.2.3. Size Exclusion Chromatography
Ultracentrifugation of small liposome samples may be performed on a Beckman Opti MaxTM ultracentrifuge or equivalent. 1. SepharoseTM 4B gel (Sigma-Aldrich) or equivalent. 2. Glass chromatography column. 3. Liposome buffer for column equilibration and elution.
2.3. Extraction and Determination of Liposomal Antigen
1. 0.15% deoxycholate (Sigma-Aldrich) in distilled water. 2. Seventy-two percent trichloroacetic acid (Sigma-Aldrich). 3. Diethyl ether/ethanol, 3:1 vol/vol (Sigma-Aldrich). Prepare and store at room temperature under a chemical hood. 4. MicroBCATM protein assay kit (Pierce). Store at 4◦ C.
Liposomal Adjuvants: Preparation and Formulation with Antigens
2.4. Equipment for the Characterization of Liposomes 2.4.1. HPLC of Lipids
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1. Gradient HPLC system equipped with an ELSD detector or variable wavelength detector and chromatographic software. 2. C18-grafted silica gel as a standard HPLC column but column should be adapted to the specific lipid composition (see Note 6). 3. Mobile phase: gradient of B [0.1% TFA in isopropanol] in A [0.1% TFA in water], as an example, but should be adapted to the specific lipid composition.
2.4.2. Particle Sizers
Liposome particle sizing may be performed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Alternatively, the Coulter N5 Submicron Particle Size Analyzer (Beckman Coulter, Inc., Fullerton, CA) or other particle sizers may be used.
2.4.3. Zetameter
Liposome surface charge may be measured by using a Malvern Zetamaster Nano Z or Nano ZS. Other zetameters may also be used.
2.4.4. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
1. Any system for SDS-PAGE may be used such as Criterion Cell Electrophoresis System with Power Pac HCTM power supply (Bio-Rad) and Ready Gel + appropriate running buffer system for SDS-PAGE (Bio-Rad). 2. Laemmli sample loading buffer containing the electrophoresis tracking dye Bromophenol Blue (Bio-Rad). 3. Quantity One software (or equivalent) for gel scanning and densitometric analysis of the bands (Bio-Rad).
3. Methods 3.1. Liposome Preparation and Association with Antigens
Depending on the manufacturing method, liposomes can be unilamellar (i.e., vesicles consisting of a single membrane bilayer) or multilamellar (i.e., vesicles consisting of a series of concentric lipid bilayers). Depending on their composition, bilayers can be in a “fluid” or “rigid” state at ambient temperature (Ta). The fluid state is generally achieved with lipids that have a gel–liquid crystalline transition temperature (Tc ) below Ta, whereas the rigid state requires lipids with a Tc above Ta. Antigens can be associated to liposomes in three ways. They can be attached to the outer surface, encapsulated within the internal aqueous spaces, or reconstituted within the lipid bilayers of the liposomes.
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3.1.1. Mixing Starting Lipid Ingredients
The preparation of liposomes generally starts with a dry lipid or mixture of lipids in the form of a powder or film obtained through the evaporation of a solution of this lipid or mixture of lipids in an organic solvent. The most commonly used solvent system to solubilize lipids is chloroform (or dichloromethane) containing 10–40% of methanol (see Note 7). To obtain a dry film of lipid 1. dissolve the lipid material in 5 mL of chloroform containing from 10 to 40% methanol in a 50 mL round-bottomed flask; 2. adapt the flask on a rotary evaporator and evaporate the solution as to leave a thin lipid film on the glass walls of the flask; 3. flush the rotavapor with nitrogen, remove the flask, and completely dry the lipid film by placing the flask overnight under high vacuum.
3.1.2. Preparation of Small Unilamellar Vesicles (SUVs) from Multilamellar Vesicles (MLVs)
1. Rehydrate the desiccated lipid material in 2–10 mL of prewarmed (>Tc ) distilled water or appropriate aqueous buffer. The total lipid concentration should be within the range of 0–400 mg/mL. 2. Place the flask in a temperature-controlled water bath and stir the mixture at >Tc until all the lipid material has transferred into a milky suspension. 3. Leave the suspension with stirring at >Tc for about 1 h, whereupon vesicles of diverse sizes, usually MLVs, are formed. Crude MLVs are usually not compatible with sterile filtration on a 0.2 μm membrane and need further processing for size reduction and homogenization. Two readily scalable processes for the size reduction of liposomes, extrusion and high-pressure homogenization (see Note 8), are described below but sonication methods (bath or probe sonication) can also be used for the size reduction of liposomes, at least at lab scale. 4. To reduce the size of MLVs by extrusion (19), transfer the MLV suspension into the prewarmed (>Tc ) chamber of a LipexTM extruder and extrude the liposomes under nitrogen pressure (100–700 psi) through two stacked polycarbonate filters of calibrated porosity (≤ 200 nm), according to the LipexTM extruder operating instructions. It may not be possible to extrude some preparations of MLVs straight through 0.2 or 0.1 μm filters. In this case, sequential extrusions through filters of decreasing porosity (0.8 μm– 0.4 μm–0.2 μm–0.1 μm) may be required. In all cases, several passes (up to seven depending on the lipid composition of the liposomes) through two stacked filters may
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be required to obtain a homogeneous suspension of mostly SUVs that is compatible with sterile filtration. 5. Alternatively, to reduce the size of MLVs by high-pressure homogenization (20), process the MLV suspension for a number of full cycles through the interaction chamber (at >Tc ) of the M-110Y MicrofluidizerTM or the EmulsiFlexTM C3 homogenizer. The number of cycles required to obtain liposomes that pass a sterile filter (≤ 0.2 μm) depends on the lipid composition of the liposomes. In some cases, a single cycle at moderate-high pressure (10,000 psi) may be sufficient to obtain sterile-filterable vesicles. 3.1.3. Preparation of SUVs Directly by Ethanol Injection
When the starting lipid or mixture of lipids is readily soluble in ethanol, it may be advantageous to use the method known as “ethanol injection technique” for the direct preparation of SUVs (21) (see Note 9). 1. Dissolve the desiccated lipid material in absolute ethanol to a concentrated stock solution. Heating (up to 60◦ C) may be necessary to increase solubility of the lipid(s) to concentrations close to the limit of saturation, as the injection step will dilute this ethanol solution by a factor of 20 (see below). 2. For the preparation of 2 mL of liposomes, place 1.9 mL of the aqueous phase (distilled water or appropriate buffer as described above) in a 5 mL jacketed glass vial and allow the temperature to equilibrate at >Tc by circulating water from a temperature-controlled water bath. 3. Place the vial on a magnetic stirrer and stir the aqueous phase vigorously at about 1,000 rpm by using a magnetic stirring bar. 4. Withdraw 100 μL of the concentrated lipid stock solution by using a standard gastight Hamilton syringe (volume <0.5 mL) with a Teflon plunger and a fine gauge needle (see Note 10). 5. Position the tip of the needle below the surface of the stirred aqueous phase in the center of the vial and inject the ethanol solution as rapidly as possible into the medium. 6. Stir the resulting liposome suspension for an additional 2 min at >Tc and collect the suspension for sterile filtration (see Note 11). 7. The lipid concentration in this liposome suspension will be 20 times less than in the ethanol stock solution and ethanol will be present in a final concentration of 5% v/v. At such low concentration, residual ethanol is compatible with parenteral pharmaceutical products (22) and should not interfere with liposome or antigen stability.
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3.1.4. Association of Antigens with Preformed Liposomes
Potent adjuvant effects can be obtained by simple mixing of antigen solutions with preformed liposomes (see Notes 12 and 13): 1. Simply mix the sterile-filtered antigen solution(s) with sterile-filtered liposomes by adding the appropriate volume of antigen to the appropriate volume of liposomes in aseptic conditions into a glass vial under a flow-hood. 2. Homogenize the mixture by gently stirring or shaking the vial.
3.1.5. Encapsulation of Soluble Antigens into Small Liposomes
Entrapment of antigens into small vesicles may be carried out by the “dehydration–rehydration” procedure in the presence of sucrose (23) (see Note 14). 1. Mix the solution of SUVs as prepared in Section 3.1.2 or 3.1.3 with the solution containing the antigen(s) and sucrose at a concentration of 3 mg per mg of lipid (see Note 15). 2. Sample the mixture into freeze-drying vials and freeze-dry overnight under vacuum (< 0.1 Torr). 3. Rehydrate the resulting powder at a temperature >Tc with as little volume of water as possible (ca. 5 μL/mg lipid) until a wet slurry is formed and allow the slurry to stand at >Tc for 60 min. 4. Dilute the slurry twice in water at >Tc and further incubate for 30 min. 5. Make up the resulting liposome suspension to its final concentration by dilution with the appropriate buffer and filter through a 0.2 μm filter for sterilization. If the reconstituted liposomes are too large for sterile filtration, they can be extruded in the presence of non-entrapped material to reduce their size to less than 200 nm in diameter, with much of the originally entrapped antigen remaining associated with the vesicles.
3.1.6. Reconstitution of Hydrophobic Antigens into Liposomes by the Detergent Removal Technique
Some antigens, especially membrane proteins, are solubilized by using detergents and chaotropic agents such as urea or arginine. Liposomes containing such antigens can be formed by the “detergent removal method” to produce a detergent-free formulation. The detergent should have a high critical micelle concentration (CMC), so that it can easily be removed by dialysis. N-octyl-β-Dglucopyrannoside (OG), with a CMC of 25 mM in water, is often used (24) (see Note 16). 1. Solubilize the SUVs as prepared in Section 3.1.2 or 3.1.3 to obtain a clear solution of mixed lipid/detergent micelles by stepwise addition under stirring of solid OG (or from a
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concentrated stock solution) up to a detergent/lipid molar ratio of 10:1 (see Note 17). 2. Add the desired amount of antigen solution to the mixed lipid/detergent micelles under gentle stirring at an appropriate temperature for antigen stability. The solution should remain clear; if not, more detergent should be added until a clear solution is obtained. 3. Place the clear solution into a Spectra/Por dialysis tubing (molecular weight cut off < molecular weight of antigen) and dialyze at 5◦ C against 100–500 volumes of the selected liposome buffer over 36 h with one change of the external buffer at 16 h (see Note 18). 4. After the dialysis, collect the detergent-free suspension of liposomes (generally SUVs with nearly 100% of the lipophilic antigens inserted into their membranes), make up to its final concentration by dilution with the liposome buffer, and filter through a 0.2 μm filter for sterilization. If the reconstituted liposomes are too large for sterile filtration, their size can be reduced by extrusion through two (stacked) polycarbonate filters of <200 nm pore size under nitrogen pressure as described above. 3.2. Separation of Liposomal Antigen from Non-associated Antigen 3.2.1. Sucrose Gradient Method
Free antigen may be separated from the liposomes by floating the vesicles through a discontinuous sucrose gradient with 0–30 and 40% steps: 1. Mix 0.5 mL of liposome suspension with 1 mL of 60% sucrose and place in the bottom of a 5 mL-ultracentrifuge tube. 2. Carefully layer 3 mL of 30% sucrose over the mixture. 3. Layer 0.5 mL of liposome buffer over the 30% sucrose. 4. Spin at 100,000×g for 30 min in a swing-out rotor at room temperature, setting acceleration to minimum. 5. After centrifugation, collect the liposomes from the interface between the upper and the middle layers. (With care, liposomes may be aspirated in 0.5 mL).
3.2.2. Ultracentrifugation Method
Free antigen may also be separated from antigen-containing liposomes by ultracentrifugation. 1. Centrifuge the liposome sample at ≥100,000×g for 1 h and remove supernatant containing free antigen. 2. Wash the liposomal pellet in liposome buffer by centrifugation as above.
3.2.3. Gel Chromatography Method
Free antigen may also be separated from the liposomes by using gel chromatography on a size exclusion column (e.g.,
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SepharoseTM 4B gel). In this procedure, liposomes which are excluded from the gel are eluted first, followed by the free antigen, which is included in the gel. 3.3. Extraction of Antigen from Liposomal Formulation
There is no unerring method for the extraction of antigens from liposomal formulations but the following method is routinely used for the extraction of proteins from lipid matrixes prior applying the microBCA protein assay (25). 1. Adjust sample volume containing between 5 and 100 μg of proteins to a final volume of 1 mL with distilled water. 2. Add 0.1 mL of 0.15% deoxycholate solution, vortex, and incubate for 10 min at room temperature (ca. 20◦ C). 3. Add 0.1 mL of 72% trichloroacetic acid, vortex, and centrifuge for 15 min in an Eppendorf bench top centrifuge at 3,000×g. 4. Discard supernatant and wash pellet in 1 mL diethyl ether/ethanol (3:1 vol/vol). 5. Recover protein by centrifugation for 45 min at maximum speed (≥ 9,000×g) in an Eppendorf bench top centrifuge.
3.4. Characterization and Quality Control of Liposomes and Liposomal Vaccines
Appropriate analytical methods are important for the documentation of the quality and stability of liposomal vaccines. Typical test methods for the characterization of liposomal vaccine formulations are listed in Table 6.1 with reference to the European Pharmacopoeia when available. Besides the characterization of appearance, pH, osmolality, endotoxin content (see Note 19), and sterility that are standard to all parenteral products, liposomal products should also be characterized through their lipid composition, particle size distribution, zeta potential, and antigen load. Stability studies comprise the documentation of the chemical stability of the antigen and of the liposomal lipids, the documentation of the physical (colloidal stability) of the liposomal product, and the documentation of the stability of the antigen– liposome association over time. Stability studies should be carried out at the normal storage temperature of the vaccine (usually 5◦ C) but accelerated stability may also be performed under temperature stress (25◦ C, 37◦ C, and 45◦ C) to select the most stable formulations. Liposomal vaccines should be stable for at least 18 months at 5◦ C.
3.4.1. Lipid Composition
Lipid composition is generally analyzed by HPLC techniques that need to be optimized for the specific lipid ingredients of the formulation. RP-HPLC is often used as the method of choice but IE-HPLC may also be useful when charged lipids are present. An ideal HPLC technique would detect not only the starting lipid
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Table 6.1 Typical test methods for the characterization of liposomal vaccine Test
Method
Appearance
Visual inspection (Eur. Ph. 2.9.20)
pH
Amperometry (Eur. Ph. 2.2.3)
Osmolality
Osmometry (Eur. Ph. 2.2.35)
Endotoxin content
LAL test (see Note 19)
Bioburden
Bacterial and fungal sterility (Eur. Ph. 2.6.1 21 CFR 610.12)
Lipid composition (and byproducts from lipid decomposition)
RP-HPLC (or IE-HPLC when charged lipids are used)
Particle size distribution
Laser light scattering
Particle surface charge
Zetametry
Residual ethanol (for liposomes prepared by the ethanol injection method) Residual octyl glucoside (for liposomes made by an octyl glucoside dialysis method)
Gas chromatography
Total antigen content
Protein assay or specific antigen assay Protein assay or specific antigen assay after purification of liposomal antigen
Amount of antigen associated to liposomes
HPLC with pulsed amperometric detection
material but also the major degradation products generated upon potential chemical decomposition of the starting lipids. 3.4.2. Particle Sizing
Particle size distribution (PSD) of liposome samples is usually analyzed by dynamic light scattering (DLS) sometimes also referred to as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS). The instruments used to perform the PSD of liposome samples operate in the submicronic to a few micrometers range and measure the hydrodynamic diameter of the particles in solution which refers to how the particles diffuse within the medium. This means that the size measured by this technique can be slightly larger than measured by electron microscopy, for example.
3.4.3. Zeta Potential
Zeta potential (i.e., particle surface charge at a given pH) is useful to predict how individual colloids will interact with one another. A charged surface will create repulsive forces between the liposomes
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and prevent liposomes from aggregating into larger, faster settling agglomerates. Zeta potential may also be used to demonstrate the electrostatic adsorption of antigens to the surface of liposomes, as the zeta potential of the liposomes is expected to change upon adsorption of antigens. The zetameters used for the determination of liposome surface charge operate by measuring the electrophoretic mobility of liposomes.
3.4.4. Total Antigen Content and Amount of Antigen Associated to the Liposomes
Antigen determination within liposomal products may be carried out by using SDS-PAGE and densitometric analysis (see Note 20). 1. Pretreat sample with 1 volume of 10% SDS for 5 min at 100◦ C. Depending on liposome composition this step may be omitted. 2. Add 1 volume of loading buffer and heat for 2 min at 100◦ C. 3. Load and run onto polyacrylamide gel. 4. Scan gel for densitometric analysis of protein bands. The amount of antigen that is actually associated to the liposomes should be measured as above but in purified liposomes: 1. Remove non-associated antigen from liposomes as described in Section 3.2. 2. Determine liposome-associated antigen by SDS-PAGE. 3. Determine the lipid content of the purified liposome sample to verify the quantitative recovery of the liposomes after the purification step. 4. Express liposomal antigen as “amount of antigen/mass of lipid.”
3.5. Use in Preclinical Studies
As for any adjuvanted vaccine, immunization with liposomal vaccines requires careful consideration of the appropriate doses and injection volumes for the selected route of administration and species to be immunized. Clinical studies with parenteral vaccines in humans usually involve injection doses of 0.5 mL by the subcutaneous (SC) and intramuscular (IM) routes and doses of 0.1 mL by the intradermal (ID) route. To ensure that the concentration of all formulation ingredients will be the same in preclinical and clinical testing a volume fraction of a human dose should be used in animal efficacy studies. In mice experiments, for instance, we typically use 1/10th of a human dose (i.e., 50 μL for SC and IM immunization and 10 μL for ID immunization). In rats and guinea pigs we use up to one-half of a human dose and in rabbits and monkeys we use up to a full human dose (see Note 21).
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For the liposomal vaccines, separation of free antigen from liposomal antigen is necessary for product characterization but may not be needed to obtain a good immune response. Wherever possible, this purification step should be avoided to facilitate product development and larger-scale manufacturing, but the manufacturing process should be robust enough to yield a consistent ratio of free antigen to liposomal antigen.
4. Notes 1. The cationic liposomes can also be used in combination with immunostimulating nucleic acids in the form of lipoplexes (i.e., small particles in the form of charge complexes between the negatively charged nucleic acid and the cationic liposome). Such lipoplexes are usually more potent adjuvants than the “naked” nucleic acids per se (26, 27). The mechanisms by which cationic liposomes interfere with the immune system have been studied in references (28, 29). 2. For the preparation of vaccine compositions, especially when human use is foreseen, the starting lipids used as liposomal ingredients will need to be obtained with a high degree of purity and with a solid batch-to-batch consistency (i.e., consistency of purity and of impurity profile). In this respect, the synthetic materials and the highly purified natural lipid extracts may be preferred, as they are produced in very controlled manners. 3. Liposome buffer should be selected with respect to lipid and antigen stability data. 4. Although M-110Y and EmulsiFlex C3 are designed as labscale equipment, they require a minimum sample volume of 10–15 mL (determined by the dead space of the pump, interaction chamber, etc.) to operate properly. Thus larger volume samples or sample dilution may be required. 5. Filtration yields will depend on both the liposome and the filter composition and should be assessed by quantitative material loss to select the best filter for a given liposome composition. 6. For charged lipids, IE-HPLC using an appropriate ionexchange column may be advantageous. 7. Special care should be given to the removal of solvents from the starting lipids as trace amounts of residual solvents, especially chlorinated solvents, may interfere with the stability of the resulting liposomes. In addition, from a regulatory standpoint, vaccine adjuvants should, as phar-
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maceutical excipients, comply with the specifications on residual solvents in accordance with ICH guideline on residual solvents: CPMP/ICH/283/95. When the starting lipid material is soluble in ethanol, ethanol should be the preferred solvent as this would allow liposome manufacturing from an ethanol-dried lipid mixture or directly through the “ethanol injection procedure.” 8. The high-pressure homogenization techniques have been scaled to industrial scales by designing large-scale homogenizers (30), and the liposome extrusion technique has been scaled up to industrial scales by the introduction of a continuous high-pressure filter extruder (31). 9. The ethanol injection method has the advantage of being simple and comes with a very low risk of inducing degradation of sensitive lipids because there is no high shearing or sonication involved. Its major shortcoming is the limitation of the solubility of lipids in ethanol. For the less soluble lipids, higher amounts of ethanol may be injected in the aqueous phase, but in this case a dialysis or diafiltration step will be required at the end of the process to remove ethanol from the bulk product. Residual ethanol can be accurately determined by gas chromatography. A variation of this technique based on “crossflow ethanol injection” useful for the production of larger batches of liposomes has been described (32). 10. For some lipids or mixture of lipids, the syringe may require to be heated to maintain a clear ethanol solution of lipids. This is achieved by placing the syringe into a glass barrel (custom-made) allowing heating of the syringe through the circulation of water from a temperature-controlled water bath. A piece of plastic tubing wound around the syringe may also be used instead of the custom-made glass barrel. 11. The ethanol injection method generally yields liposomes that pass through 0.2 μm filters without further processing. However, the liposome characteristics will also depend on the type of lipid(s), ionic strength of the medium, speed of injection, and degree of mechanical agitation during injection. 12. To provide the physical association of antigen with liposomes that is necessary for optimal delivery and adjuvantation, the liposome composition is selected in such a way as to favor the interaction with the antigens. For instance, if the antigen is negatively charged, it will be advantageous to have a cationic liposome to associate most of this antigen with the liposomes.
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Fluid liposomes, made of lipids with low Tc , may also be more prone than “rigid” membranes to accommodate antigens through hydrophobic interactions. In some specific cases, where the antigenic material is composed of proteins/peptides bearing a 6His-tag, an active binding can be promoted by inclusion of a small proportion (1–5%) of the chelator lipid, NTA3-DTDA (Avanti Polar Lipids), in the liposome. Active binding of the His-tagged antigen to the chelator lipid-containing liposomes occurs upon simple incubation in a buffer containing an appropriate amount of Ni2+ , usually in the form of NiSO4 . Methods for the covalent coupling of antigens to preformed liposomes have also been developed. These methods are reviewed in reference (33). 13. The simple mixing procedure is advantageous in terms of product development as it facilitates dose-finding studies by simply mixing different doses of liposomes with a selected dose of antigen (and vice versa) in preclinical and clinical development. It is also advantageous in terms of manufacturing and stability as antigens and liposomes are processed independently and the antigens are not submitted to liposome processing methods that may involve conditions that are detrimental to antigen stability. 14. The dehydration–rehydration procedure, in the presence of sugar at concentrations which are below those known to preserve the stability of vesicles during rehydration, has been described to destabilize SUVs just enough to allow the entry of solutes while they reform essentially to their original state or as modestly larger vesicles. When used for peptide or protein encapsulation, this method usually yields encapsulation rates from 20 to 90% (depending on the nature of the lipid and antigen used) in liposomes compatible with sterilization by filtration (23). 15. Optimization of the sucrose/lipid ratio from 0 to 10 mg sucrose/mg lipid may be required to obtain high encapsulation rates. Typically, 3 mg sucrose per mg lipid is found as a ratio compromising high solute entrapment with preservation of liposome size. 16. The detergent removal method is certainly the best general method for preparing liposomes with lipophilic proteins inserted into the membranes. At the same time this formulation process allows for the removal of chaotropic or denaturizing agents from some antigen solutions and for the refolding of the membrane associated-protein antigens into lipid bilayers, which is important with respect to exposing the immune system to antigens in their native conformation. However, the factors determining the size of
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liposomes using this method are quite complex, and when using new combinations of lipids and antigens, a process of optimization with different lipid/detergent ratios, different buffers, different types of semi-permeable membranes, and potentially different detergents (e.g., different alkyl glycosides), is advisable in order to obtain vesicles of the desired size. When using this technique for new lipid–antigen combinations, high detergent to lipid ratio in the range of 10:1 and above should be preferred to start with, as the excess detergent should help in the solubilization of antigen and prevent the formation of lipid aggregates and MLVs upon non-controlled dialysis. Upon optimization of the dialysis conditions (dialysis flow rate, stirring, temperature, etc.), the detergent to lipid ratio may be lowered as to spare on detergent. Trace amounts of residual OG in liposomal formulations are accurately determined by highperformance anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection such as available from Dionex (Sunnyvale, CA, USA). The detergent dialysis technique can easily be upscaled by using diafiltration instead of dialysis for detergent removal (34). The two marketed liposomal vaccines, Epaxal BernaTM (against Hepatitis A) and Inflexal Berna VTM (against influenza) (Crucell, Leiden, The Netherlands), both based on influenza virosomes, are manufactured by the detergent removal technique (35). 17. Solubilization of the liposomes may sometimes require less or more detergent and heating to 60◦ C or bath sonication, depending on the lipid composition and concentration. It can be followed by eye or by turbidity measurements with a spectrophotometer at 500 nm for instance. 18. Removal of detergent may also be performed by column techniques, either by standard gel filtration chromatography or by affinity chromatography using SM2 Biodeads (Bio-Rad) to adsorb the detergent. 19. LAL assay may not always be applicable as incorporation of the lipopolysaccharide into liposome membranes may lead to false-negative results (36). 20. Antigen-specific capture ELISAs may also be developed. These can be used directly for antigen quantitation without prior treatment of the liposome samples as ELISA incubation buffers and washing solutions contain sufficient amounts of detergent (e.g., Tween 20) to destroy the liposomes (17).
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Antigen content may also be determined after extraction from the lipid matrix as described in Section 3.3 by using the microBCA protein assay, or by the absorbance of aromatic side chains at 280 nm, or other more specific assays such as antigen-specific HPLC or capillary electrophoresis (CE) methods. 21. Using a volume fraction of a human dose in animal experiments allows for the careful optimization of the antigen/adjuvant/excipient ratios in preclinical studies. As the standard calculation for dose extrapolation used in the drug industry (mg drug/body weight) does not apply in the vaccine field, the antigen/adjuvant/excipient ratio remains the sole parameter that can be transposed directly from preclinical testing to clinical trials. References 1. Pashine, A., Valiante, N. M., Ulmer, J. (2005) Targeting the innate immune response with improved vaccine adjuvants. Nat Med 11, S63–S68. 2. Guy, B. (2007) The perfect mix: recent progress in adjuvant research. Nat Rev Microbiol 5, 505–517. 3. Alving, C. R. (1991) Liposomes as carriers of antigens and adjuvants. J Immunol Methods 140, 1–13. 4. Glück, R. (1995) Liposomal presentation of antigens for human vaccines, in (Powell, M. F., Newman, M. J., eds.) Vaccine Design: The Subunit and Adjuvant Approach. Plenum Press, New York, pp 325–343. 5. Mori, M., Nishida, M., Maekawa, N., Yamamura, H., Tanaka, Y., Kasai, M., Taneichi, M., Uchida, T. (2005) An increased adjuvanticity of liposomes by the inclusion of phosphatidylserine in immunization with surface-coupled liposomal antigen. Int Arch Allergy Immunol 136, 83–89. 6. Perrin-Cocon, L., Agaugué, S., Coutant, F., Saint-Mezar, P., Guironnet-Paquet, A., Nicolas, J. F., Andre, P., Lotteau, V. (2006) Lysophosphatidylcholine is a natural adjuvant that initiates cellular immune responses. Vaccine 24, 1254–1263. 7. Lipford, G., Wagner, H., Heeg, K. (1994) Vaccination with immunodominant peptides encapsulated in Quil A-containing liposomes induces peptide-specific primary CD8+ cytotoxic T-cells. Vaccine 12, 73–80. 8. White, K., Rades, T., Kearns, P., Toth, I., Hook, S. (2006) Immunogenicity of liposomes containing lipid core peptides and the adjuvant Quil A. Pharm Res 23, 1473–1481. 9. Garcon, N., Chomez, P., Van Mechelen, M. (2007) GlaxoSmithKline adjuvant sys-
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Haensler immunostimulatory vaccine delivery system. Curr Drug Deliv 2, 407–421. Guy, B., Pascal, N., Francon, A., Bonnin, A., Gimenez, S., Lafay-Vialon, E., Trannoy, E., Haensler, J. (2001) Design, characterization and preclinical efficacy of a cationic lipid adjuvant for influenza split vaccine. Vaccine 19, 1794–1805. Chen, W., Yan, W., Huang, L. (2008) A simple but effective cancer vaccine consisting of an antigen and a cationic lipid. Cancer Immunol Immunother 57, 517–530. Mayer, L. D., Hope, M. J., Cullis, P. R. (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochem Biophys Acta 858, 161–168. Mayhew, E., Lazo, R., Vail, W. J., King, J., Green, A. M. (1984) Characterization of liposomes produced using a microemulsifier. Biochim Biophys Acta 775, 169–174. Batzri, S., Korn, E. D. (1973) Single bilayer liposomes prepared without sonication. Biochim Biophys Acta 298, 1015–1019. Zadi, B., Gregoriadis, G. (2000) A novel method for high-yield entrapment of solutes into small liposomes. J Liposome Res 10, 73–80. Schwarz, D., Zirwer, D., Gast, K., Meyer, H. W., Lachmann, U. (1988) Preparation and properties of large octylglucoside dialysis/adsorption liposomes. Biomed Biochim Acta 47, 609–621. Brown, R. E., Jarvis, K. L., Hyland, K. J. (1989) Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal Biochem 180, 136–139. Zaks, K., Jordan, M., Guth, A., Sellins, K., Kedl, R., Izzo, A., Bosio, C., Dow, S. (2006) Efficient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes. J Immunol 176, 7335–7345. Dow, S. W. (2008) Liposome-nucleic acid immunotherapeutics. Expert Opin Drug Deliv 5, 11–24.
27. Vangasseri, D. P., Cui, Z., Chen, W., Hokey, D. A., Falo, L. D., Jr., Huang, L. (2006) Immunostimulation of dendritic cells by cationic liposomes. Mol Membr Biol 23, 385–395. 28. Lonez, C., Vandenbranden, M., Ruysschaert, J.-M. (2008) Cationic liposomal lipids: from gene carriers to cell signaling. Prog Lipid Res 47, 340–347. 29. Brandl, M., Bachmann, D., Drechsler, M., Bauer, K. H. (1993) Liposome preparation using high pressure homogenizers, in (Gregoriadis, G., ed.) Liposome Technology, 2nd edn. CRC Press, Boca Raton, vol I, pp 49–65. 30. Schneider, T., Sachse, A., Rössling, G., Brandl, M. (1994) Large-scale production of liposomes of defined size by a new continuous high-pressure extrusion device. Drug Dev Ind Pharm 20, 2787–2807. 31. Wagner, A., Vorauer-Uhl, K., Katinger, H. (2002) Liposomes produced in a pilot scale: production, purification and efficiency aspects. Eur J Pharm Biopharm 54, 213–219. 32. Lewis, S. J. (1994) (Wade, A., Weller, P. J., eds.) Handbook of Pharmaceutical Excipients, 2nd edn. The Pharmaceutical Press, London. 33. Altin, J. G., Parish, C. R. (2006) Liposomal vaccines – targeting the delivery of antigen. Methods 40, 39–52. 34. Weder, H. G., Zumbuehl, O. (1984) The preparation of variably sized homogeneous liposomes for laboratory, clinical and industrial use by controlled detergent dialysis, in (Gregoriadis, G., ed.) Liposome Technology. CRC Press, Boca Raton, vol III, pp 79–106. 35. Glück, R. (1999) Adjuvant activity of immunopotentiating reconstituted influenza virosomes (IRIVs). Vaccine 17, 1782–1787. 36. Schmidtgen, M., Brandl, M. (1995) Detection of lipopolysaccharides in phospholipids and liposomes by using the limulus test. J Liposome Res 5, 109–116.
Chapter 7 Micro/Nanoparticle Adjuvants: Preparation and Formulation with Antigens Padma Malyala and Manmohan Singh Abstract Recombinant proteins are increasingly being used as a novel approach for antigens in vaccines. These genetically engineered antigens are poorly immunogenic and require a delivery system and adjuvant to elicit their effect at targeted site of action. A delivery system transports the antigen to site of action and an adjuvant activates the cells via interaction with cell receptors and enhances the potency of the antigen. Micro/nanoparticles made from biodegradable and biocompatible polyesters, polylactide-co-glycolides (PLG), have been extensively used as an adjuvant and delivery system. This chapter discusses the applications of PLG micro/nanoparticles as delivery systems and adjuvant for antigens. PLG microparticles are prepared by a solvent evaporation method while nanoparticles are prepared by solvent displacement method. Synthesis of PLG nanoparticles is simpler in comparison to microparticles and unlike microparticles, it also enables particles to be sterile filtered. In a direct comparison using mouse animal model, our group found that microparticles and nanoparticles exhibited similar immunogenic responses. Materials and methods for synthesis and characterization of micro/nanoparticles with adsorbed antigens are discussed in detail. Key words: PLG, microparticles, nanoparticles, antigen, adjuvant, adjuvants, delivery vehicle.
1. Introduction Recombinant proteins are increasingly being used as a novel approach for antigens in vaccines. While these genetically engineered antigens are safer when compared to traditional vaccines, they are poorly immunogenic and require a delivery system and adjuvant to elicit their effect at targeted site of action. A delivery system transports the antigen to site of action, which are the cells responsible for induction of immune responses. An adjuvant G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_7, © Springer Science+Business Media, LLC 2010
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activates the cells via interaction with cell receptors and enhances the potency of the antigen. Several particulate systems have been evaluated as vaccine delivery systems and adjuvants (1). Particulate systems prepared from biodegradable polymers such as hyaluronic acid and poly (lactide-co-glycolide) (PLG), chitosan, and starch are a few examples that have been investigated as delivery systems (2). Micro/nanoparticles made from biodegradable and biocompatible polyesters, poly (lactide-co-glycolide) (PLG), have been extensively used as an adjuvant and delivery system. This polymer has a long safety profile in humans as it has been used in resorbable sutures and controlled-release drug delivery. These particulate systems function effectively as delivery systems due to their uptake by antigen presenting cells (3). PLG particles of size 5 μm and lesser are similar in size to pathogens and are taken up by phagocytic antigen presenting cells (APCs) (4–6). Particles are then carried to lymph nodes by these macrophages, which mature into dendritic cells (DCs), in addition to direct uptake by DCs (4–6). The particles not only promote trapping and retention of antigens in local lymph nodes but also present multiple copies of antigens to the immune system by particulate delivery systems (7). Several groups have reported that antigens adsorbed or encapsulated with PLG micro/nanoparticles induce potent antibody and CTL responses in mice, rodents, and nonhuman primates (1, 7–10). Our group has used PLG micro/nanoparticles as a delivery system by preparing charged micro/nanoparticles and adsorbing antigen on surface of the particles (7, 11–14). Depending on the charge of antigen, cationic or anionic particles are used for adsorption by electrostatic attraction (14). Antigen adsorption is influenced by several factors including electrostatic attraction and hydrophobic forces (14). Adsorption on surface of particles instead of encapsulation helps preserve the structure of the antigen and prevents degradation and denaturation of antigen during the synthesis of micro/nanoparticles (2). Some antigens we evaluated with PLG particles are HIV gp140, recombinant proteins from Neisseria meningitides, and HIV gag DNA (5, 15–17). Microparticles are prepared by double emulsification using solvent evaporation method (17). Briefly, primary emulsion of water in oil is prepared by homogenization with a probe, which is then added to a water phase and emulsified by homogenization, resulting in water-in-oil-in-water emulsion. The solvent is then allowed to evaporate resulting in a suspension of PLG microparticles in water. In comparison to microparticles, synthesis of PLG nanoparticles is simpler and prepared by solvent displacement method (8). Nanoparticles are prepared under magnetic stirring by addition of oil phase to water. Organic solvent is allowed to evaporate, yielding PLG nanoparticles. This process also enables
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particles to be sterile filtered through a 0.2 μm filter. Size of PLG nanoparticles prepared by our group is typically around 100 nm while microparticles are around 1 μm. A direct comparison of PLG microparticles and nanoparticles was made by several groups using systemic and mucosal administrations, and with different antigens (8). Conclusions from these studies were varied; some groups showed microparticles were better than nanoparticles while some showed the latter is better (7). Our group conducted three studies with two protein antigens and found that microparticles and nanoparticles exhibited similar responses (7). Formulation process for PLG micro/nanoparticles as a delivery system for antigens is discussed in the sections below. Antigens described in the methods are Escherichia coli-derived recombinant meningococcal vaccine candidate MB1 for Neisseria meningitides serotype B (IRIS, Novartis Corporation, S.r.l., Siena, Italy) for protein antigens (7) and HIV-1 pCMVkm p55 gag plasmid (made at Chiron) for DNA plasmid antigens (5). In the description for encapsulated PLG microparticles, CpG is used as an example for encapsulated adjuvant (17). Instruments specified are those that have been used by our group and can be replaced with other suitable instruments. A detailed description of the materials and methods for synthesis and characterization of micro/nanoparticles along with critical notes is given.
2. Materials 2.1. Synthesis of Anionic Blank PLG Microparticles
1. RG503, poly (D,L-lactide-co-glycolide) 50:50 copolymer composition (intrinsic viscosity 0.4 from manufacturer’s specifications) (Boehringer Ingelheim) 2. Dioctylsulfosuccinate (Sigma Chemical, St. Louis, MO) 3. PBS buffer 4. Methylene chloride (Sigma Chemical, St. Louis, MO) 5. Water 6. Ultra-Turrax Germany)
T25
homogenizer
(IKA-Labortechnik,
7. ES-15 (Omni International, Jarrenton, VA) 8. Ice 2.2. Synthesis of Cationic Blank PLG Microparticles
1. RG504, poly (D,L-lactide-co-glycolide) 50:50 copolymer composition (molecular weight of 48,000 Da from manufacturer’s specifications) (Boehringer Ingelheim) 2. Methylene chloride (Sigma Chemical, St. Louis, MO)
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3. TE buffer: 10 mM Tris, Na+ , Cl– counterions (pH 8.0), 1 mM EDTA 4. Ultra-Turrax T25 homogenizer (IKA-Labortechnik, Germany) 5. Water 6. Cetyl trimethyl ammonium bromide (CTAB) (Sigma Chemical, St. Louis, MO) 7. ES-15 (Omni International, Jarrenton, VA) 8. Ice 2.3. Synthesis of Encapsulated PLG Microparticles
1. CpG oligonucleotide 1826, 5-TCC ATG ACG TTC CTG ACG TT-3, synthesized with phosphorothioate backbones (Oligos Etc., Wilsonville, OR) 2. Low molecular weight chitosan (Fluka—Sigma-Aldrich Chemie GmbH Industriestrasse 25 CH-9471 Buchs SG Switzerland) 3. TE buffer: 10 mM Tris, Na+ , Cl– counterions (pH 8.0), 1 mM EDTA 4. RG503, poly (D,L-lactide-co-glycolide) 50:50 copolymer composition (intrinsic viscosity 0.4 from manufacturer’s specifications) (Boehringer Ingelheim) 5. Methylene chloride (Sigma Chemical, St. Louis, MO) 6. Ultra-Turrax Germany)
T25
homogenizer
(IKA-Labortechnik,
7. Water 8. Dioctylsulfosuccinate (Sigma Chemical St. Louis, MO) 9. ES-15 homogenizer (Omni International, Jarrenton, VA) 2.4. Synthesis of Blank PLG Nanoparticles
1. RG503, poly (D,L-lactide-co-glycolide) 50:50 copolymer composition (intrinsic viscosity 0.4 from manufacturer’s specifications) (Boehringer Ingelheim) 2. Acetone 3. Water 4. Acrodisc 0.2 μm Supor membrane syringe filters (Pall, East Hills, NY)
2.5. Adsorption of Protein Antigens
1. Escherichia coli-derived recombinant meningococcal vaccine candidate MB1 for Neisseria meningitides serotype B (IRIS, Novartis Corporation, S.r.l., Siena, Italy) 2. Recombinant glycoprotein from HIV (Novartis Corporation) 3. Concentrated histidine buffer: 100 mM counterion, pH 5.0
L -histidine,
Na+
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4. Concentrated (10x) PBS (Novartis Corporation) 5. Lab rocker (aliquot mixer, Miles Laboratories) 2.6. Adsorption of DNA Plasmid Antigens 2.7. Lyophilization of PLG/Antigen Formulations
1. HIV-1 pCMVkm p55 gag plasmid (made at Novartis Corporation) 1. Sucrose 2. Mannitol 3. Polyvinyl alcohol (PVA) (MW = 15,000) (MP Biomedicals, Irvine, CA) 4. FreeZone 4.5 L benchtop freeze dry system (LABCONCO, Kansas City, MO)
2.8. Characterization of Formulations
1. Horiba LA-930 (Irvine, CA) 2. Malvern zeta analyzer 3000 HSA (Malvern Instruments, UK) 3. Wescor osmometer 4. Endotoxin materials as specified by USP 5. SDS-PAGE and agarose gel electrophoresis 6. HPLC
3. Methods 3.1. Synthesis of Anionic PLG Microparticles
Microparticles are prepared by the solvent evaporation method (Fig. 7.1). 1. Homogenize 30 mL of 6% w/v PLG polymer solution in methylene chloride with 6 mL of PBS using a clean 10-mm probe in the Ultra-Turrax T25 homogenizer (see Notes 1 and 2). The water to oil ratio in the primary emulsion is 1:5 and homogenization is at 24,000 rpm for 2 min. 2. Add the water-in-oil emulsion thus formed to 150 mL of distilled water containing dioctylsulfosuccinate surfactant (0.5% w/w PLG) and homogenize the mixture at 7,600 rpm with a 20-mm probe in the ES-15 homogenizer for 20 min in an ice bath. The oil to water ratio in the secondary emulsion process is 1:5. 3. This procedure results in the formation of water-in-oilin-water emulsion. Stir at 1,000 rpm for 12 h at room temperature. Allow the methylene chloride to evaporate (see Note 3). 4. Cap the bottle and store at 4◦ C with stirring.
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PLG-DCM (Organic Phase)
Homogenisation (w/o emulsion) 0.05 % DSS solution
w/o/w emulsion
Solvent Evaporation
PLG/ DSS Microparticles
Fig. 7.1. Preparation of blank PLG microparticles.
3.2. Synthesis of Cationic Blank PLG Microparticles
Microparticles are prepared by solvent evaporation method. 1. Homogenize 75 mL of 6% w/v PLG polymer solution in methylene chloride with 7.5 mL TE buffer using a clean 10-mm probe in the Ultra-Turrax T25 homogenizer (see Notes 1 and 2). The water to oil ratio in the primary emulsion is 1:10 and homogenization is at 24,000 rpm for 3 min. 2. Add the water-in-oil emulsion thus formed to 300 mL of distilled water containing CTAB surfactant (1% w/w PLG) and homogenize the mixture at 7,600 rpm with a 20-mm probe in the ES-15 homogenizer for 20 min in an ice bath. The oil to water ratio in the secondary emulsion process is 1:4. 3. This procedure results in the formation of water-in-oilin-water emulsion. Stir at 1,000 rpm for 12 h at room temperature. Allow the methylene chloride to evaporate (see Note 3). 4. Cap the bottle and store at 4◦ C with stirring.
3.3. Synthesis of Encapsulated PLG Microparticles
Encapsulated microparticles (Fig. 7.2) are prepared by solvent evaporation method. 1. Add 0.5% CpG (w/w PLG) to chitosan in a ratio of 1.4:1 and add to 6 mL of TE buffer (see Note 2). Homogenize 30 mL of 6% w/v PLG polymer solution in methylene chloride with 6 mL of TE buffer containing CpG–chitosan complex using a clean 10-mm probe in the Ultra-Turrax T25 homogenizer (see Note 1). The water to oil ratio in the primary emulsion is 1:5 and homogenization is at 24,000 rpm for 2 min.
Micro/Nanoparticle Adjuvants: Preparation and Formulation with Antigens
+ + Protei Protei + - -+ + - +n n + + + 1µm -+ +PLG -+ Protein +p Microparticle + +-+ +Prote + - - - -+ + Protei + + in n Protein
+ Protei + n IP + + - IP + - IP IP +IP IP + IP ++- - Protei + + n
PLG / Protein / IP PLG/Protein + IP The IP is added before or after the lyophilization l hili ti process
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+ Protei - -+ + - +n -+ -+ IP -+ Protein IP - + -+ Prote -+ -+ in
PLG /IP/ Protein The IP gets incorporated p into the PLG microparticles
Fig. 7.2. Anionic PLG microparticles with protein and immunopotentiator (IP).
2. Add the water-in-oil emulsion thus formed to 150 mL of distilled water containing surfactant (0.05% w/w PLG). Homogenize the mixture at 7,600 rpm with a 20-mm probe in the ES-15 homogenizer for 8 min in an ice bath. The oil to water ratio in the secondary emulsion process is 1:5. 3. This procedure results in the formation of water-in-oilin-water emulsion. Stir at 1,000 rpm for 12 h at room temperature. Allow the methylene chloride to evaporate (see Note 3). 4. Cap the bottle and store at 4◦ C with stirring. 3.4. Synthesis of Blank PLG Nanoparticles
PLG nanoparticles were prepared by the solvent displacement method (Fig. 7.3). 1. Add dropwise the organic phase comprising PLG dissolved in acetone to 50 mL of pure, distilled water with magnetic stirring. The concentration of polymer solution is chosen based on PLG content required. 2. Allow the acetone to evaporate overnight (see Note 3). 3. There was no surfactant present in the aqueous phase. 4. Sterilize particles by filtration.
3.5. Adsorption of Protein Antigens
1. Incubate a suspension containing 100 mg of blank anionic PLG microparticles or encapsulated microparticles or nanoparticles with protein antigen (1% w/w PLG). 2. Add concentrated buffer solution to affect a final concentration of 10 mM histidine buffer pH 5 for MB1 protein, and 1× PBS for gp120, in a 10 mL total volume. 3. Allow the suspension to mix overnight on a lab rocker at 4◦ C (see Note 4). 4. Remove about 1 mL of suspension for the determination of adsorption efficiency.
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Nanoparticles
Microparticles solvent evaporation
solvent displacement
W/O/W emulsion
O/W emulsion
dropwise addition PLG +
Acetone
Water 500 rpm
homogenize @ 2,000 rpm for 2 min
+
allow solvent to evaporate
aqueous phase: PBS nanoparticle suspension
oil phase: PLG in dichloromethane
add O/W to external water phase
homogenize @ 12,000 rpm for 8 min
microparticle suspension
Water + DSS
dioctyl sodium sulfosuccinate (DSS) at 0.05 % wt/ wt PLG
on ice bath allow solvent to evaporate
Fig. 7.3. Particle preparation methods: a comparison.
5. Aliquot volumes containing the required dose of protein into small glass vials. 3.6. Adsorption of DNA Plasmid Antigens
1. Take a suspension containing 100 mg of cationic blank PLG microparticles in a beaker. 2. Add DNA plasmid antigen at a typical load of 4% w/w PLG in a dropwise fashion to the PLG microparticles under constant stirring (see Note 5). 3. Allow the suspension to stir for 30 min after complete addition of the antigen. 4. Remove about 1 mL of suspension for the determination of adsorption efficiency. 5. Aliquot volumes containing required dose of antigen into small glass vials.
3.7. Lyophilization of PLG/Antigen Formulations
1. To the aliquots from Sections 3.5 and 3.6, Step 5, add mannitol and sucrose to affect a final concentration of 4.5 and 1.5%, respectively, upon reconstitution. 2. Cap the vials with a stretch plastic film. Prepare the vials for lyophilization by providing vents for evaporation on the film. 3. Freeze the vials at –20◦ C. 4. Lyophilize the vials at −50◦ C and 90×10−3 mbar. 5. Cap the vials after lyophilization and store at 4◦ C.
3.8. Characterization of Formulations (see Notes 6 and 7) 3.8.1. Sizing
1. Take 1 mL of PLG microparticles and add dropwise in a particle size analyzer until the particles are in the required range for transmission and obscuration of the instrument. Measure
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size of the particles following directions of the instrument. Record the size distribution of the microparticles. 2. Take about 20 μL of PLG nanoparticles and dilute up to 2 mL with purified water and place in sample cell of a Malvern zeta analyzer. Measure the size of the particles following the directions of the instrument. 3.8.2. Osmolarity
1. Calibrate the osmometer before sample analysis. 2. Take 10 μL of the formulation and add to sample holder. 3. Measure osmolarity as directed in the manual. 4. Osmolarity range should be 270–330 mOsm/kg.
3.8.3. Endotoxin
1. Use gel clot method as directed by USP.
3.8.4. SDS-PAGE and Agarose Gel Electrophoresis
1. Determine the antigen loading levels and integrity of antigen using SDS-PAGE and agarose gel electrophoresis. 2. Centrifuge 1 mL of samples in replicates at approximately 9,660×g. 3. Separate the supernatant from the pellet. 4. For protein antigens, load samples of supernatant and pellet, extracted with SDS sample buffer, along with controls in SDS-PAGE gels (4–20% gradient tris/glycine polyacrylamide gels). 5. For DNA plasmids, load samples of supernatant and pellet and follow standard protocol for agarose gel electrophoresis. 6. For protein samples, stain with a Coomassie stain as instructed in a manual for SDS-PAGE. 7. Compare with control after destaining and confirm integrity of protein as well as semiquantification of protein associated with supernatant and pellet.
3.8.5. HPLC
1. Determine the protein loading levels and integrity of protein using HPLC. 2. Centrifuge 1 mL of samples in replicates at approximately 9,660×g for 20 min. 3. Separate the supernatant from the pellet. 4. Load supernatant samples in HPLC along with relevant controls by a method suitable to protein in question. 5. Quantify amount of protein present in supernatant to determine protein adsorbed on particles.
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4. Notes 1. Take adequate care to ensure probes are clean before and after use. Failure to do so might result in a poor formation of microparticles (Sections 3.1, 3.2, and 3.3). 2. Buffers in the primary emulsion can be changed, based on formulation parameters (Sections 3.1, 3.2, and 3.3). 3. After evaporation of organic solvent, measure the volume of suspension. Add water, if required, to compensate for water evaporation losses (Sections 3.1, 3.2, 3.3, and 3.4). 4. Ensure gentle mixing of particles during protein adsorption to prevent aggregation (Section 3.5). 5. During DNA adsorption, add DNA slowly to ensure complete adsorption on to microparticles (Section 3.6). 6. Follow instructions set up by the instruments for analytical characterization. 7. Use relevant controls for all analytical characterization.
Acknowledgments The authors deeply acknowledge the contributions of Janet Wendorf and Aravind Chakrapani who worked extensively on formulation and characterization of PLG nanoparticles in their post doctoral tenure with us. Thanks are also due to the Vaccine Formulation and Delivery group in Novartis Corporation. References 1. O’Hagan, D. T., Singh, M., Ulmer, J. B. (2006) Microparticle-based technologies for vaccines. Methods 40, 10–19. 2. Malyala, P., Singh, M. (2008) Formulations and delivery systems for mucosal vaccines, in (Vajdy, M. ed.) Immunity Against Mucosal Pathogens. Springer Publications, Netherland, pp 499–512. 3. Maloy, K. J., Donachie, A. M., O’Hagan, D. T., Mowat, A. M. (1994) Induction of mucosal and systemic immune responses by immunization with ovalbumin entrapped in poly(lactide-co-glycolide) microparticles. Immunology 81, 661–667. 4. O’Hagan, S., Ulmer, J. (2004) Microparticles for the delivery of DNA vaccines. Immunol Rev 199, 191–200.
5. Singh, M., Fang, J. H., Kazzaz, J., et al. (2006) A modified process for preparing cationic polylactide-co-glycolide microparticles with adsorbed DNA. Int J Pharm 327, 1–5. 6. Tabata, Y., Ikada, Y. (1988) Macrophage phagocytosis of biodegradable microspheres composed of L-lactic acid/glycolic acid homo- and copolymers. J Biomed Mater Res 22, 837–858. 7. Wendorf, J., Chesko, J., Kazzaz, J., Vajdy, M., O’Hagan, D. T., Singh, M. (2008) A comparison of anionic nanoparticles and microparticles as vaccine delivery systems. Hum Vaccin 1, 43–48. 8. Diwan, M., Elamanchili, P., Cao, M., Samuel, J. (2004) Dose sparing of CpG
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10.
11.
12.
oligodeoxynucleotide vaccine adjuvants by nanoparticle delivery. Curr Drug Deliv 1, 405–412. Xie, H., Gursel, I., Ivins, B. E., et al. (2005) CpG oligodeoxynucleotides adsorbed onto polylactide-co-glycolide microparticles improve the immunogenicity and protective activity of the licensed anthrax vaccine. Infect Immun 73, 828–833. Hunter, S. K., Andracki, M. E., Krieg, A. M. (2001) Biodegradable microspheres containing group B Streptococcus vaccine: immune response in mice. Am J Obstet Gynecol 185, 1174–1179. Kazzaz, J., Singh, M., Ugozzoli, M., Chesko, J., Soenawan, E., O’Hagan, D. T. (2005) Encapsulation of the adjuvants MPL and RC529 in PLG microparticles enhance their potency. J Control Release 110, 566–573. Singh, M., Chesko, J., Kazzaz, J., Ugozzoli, M., Malyala, P., O‘Hagan, D. T. (2007) Surface-charged poly(lactide-co-glycolide) microparticles as novel antigen delivery systems, in (Singh M., ed.) Vaccine Adjuvants and Delivery Systems. John Wiley & Sons Inc., New York, pp 223–247.
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13. Singh, M., Kazzaz, J., Ugozzoli, M., Malyala, P., Chesko, J., O’Hagan, D. T. (2006) Polylactide-co-glycolide microparticles with surface adsorbed antigens as vaccine delivery systems. Curr Drug Deliv 3, 115–120. 14. Chesko, J., Kazzaz, J., Ugozzoli, M., O’Hagan, D. T., Singh, M. (2005) An investigation of the factors controlling the adsorption of protein antigens to anionic PLG microparticles. J Pharm Sci 94, 2510–2519. 15. Chesko, J., Kazzaz, J., Ugozzoli, M., et al. (2004) Adsorption of a novel recombinant glycoprotein from HIV (env gp120dv2) to anionic PLG microparticles retains structural integrity, while encapsulation in PLG microparticles does not. Pharm Res 21, 2148–2152. 16. Singh, M., Kazzaz, J., Ugozzoli, M., Chesko, J., O’Hagan, D. T. (2004) Charged polylactide co-glycolide microparticles as antigen delivery systems. Expert Opin Biol Ther 4, 483–491. 17. Malyala, P., Chesko, J., Ugozzoli, M., et al. (2008) The potency of the adjuvant, CpG oligos, is enhanced by encapsulation in PLG microparticles. J Pharm Sci 97, 1155–1164.
Chapter 8 Adjuvant Activity on Human Cells In Vitro Dominique De Wit and Michel Goldman Abstract Efficient vaccines against intracellular microbes or tumors will be based on innovative adjuvants able to induce efficient activation of dendritic cells. Indeed, natural or synthetic products activating Toll-like receptors (TLR) on dendritic cells (DCs) are currently in development for this purpose. Herein, we describe in vitro assays on human cells which might be useful for the preclinical screening and assessment of potential DC activators. Key words: Dendritic cells, TLR ligands, whole blood cells, HEK 293 cell line, adjuvants.
1. Introduction Antigens contained in vaccines are presented to lymphocytes in lymph nodes by specialized antigen-presenting cells named dendritic cells (because of their multiple long cytoplasmic expansions) (1). In order to elicit protective immune responses, dendritic cells (or their precursors) loaded with antigen at the site of vaccine injection must undergo a phenomenon of maturation followed by their migration to draining lymph nodes. Dendritic cell (DC) maturation and migration can be induced by microorganisms. For vaccines based on isolated antigens, these processes require the presence of a substance named adjuvant (2). Aluminum hydroxide (alum) has been used for this purpose for a long time, but it is only very recently that its mode of action has been clarified (3). Aluminum hydroxide induces the activation of a protein complex named inflammasome in antigen-presenting cells (4, 5). Although alum is quite efficient for the induction of antibody G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_8, © Springer Science+Business Media, LLC 2010
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responses in healthy individuals, it does not induce the CD8+ T cell-dependent and Th1-type responses needed to fight intracellular pathogens (6). Therefore, future advances in the field of vaccines require the identification of novel adjuvants able to elicit protective cell-mediated immune responses depending on CD8+ and CD4+ T lymphocytes (2). Since the instruction of T lymphocytes mainly depends on DCs, research on adjuvant candidates is primary focused on ligands of DC receptors (7). Among DC activators, ligands of the Toll-like receptors (TLR) are currently considered as the most attractive adjuvant candidates (8, 9). Receptors of the TLR family recognize microbial products and endogenous ligands released under stress conditions. In humans, 10 functional TLRs have been identified so far. They differ in their pattern of expression and the signaling pathways they are coupled to (10). Thus, TLR3 and TLR4 are expressed on DCs of myeloid origin, whereas TLR9 is expressed on plasmacytoid DCs (pDCs), the latter being specialized in the production of type I interferons upon viral infections. TLR7 and TLR8 are expressed on both types of DCs and like TLR9 agonists TLR7/8 ligands are potent type I interferon (IFN) inducers (11). Indeed, the responses of DCs to TLR engagement depend on signaling cascades which differ according to the TLR and the DC type considered (12). All TLRs except TLR3 are coupled to the adaptor proteins named myeloid differentiation primary-response protein 88 (MyD88) and MyD88 adapter-like protein (Mal) which initiate activation of mitogen-activated protein (MAP) kinases and nuclear translocation of the transcription factor NF-κB, resulting in transcription of genes controlling synthesis of key mediators of inflammatory responses. TLR3 and TLR4 are coupled to TIR domain—containing adaptor protein inducing interferon (IFN)-β (TRIF) and TRIF-related adaptor molecule (TRAM). In myeloid DCs (mDCs), the TRIF pathway downstream of TLR3 and TLR4 is critically involved in the activation of genes controlled by IFN-regulatory factors (IRF) 3 and 7, which include the genes encoding type I interferons and the p35 chain of IL-12 (13). Some associations of TLR ligands strongly synergize in the activation of DCs such as the combination of TLR4 and TLR7/8 ligands which might prove useful to enhance the activity of therapeutic vaccines against cancer or chronic infections. Indeed, recent observations suggest that adjuvants targeting TLR will be especially efficient in stimulating T lymphocytes with high avidity for target antigens (14). Several vaccines based on attenuated live or heat-killed viruses or bacteria contain TLR ligands acting as natural intrinsic adjuvants (8). For vaccines based on purified antigens, TLR ligands are currently considered as candidate adjuvants in replacement of or in addition to alum (15). Monophosphoryl lipid A (MPL), a detoxified form of lipopolysaccharide (LPS) which is included in one of the commercially available human papilloma virus (HPV)
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vaccines, is the prototypical example of new adjuvants targeting TLR4 (16). More recently, synthetic aminoalkyl glucosaminide phosphates (AGP) and synthetic triacylated lipid A mimetics targeting TLR4 were developed as potential MPL substitutes (17, 18). Since the development of future vaccines will depend on reliable assays to assess efficacy and safety of DC activators, it is important to define preclinical methods to screen candidate adjuvants on human cells. In this chapter, we will describe three distinct in vitro systems for the analysis of adjuvant effects on human dendritic cells, focusing on assays suitable for the assessment of TLR agonists. These systems are based on the use of either whole blood or monocytederived DCs generated in vitro or the HEK 293 human embryonic kidney cell line as shown in Fig. 8.1.
Adjuvant activity on circulating human DCs
Whole blood assays
« Ex vivo »
Monocyte-derived DCs
293 HEK cells
« myeloid DC model »
«TLR pathways»
Phenotype Cytokine production mRNA expression Signaling pathway analysis
Purification of mDCs, pDCs and monocytes
Signaling pathway analysis
Whole blood cells
Phenotype Cytokine production mRNA expression
Fig. 8.1. Three distinct in vitro systems for the analysis of adjuvant effects on human dendritic cells, focusing on assays suitable for the assessment of TLR agonists.
2. Materials 2.1. Monocyte-Derived Dendritic Cells 2.1.1. Preparation of Monocyte-Derived DCs and Incubation with TLR Ligands
1. DC culture medium: RPMI 1640 (BioWhittaker), supplemented with 2 mM L-glutamine, 20 μg/mL gentamicin, 50 μM 2-mercaptoethanol, 1% non-essential amino acids, and 10% fetal bovine serum (PAA) (see Note 1) 2. Human buffy coats or fresh blood
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3. Lymphoprep (Nycomed) 4. Anti-CD14 mAb coupled to magnetic beads and Automacs system (Miltenyi Biotec) 5. Recombinant GM-CSF (Gentaur) 6. Recombinant IL-4 (Gentaur) 7. FITC-conjugated anti-CD14 monoclonal antibody (Becton Dickinson) 8. Ultra-pure LPS from Escherichia coli 111:B4 (InvivoGen) 9. Resiquimod R-848 (Pharma Technologies) 10. Polyinosine–polycytidylic acid poly I:C (Roche Diagnostics). 2.1.2. Analysis of Ligand Activities on Monocyte-Derived DC Maturation
1. PBS with 0.1% (w/v) bovine serum albumin (BSA) 2. PE-conjugated monoclonal antibodies to CD80, CD83, CD86, CD40, CD54, and HLA-DR (Becton Dickinson) 3. PE-conjugated monoclonal antibodies to CCR7, DC-SIGN, DEC-205, and ICOS-L (Becton Dickinson)
2.1.3. Analysis of Ligand Activities on Cytokine Production by Monocyte-Derived DCs
1. IL-12p40, IL-12p70, IL-8, and IP-10 levels are determined by Duoset ELISAs (R&D Systems) 2. IL-6, TNF-α, and IL-10 production is tested by Cytoset ELISAs (Biosource) 3. IFN-α and IFN-β secretions are assayed using ELISA kits (Invitrogen) 4. IL-23 production is tested by a Quantikine ELISA kit from (R&D Systems) 5. A broader panel of cytokines and chemokines will be tested by multiplex bead array Luminex technique (Biosource) 6. TriPure reagent (Roche Diagnostics) 7. LightCycler-RNA Master Hybridization Probes (Roche Diagnostics) 8. LightCycler device (Roche Diagnostics)
2.2. Whole Blood Assays
1. Human heparinized whole blood
2.2.1. Stimulation of Human Blood Cells
3. CpG A 2006 for DC maturation: TCgTCgTTTTgTCgTTTTgTCgTT-3 (Tib MolBiol)
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4. CpG B 2216 for type I IFN production: ggGGGACGATCGTCgggggG-3 (Tib MolBiol)
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2. TLR ligands (see Section 2.1.1)
5. CpG C 2395 (combines the effects of CpG A and CpG B) 6. 5 -TCGTCGTTTTCGGCGCGCGCCG-3 (Tib MolBiol)
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1. FcR blocking antibodies (Miltenyi Biotec) 2. FITC-conjugated mAbs specific for CD3, CD16, CD19, CD56, and CD20 receptors (Becton Dickinson) 3. APC-conjugated anti-CD11c mAb (Becton Dickinson) 4. EDC-conjugated anti-HLADR mAb (Becton Dickinson) 5. PeCy5-conjugated anti-CD123 mAb (Becton Dickinson) 6. Pacific Blue-conjugated anti-CD14 mAb (Becton Dickinson) 7. PE-conjugated mAbs for CD80, CD83, CD86, CD40, and CD154 (Becton Dickinson) 8. FACS lysis buffer (Becton Dickinson) 9. PBS with 0.1% (w/v) BSA 10. Cell Fix solution (Becton Dickinson)
2.2.3. Cytokine Production in Whole Blood
1. ELISA or LUMINEX kits as described in Section 2.1.3 above 2. High sensitivity kit with a detection limit of less than 1 pg/mL for some cytokines, such as IL-12p70 (R&D) 3. Fluorochrome-conjugated mAb for IL-12p40, TNF-α, IL10, IL-6, and IP-10 4. PAXgene reagent (Qiagen) 5. MagNaPure LC mRNA Isolation kit (Roche Diagnostics)
2.2.4. Determination of Cell Types in Whole Blood Responsible for Cytokine Production
1. Ammonium chloride buffer (Becton Dickinson) 2. Brefeldin A (GolgiPlug, Becton Dickinson) 3. Cytofix/Cytoperm solution (Becton Dickinson) 4. FITC-conjugated monoclonal antibodies for cell membrane molecules (Becton Dickinson) 5. PE-conjugated monoclonal antibodies for IL-12p40, TNFα, IL-6, and IP-10 (Becton Dickinson)
2.2.5. Stimulation of Freshly Isolated DCs
1. Monoclonal antibodies for CD16, CD19, CD14, CD15, CD33, CD3, and Glycophoryn A coupled to magnetic beads (Miltenyi Biotec) 2. LD columns (Miltenyi Biotec) 3. Monoclonal antibody for BDCA-4 coupled to magnetic microbeads (Miltenyi Biotec) 4. MS columns (Miltenyi Biotec) 5. FITC-conjugated monoclonal antibody for BDCA-2 6. PE-conjugated monoclonal antibody for CD123
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7. DC culture medium: RPMI 1640 (BioWhittaker), supplemented with 2 mM L-glutamine, 20 μg/mL gentamicin, 50 μM 2-mercaptoethanol, 1% non-essential amino-acids, and 10% fetal bovine serum (PAA) 8. TLR ligands (see Section 2.1.1) 9. CpG A 2006 for DC maturation: 5 TCgTCgTTTTgTCgTTTTgTCgTT-3 (Tib MolBiol) 10. CpG B 2216 for type I IFN production: ggGGGACGATCGTCgggggG-3 (Tib MolBiol)
5 -
11. CpG C 2395 (combines the effects of CpG A and CpG B): 5 -TCGTCGTTTTCGGCGCGCGCCG-3 (Tib MolBiol) 12. Monoclonal antibodies for CD16, CD19, CD14, CD15, CD3, Glycophoryn A, and BDCA-4 coupled to magnetic beads (Miltenyi Biotec) 13. Biotin-conjugated monoclonal antibody for BDCA-1 14. Monoclonal antibody for biotin coupled to magnetic beads 15. FITC-conjugated monoclonal antibody for CD11c 16. PE-conjugated antibody for CD14 2.3. HEK 293 Cells
1. Complete DMEM medium: DMEM medium supplemented with 10% FCS (Gibco), 2 mM glutamine, and antibiotics (BioWhittaker) 2. Transfection with reporter luciferase gene activated by the nuclear transcription factor studied: NF-κB (pIL8-κB-Luc), IRFs (IRF3: pIFNb-Luc), or AP-1 (InvivoGen) 3. Accutase (PAA) 4. Dual luciferase assay system (Promega) 5. pRL-TK plasmid (Promega)
3. Methods 3.1. Monocyte-Derived DCs
3.1.1. Preparation of Monocyte-Derived DCs and Incubation with TLR Ligands
Romani et al. (19) described a now well-established method for the generation of significant numbers of DCs from human monocytes cultured for 6 days in the presence of IL-4 and GM-CSF. These DCs are considered as “myeloid” DCs. 1. Isolate peripheral blood mononuclear cells (PBMC) from buffy coats or fresh blood samples by density centrifugation on Lymphoprep and suspend in DC culture medium. 2. Isolate CD14+ monocytes from PBMC by positive selection using anti-CD14 mAb coupled to magnetic beads through the Automacs system.
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3. Culture 15 × 106 purified cells in 30 mL DC culture medium containing 800 U/mL recombinant GM-CSF and 500 U/mL rIL-4 in large Falcon flasks. After 3 days, resupplement the cultures with 800 U/mL rGM-CSF and 500 U/mL rIL-4. 4. Three days later, harvest non-adherent cells corresponding to the DC fraction, wash, and resuspend in DC culture medium. 5. Test for DC generation by staining with FITC-conjugated anti-CD14 mAb and analyze by flow cytometry. After a 6day culture in the presence of GM-CSF and IL-4, DCs normally lose CD14 expression. 6. Seed DCs (5 × 105 DCs/mL) in 24-well plates for 24 h stimulation in the presence of the TLR ligand (LPS 1 μg/mL, R-848 10 μg/mL, poly I:C 20 μg/mL). Of note, ligands are tested on monocyte-derived DCs alone or in combination with other ligands or recombinant cytokines (20). 3.1.2. Analysis of Ligand Activities on Monocyte-Derived DC Maturation
1. Harvest cells and wash in PBS BSA 0.1% 2. Stain DCs with 3 μL PE-conjugated mAbs specific for CD80, CD83, CD86, CD40 (costimulatory molecules on DCs), and HLA-DR (MHC class II molecules). 3. Analyze by flow cytometry. 4. If required, stain using a broader panel of monoclonal antibodies detecting DC adhesion molecules (CD54), chemokine receptors (CCR7), DC-SIGN, DEC-205, and ICOS-L molecules. This could be useful to predict the immune response direction which could be engaged by adjuvant-stimulated DCs presenting a particular phenotype (21).
3.1.3. Analysis of Ligand Activities on Cytokine Production by Monocyte-Derived DCs
Upon TLR triggering, monocyte-derived DCs produce large amounts of cytokines which are detectable in the supernatants following 24 h of stimulation by ELISA or Luminex. For some others such as IL-27 (no ELISA available for the detection of human IL-27) or IFN-α/β (type I IFN levels produced are in some conditions below the detection threshold), quantitative RTPCR technique is useful for the quantification of their mRNA expression. If required, molecular analysis of the signaling pathways triggered by TLR ligands is feasible using monocyte-derived DCs (see Note 2). 1. Centrifuge cells and collect the supernatants 2. As an example, for the prototype TLR4 ligand, LPS, determine the production of IRF3-independent cytokines by DCs such as IL-12p40, TNF-α, IL-6, IL-8, IL-23, and IL-10 and
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IRF3-dependent factors such as IFN-β, IL-12p70, IP-10, and IL-27 using ELISA assays or LUMINEX technique 3. For IL-27 or IFN-α/β determination, stimulate DCs in DC culture medium for 3, 6, or 12 h at the concentration of 2.5 × 105 /500 μL in the presence of the ligand. At the end of the culture, extract total cellular RNA using the TriPure reagent according to the manufacturer’s instructions. Carry out reverse transcription (RT) and real-time polymerase chain reaction (PCR) using LightCycler-RNA Master Hybridization Probes (one step procedure) on a LightCycler device 3.2. Whole Blood Assays
Studies using fresh whole blood samples present some advantages (22): (a) they allow “ex vivo” assessment of immune cell functions, reducing possible isolation artifacts; (b) they take into account interactions between immune cells and also assess the role of soluble circulating factors present in the plasma; and (c) they can be performed with small amounts of blood. This is particularly useful for the analysis of immune responses developed by human newborns, young infants, or elderly people (23). There are also some limitations: ex vivo whole blood is a closed system, in which primary and secondary mediators are released and amplified as compared to in vivo situations where circulating immune cells interact with the endothelium and migrate to the adjacent tissues. It is therefore impossible to discriminate between initial activation by the TLR ligand and secondary feedback loops.
3.2.1. Stimulation of Human Blood Cells
1. For TLR stimulation of human blood cells, incubate heparinized whole blood samples (1 mL/condition) at 37◦ C in the presence of the ligand (10 ng/mL LPS, 5 μg/mL CpG, 20 μg/mL poly I:C, 10 μg/mL R-848). 2. Incubate overnight and harvest blood cells by centrifugation for flow cytometry analysis and collect plasma for determination of cytokine levels.
3.2.2. Analysis of the Phenotype of Circulating DCs and Monocytes
1. Incubate stimulated and control whole blood samples with 15/500 μL blood of FcR blocking antibodies. 2. Incubate 150 μL of blood from each condition for 30 min at 4◦ C with 2 μL of a cocktail of FITC-conjugated mAbs specific for CD3, CD16, CD19, CD56, and CD20 receptors (lineage cells), 3 μL of APC-conjugated anti-CD11c mAb, 3 μL of EDC-conjugated anti-HLADR mAb, 3 μL of PeCy5-conjugated anti-CD123 mAb, 3 μL of Pacific Blue-conjugated anti-CD14 mAb, and one of the following PE-conjugated mAbs for the APC phenotype determination: CD80, CD83, CD86, CD40, and CD154 (3 μL/condition).
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3. Lyse red blood cells by incubating blood samples for 10 min with the FACS lysis buffer in the dark (3 mL/tube). 4. Wash whole blood cells in PBS BSA 0.1% and resuspend in 300 μL Cell Fix solution. 5. Identify circulating APC populations after appropriate gating using a six-color flow cytometer as follows: – pDCs are identified as lineage– /HLADR+ /CD11c– / CD123++ cells – mDCs are identified as lineage– /HLADR+ /CD11c+ / CD123+/– cells – Monocytes are identified CD11C+ / CD14+ cells
as
lineage– /HLADR+ /
6. Quantify expression of HLA-DR and costimulatory molecules for each APC population for phenotypical maturation determination. 3.2.3. Cytokine Determination
1. Determine cytokine production in plasma (from Section 3.2.1) using ELISA or LUMINEX kits. 2. For some cytokines, such as IL-12p70, use of a high sensitivity kit with a detection limit of less than 1 pg/mL is recommended (R&D). 3. Quantify cytokine mRNA using real-time PCR. Incubate blood samples (1 mL) with the appropriate ligand for 3–6 h. Stop the reaction and stabilize the mRNA by the addition of 250 μL PAXgene reagent, which has been designed for whole blood samples (24). Extract mRNA using the MagNaPure LC mRNA Isolation kit following manufacturer’s instructions. Briefly, reverse transcription (RT) and real-time polymerase chain reaction (PCR) are carried out using LightCycler-RNA Master Hybridization probes (one step procedure) on a LightCycler devise (Roche diagnostics).
3.2.4. Determination of Cell Types in Whole Blood Responsible for Cytokine Production
1. Activate whole blood cells overnight with TLR ligands (see Section 3.2.1) 2. Add Brefeldin A (10 μg/mL) for IFN-γ or TNF-α detection or Monensin (2 μM)for IL-4 or IL-10 detection for the last 4 h of incubation 3. Wash the cells in 3 mL PBS/0.1% BSA 4. Stain cells with FITC-conjugated mAbs specific for cell membrane molecules and incubate for 15 min at 4◦ C 5. Add 3 mL RBC lysis buffer (1/10) (Becton Dickinson) 6. Incubate 15 min at room temperature and wash with 3 mL PBS/0.1% BSA
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7. Add 250 μL Perm 2 solution (1/10) (Becton Dickinson) 8. Incubate for 15 min at room temperature in the dark and wash the cells in 3 mL PBS/0.1% BSA 9. Add intracellular PE-conjugated mAbs specific for cytokines (IL-12p40, TNF-α, IL-6, IL-10, and IP-10) and incubate for 15 min at 4◦ C 10. Wash the cells in PBS/0.1% BSA and resuspend in 200 μL Cell Fix solution (1/10) (Becton Dickinson) 3.2.5. Stimulation of Freshly Isolated DCs
In order to study intrinsic properties of DCs, these cells have to be isolated. Indeed, DC function in whole blood or among other mononuclear cells is influenced by soluble mediators such as TNF-α, IFN-γ, or type I IFNs, or interactions with other cell types such as CD4+ or CD8+ T lymphocytes, NKT cells, or γδ T cells. As mentioned above, human circulating DCs are present at a very low frequency in peripheral blood and are extremely fragile cells. This imposes the use of fresh blood samples with a minimum volume of 100 mL. 1. Isolate human pDCs from PBMC by MACS, first by a negative depletion using a cocktail of antibodies (CD16, CD19, CD14, CD15, CD33, CD3, and Glycophoryn A) directly coupled to magnetic beads passed on a LD column, followed by a positive selection using the anti-BDCA-4conjugated magnetic microbeads mAb passed over two MS columns. Check the purity of the pDC fraction by staining with the FITC-conjugated anti-BDCA-2 mAb and the PEconjugated anti-CD123 mAb. 2. Stimulate freshly isolated pDCs for 48–72 h in RPMI 1640 complete medium, in the presence of the ligand, such as 10 μg/mL R-848 (TLR-7) or 10 μg/mL CpG (TLR-9). Of note, different classes of CpG-ODN have been described (see Note 3). 3. Isolate circulating myeloid DCs from PBMC by MACS, first by a negative depletion using a mix of antibodies (CD16, CD19, CD14, CD15, CD3, Glycophoryn A, and BDCA-4) coupled to magnetic beads; biotin-conjugated anti-BDCA-1 mAb is added to the pellet before passage on a LD column. Perform a positive selection using anti-biotin microbeadconjugated mAb with a passage on a MS column. Test mDC purity using FITC-conjugated anti-CD11c mAb and PEconjugated anti-CD14 mAb. 4. Stimulate purified mDCs in RPMI 1640 complete medium in the presence of the ligand, such as 1 μg/mL LPS (TLR4), 10 μg/mL poly I:C (TLR-3), 10 μg/mL R-848 (TLR8), or both for 24–48 h.
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5. Test the effects of the ligand on purified DC subpopulations. All the techniques described previously for monocytederived DCs or whole blood cells are applicable provided that concentration of harvested cells is sufficient (105 ) (25) (see Note 4). 3.3. HEK 293 Cell System
From the 1980s, embryonic kidney HEK 293 cell line has been extremely useful to decipher molecular signaling pathways in multiple diverse models. Indeed, those cells grow fast, remain stable, and unlike human DCs are easily transfectable. TLR expressing HEK cells have been generated allowing analysis of TLR responses at the molecular level (see Note 5). 1. Culture TLR expressing HEK cells in complete DMEM medium. 2. Transfect the HEK cells transiently with a reporter luciferase gene activated by a nuclear transcription factor under study such as NF-κB (pNF-κB-Luc), IRFs (IRF3: pIFNb-Luc), or AP-1 (26) in 24-well plates (2 × 105 cells/well). 3. After 2 days, stimulate HEK cells with TLR ligands for 24 h. 4. Analyze promoter activities using the dual luciferase assay system and normalize to renilla luciferase activities using the pRL-TK plasmid (see Note 6).
4. Notes 1. Importantly, human DCs are very sensitive to endotoxins (LPS). The presence of LPS during their generation may alter their development or render them in a state of “tolerance” and thus unresponsive to a second challenge with LPS. Prior exposure to LPS induces a transient state of cell refractoriness to subsequent LPS restimulation known as endotoxin tolerance (27). For this reason, all the products used for DC generation and stimulation such as FCS, cytokines, medium, or ligands have to be tested for their endotoxin contamination. Endotoxin levels below 10 pg/mL are acceptable for human DC cultures. Endotoxin levels are determined using the limulus assay (Limulus Amebocyte Lysate QCL 1000, Cambrex). 2. As an example, we have been recently investigating the effects of three distinct TLR-4 ligands, such as LPS, MPL (15), and an AGP (CRX527) (17). On human DCs, cytokine profiles induced by LPS, MPL, and CRX527 were shown to be distinct, providing evidence that the mode of action of these molecules through TLR4 is related to
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their structure. To get a broader view of the distinct TLR4mediated responses in human DCs, we are now comparing the gene expression profile induced by TLR4 agonists using oligonucleotide microarrays or microfluidic card techniques (28). Using western blot techniques, the phosphorylation of triggered molecules playing an important role in the signaling pathways triggered by TLRs can be also tested in human DCs, as well as their nuclear translocation (NFκB, IRFs) by confocal microscopy or Image Stream techniques (25). 3. Different classes of CpG have been described, showing differential effects on pDCs (29). Type A CpG, such as CpG 2216, triggers the production of high levels of type I IFNs by pDCs, whereas type B CpG, such as CpG 2006, elicits only low levels of type I IFNs but induces efficient pDC maturation. Finally, type C CpG is able to induce pDC phenotypical maturation as well as their production of type I IFNs. 4. Importantly, aluminum salts (alum), which is currently the most used vaccine adjuvant, is unable to act as a TLR agonist. Recent data have demonstrated that alum instead activates a multi-molecular complex called the inflammasome. Through NOD-like receptors (NLR) such as Nalp3, the inflammasomes would sense aluminum salts or more precisely crystal-induced lysosomal damage and leakage of lysosomal content into the cytosol. After its triggering, activated Nalp3 recruits the adaptor molecule ASC that binds to the Caspase-1, which in turn will cleave pro IL-1β, IL-18, and IL-33 allowing their release (4, 5). The precise mechanisms by which alum activates innate immunity in vivo are still not fully understood, and especially, how alum favors Th2-type immune responses over Th1 differentiation. Multiple novel adjuvant systems are therefore evaluated combining alum to other ligands to promote the induction of Th1 cell-mediated immunity (15). In this regard, MPL is mixed with alum in certain novel vaccines for several human infectious agents (hepatitis B and papilloma viruses) (16). Human monocytederived DCs, PBMC, or whole blood samples are useful in vitro models to evaluate the effects of alum in combination with other adjuvant molecules, such as TLR ligands. To test inflammasome activity, IL-1β, IL-18, and IL-33 levels are measured in the supernatants by ELISA techniques (R&D). A larger panel of molecules and time combinations can therefore be evaluated to identify synergy or antagonism mediated by TLR activation on inflammasome-dependent cytokine expression at both cytokine production and intracellular signaling pathway levels.
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5. We have been using HEK cells stably transfected with TLR4 and MD2, TLR2, and TLR3 (named HEK-TLR4/MD2, HEK-TLR2, and HEK-TLR3 cells, respectively), kindly provided by D. Golenbock (University of Massachusetts Medical School, Worcester, USA) (30). 6. Using TLR4/MD2 expressing HEK 293 cells, we recently analyzed NF-κB and IRF3 activities following stimulation in the presence of various TLR4 ligands. We could confirm that TLR4 ligands activate differently TLR4-dependent signaling pathways (IRF3-independent and IRF3-dependent pathways). Second, using HEK cells transfected or not with the CD14 gene, we observed that CD14 requirement also depends on the TLR4 ligand structure (31). References 1. Steinman, R. M. (2003) Some interfaces of dendritic cell biology. Apmis 111, 675–697. 2. Wilson-Welder, J. H., Torres, M. P., Kipper, M. J., Mallapragada, S. K., Wannemuehler, M. J., Narasimhan, B. (2008) Vaccine adjuvants: current challenges and future approaches review. J Pharm Sci 10, 1–39. 3. De Gregorio, E., Tritto, E., Rappuoli, R. (2008) Alum adjuvanticity: unraveling a century old mystery. Eur J Immunol 38, 2068– 2071. 4. Li, H., Nookala, S., Re, F. (2007) Aluminium hydroxide adjuvants activate caspase1 and induce IL-1beta and IL-8 release. J Immunol 178, 5271–5276. 5. Eisenbarth, S. C., Colegio, O. R., O’Connor, W., Sutterwala, F. S., Flavell, R. A. (2008) Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 453, 1122–1126. 6. McKee, A. S., Munks, M. W., Marrack, P. (2007) How do adjuvants work? Important considerations for new generation adjuvants. Immunity 27, 687–690. 7. Fajardo-Moser, M., Berzel, S., Moll, H. (2008) Mechanisms of dendritic cell-based vaccination against infection. Int J Med Microbiol 298, 11–20. 8. Kanzler, H., Barrat, F. J., Hessel, E. M., Coffman, R. L. (2007) Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nat Med 13, 552–559. 9. van Duin, D., Medzhitov, R., Shaw, A. C. (2006) Triggering TLR signaling in vaccination. TRENDS Immunol 27, 49–55. 10. Kawai, T., Akira, S. (2006) TLR signaling review. Cell Death Differ 13, 816–825.
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Bocarsly, P., Marchant, A., Goldman, M., Willems, F., De Wit, D. (2008) Interferon regulatory factor7-mediated responses are defective in cord blood plasmacytoid dendritic cells. Eur J Immunol 38, 507–517. Johnson, J., Albarani, V., Nguyen, M., Goldman, M., Willems, F., Aksoy, E. (2007) Protein kinase Cα is involved in interferon regulatory factor 3 and type I interferon-β synthesis. J Biol Chem 282, 15022–15032. Medvedev, A. E., Lentschat, A., Whal, L. M., Golenbock, D. T., Vogel, S. N. (2002) Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex formation and IL1 receptor-associated kinase 1 activation in endotoxin-tolerant cells. J Immunol 169, 5209–5216. Aksoy, E., Albarani, V., Nguyen, M., Laes, J. F., Ruelle, J. L., De Wit, D., Willems, F., Goldman, M., Goriely, S. (2007) Interferon regulatory factor-3-dependent responses to lipopolysaccharide are selectively blunted in cord blood cells. Blood 109, 2887–2893. Vollmer, J., Weeratna, R., Payette, P., Jurk, M., Schetter, C., Laucht, M., Wader, T., Tluk, S., Liu, M., Davis, H. L., Krieg, A. M. (2004) Characterization of three CpG oligodeoxynucleotides classes with distinct immunostimulatory activities. Eur J Immunol 34, 251–262. Latz, E., Visintin, A., Lien, E., Fitzgerald, K. A., Monks, B. G., Kurt-Jones, E. A., Golenbock, D. T. (2002) Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the Toll-like receptor 4-MD-2CD14 complex in a process that is distinct from the initiation of signal transduction. J Biol Chem 277, 47834–47843. Jiang, Z., Geogel, P., Du, X., Shamel, L., Sovath, S., Mudd, S., Huber, M., Kalis, C., Keck, S., Galanos, C., Freudenberg, M., Beutler, B. (2005) CD14 is required for MyD88-independent LPS signalling. Nat Immunol 6, 565–570.
Chapter 9 Adjuvant Activity on Murine and Human Macrophages Valerie Quesniaux, Francois Erard, and Bernhard Ryffel Abstract Activation of cells of the innate immunity such as macrophages and dendritic cells is critical to mount an adaptive immune response. Recent advances on the understanding of innate immune receptors such as the Toll-like receptors (TLR) and NOD-like receptors (NLR) and the demonstration that microbial products activate specific receptors. This discovery represented a major advance and provided tools to test novel adjuvants in vitro to investigate activation on innate immune cells. Here the isolation and culture of murine macrophages is described, and the use of macrophages derived from gene-deficient mice is proposed to define receptor usage. Novel adjuvants may be tested for their capacity to induce cytokines, chemokines and the expression of costimulatory molecules. The basic methods to assess macrophage activation are given, which may predict an in vivo activity of a novel adjuvant. Key words: Macrophages, monocytes, Toll-like receptors, TNF, IL-12, cell activation, mycobacteria.
1. Introduction Recent insights into the activation of the innate immunity such as macrophages and dendritic cells provided new understanding of the mechanism of the action of adjuvants. A short list of ligand and their specificity to activate TLRs is given in Table 9.1. Several bacterial components typically activate macrophages, but also endogenous molecules have been identified (1, 2). In addition to microbial-derived products one of the commonly used adjuvants, alum, also activates the macrophages and causes IL-1-dependent inflammation (3). Engagement of the TLRs induces inflammation and favours the development of a protective Th1-biased T cell response. Interruption of TLR recognition or signalling has profound effects on G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_9, © Springer Science+Business Media, LLC 2010
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Table 9.1 Overview of the Toll-like receptors and their ligands TLR1 TLR2
Triacyl lipopeptides (mycobacteria, bacteria) Lipoproteins and lipopeptides Peptidoglycan (Gram-positive bacteria) Lipoarabinomannan Lipoteichoic acid (Gram-positive bacteria) Zymosan Lipopolysaccharides, atypical (Leptospira and others) Porins (Neisseria) Glycoinositol phospholipids (Trypanosoma cruzi) Hsp70
TLR3 TLR4
Double-stranded RNA, poly-inositol-cytosine Lipopolysaccharide (Gram-negative bacteria) Taxol Viral proteins (RSV, MMTV) HMGB1 Hsp60 and 70 Hyaluronic acid and Type III repeat extradomain A of fibronectin Heparan sulphate (fragments) Fibrinogen
TLR5
Flagellin (bacteria)
TLR6
Diacyl lipopeptides (mycoplasma)
TLR7
Imidazoquinoline derivatives
TLR8
Imidazoquinoline derivatives
TLR9
CpG DNA (bacteria)
TLR10
?
TLR11
Profilin form apicomplexa
Abbreviations: Hsp: heat shock protein; HMGB1 protein: high mobility group box1; poly-IC: poly-inositol-cytosine; RSV: respiratory syncytial virus (1, 2).
innate immunity. Agonists or antagonists of specific TLRs modulate the host response to microbial infections with effects beyond infection control and may be used as immunostimulators in vaccines, cancer, inflammatory disorders and allergy. However, TLR agonists are now increasingly used as vaccine adjuvants (4). Here emphasis is given to the culture of bone marrow-derived macrophages, while alternative sources such as resident/resting and elicited peritoneal macrophages and BAL macrophages are briefly described. Finally, a short section on the preparation of human blood-derived monocytes/macrophages is given.
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2. Materials 2.1. Material for Bone Marrow-Derived Macrophages (BMDM) in Mice
1. Mice: any strain, we usually use C57BL6 2. Carbon dioxide 3. Ethanol 70% in water 4. Sterile scissors 5. Pincettes 6. 22 G Needles 7. 1 mL syringes 8. Sterile culture flasks (see Note 1) 9. Petri dish (Sterilin Ltd.) (see Note 2) 10. Pipette 11. Complete DMEM medium: DMEM supplemented with L-glutamine (10 nM), penicillin and streptomycin (100 U/mL) and HEPES (25 nM) 12. Complete DMEM medium as above which is enriched with 20% horse serum (GIBCO) and 30% supernatant from L929 cells (see below) 13. The L929 cells in complete DMEM medium containing 10% FCS are grown to confluence during 14 days (5), as a supply of monocyte colony-stimulating factor (M-CSF) 14. Ca2+ -free phosphate buffered saline (Ca2+ -free PBS)
2.2. Activation of BMDM in Culture and Measurement of Cytokine Production
1. Sterile microtitre plates 2. Complete DMEM medium (as above) which is supplemented with 0.1% FCS medium 3. TLR ligands such as bacterial lipopeptide (Pam3CK4), poly-IC, LPS, flagellin, etc. (Invivogen). Alternatively, use whole bacteria including Mycobacterium bovis BCG, lipomannan (LM) and mannosylated lipoarabinomannan (LAM) which are potent immunostimulating TLR agonists (6) 4. Inverted microscope 5. PBS 6. Elisa kit for TNF, IL-1 and other cytokines and chemokines (R&D Systems) 7. Microtitre plate reader 8. C57BL/6 mice and TLR-deficient mice are available and can be obtained through our research unit, with an MTA from Professors Akira or Beutler (Table 9.2). These mice have no major phenotype under steady state conditions,
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Table 9.2 Source and phenotype of TLR-deficient mice (reference) TLR1
Co-receptor for TLR2 (12)
TLR2
Reduced inflammatory response to bacterial lipopeptide (13)
TLR3
Reduced inflammatory response to poly-IC (14)
TLR4
LPS/endotoxin resistance (15)
TLR5
Unresponsiveness to bacterial flagellin (16)
TLR6
Co-receptor for TLR2 (17)
TLR7
Reduced response to DNA (18)
TLR9
Reduced response to DNA (19)
MyD88
Profound defect in response to bacteria and bacterial products (20)
TRIF
Reduced endotoxin response (21)
TIRAP
Adaptor for TLR2-4 receptor (22)
but some are very sensitive to infections such as Listeria and mycobacterial infections (7–11) 9. 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) 2.5 mg/mL 10. Sodium dodecyl sulphate (SDS) 10% solution
2.3. Assessment of Cell Activation by Flow Cytometric Analysis
1. Twenty-four well culture plate 2. Complete DMEM medium supplemented with 0.1% FCS (as above) 3. TLR ligands (see Section 2.2) 4. Fluorochrome-labelled rat monoclonal antibodies to mouse CD antigens (BD Pharmingen, San Diego, CA) 5. Flow cytometry analyser (e.g. Calibur or Canto) using the standard protocols
2.4. Preparation of Resting and Elicited Peritoneal and BAL Macrophages from Mice
1. Mice, any strain, usually C57BL6 mice 2. PBS 3. Complete DMEM medium supplemented with 0.1% FCS (as above) 4. Thioglycollate solution: thioglycollate prepared at 3% (w/v) in distilled water and left 1 month 5. TLR ligands (see Section 2.2)
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1. EDTA R , Axis-Shield, Oslo) 2. Iodixanol (Optiprep
3. RPMI/FCS medium: RPMI 1640 (Life Technologies, Rockville, MD) plus 10% fetal calf serum (endotoxin free) 4. Swinging bucket centrifuge 5. RPMI/gentamycin medium: RPMI 1640 supplemented with gentamicin 10 μg/mL, L-glutamine 10 mM and pyruvate 10 mM 6. Magnetic beads and columns for monocyte purification (Miltenyi Biotec, Bergisch Gladbach, Germany) 7. Six-well plates (Falcon, Becton Dickinson, Franklin Lakes, NJ) 8. RPMI/HEPES medium: RPMI 1640 medium supplemented with 0.6 μg/mL penicillin, 60 μg/mL streptomycin, 2 mM L-glutamine and 20 mM HEPES 9. Phosphate buffered saline (PBS) 10. Macrophage/GM-CSF medium: macrophage serum-free medium (Life Technologies, Grand Island, NY) supplemented with antibiotics and human recombinant (hr)GMCSF, 10 ng/mL (Nordic Biosite, Täby, Sweden) 11. RPMI/ATP medium: RPMI 1640 medium containing 1 mM ATP 12. IL-1β ELISA kit 13. oxATP 14. RNeasy Mini kit (Qiagen) 15. RT medium: TaqMan RT buffer with 5.5 mM MgCl2 , 500 μM dNTPs, 2.5 μM oligo d(T)16, 0.4 U/μL RNase inhibitor and 1.25 U/mL MultiScribe RT (Applied Biosystems, Foster City, CA) 16. TaqMan universal PCR master mix buffer (Applied Biosystems) 17. Gene expression system assay mix oligonucleotides (Applied Biosystems) to analyse mRNA levels, for example IL-6 (Hs00174131_m1), IL-10 (Hs00174086_m1), IL-12p35 (Hs00168405_m1), IL-12p40 (Hs00233688_m1), IL-23p19 (Hs00372324_m1), IL-27p28 (Hs00377366_m1), IL-27 EBI3 (Hs00194957_m1), TNF-α (Hs00174128_m1) and β-actin (Hs99999903_m1) 18. GeneAmp 5700 sequence detector (Applied Biosystems) 19. β-Actin mRNA
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3. Methods 3.1. Preparation of Murine Bone Marrow-Derived Macrophages (BMDM)
This work has to be performed in a PSM flow bench under sterile conditions. 1. Mice are killed by CO2 exposure, transferred in a laminar flow bench and the mouse is sprayed with 70% alcohol. 2. Then with sterile instruments an incision of the skin and muscle of the thighs is performed, the femur is liberated from the muscles with a scissor and transferred into a Petri dish and the manoeuvre will be repeated for the second femur. 3. The content of the bone marrow cavity of the femur is then flushed with a 1 mL syringe containing sterile PBS into a sterile tube, then the bone marrow is disaggregated with a 22G needle, and the debris is allowed to sediment for a few minutes. 4. The supernatant containing the dissociated cells is recovered, the cells are gently sedimented by centrifugation and resuspended in complete DMEM medium and counted with a microscopic counting chamber (see Note 3). 5. The cells are then transferred into Sterilin type Petri dishes at 1 × 106 cells/mL in 5 mL in complete DMEM medium enriched with 20% horse serum and 30% L929 supernatant. 6. At day 7 the supernatant is removed, the adherent cells are incubated with cold Ca2+ -free PBS for 20 min at 4◦ C and the cells are then removed by vigorous pipetting, transferred into tubes, gently centrifuged and resuspended in the complete DMEM medium enriched with 20% horse serum and 30% L929 supernatant and recultured in Sterilin type Petri dishes as before for an additional 3 days when a homogenous monolayer of adherent mature macrophages is formed. 7. The mature macrophages are then recovered as before, counted, resuspended in complete DMEM medium in the absence of serum and transferred into microtitre plate usually at 100,000 cells/well in 100 μL and stimulated (see Notes 4 and 5).
3.2. Activation of BMDM in Culture and Measurement of Cytokine Production
1. Plate cells at 105 cells/well in 100 μL complete DMEM medium enriched with 0.1% FCS in sterile microtitre plates. 2. Dilute the TLR agonists from 20 to 2 μg/mL and add in 100 μL to the culture. 3. Set up control wells containing medium but no TLR agonist.
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4. Culture for 6 and 24 h is sufficient for most cytokines and chemokines. 5. Assess the appearance of the cultured cells, viability and absence of microbial contamination of the culture with an inverted microscope. 6. Collect 150 μL of the supernatant and either freeze or use directly for cytokine determination by ELISA. 7. Transfer serial twofold dilutions of the supernatant and serial dilution of recombinant standard cytokine to precoated commercial ELISA microtitre plates such as for TNF, IL-12, or other cytokines or chemokines. 8. Leave the microtitre plates overnight at 4◦ C, then wash three times with 1 mL PBS. 9. Add 100 μL of the enzyme-coupled secondary antibody at dilutions suggested by the manufacturer. 10. Wash the plates three times with PBS and incubate with substrate for 10–30 min. 11. As soon as the wells with the standard cytokine dilutions show a graded colour change, read the OD of the plates with a microplate reader in order to quantify the cytokine production relative to the dilution of the recombinant standard protein. 12. Plot the OD results versus the standard in ng/mL. 13. Assess cell viability by the MTT assay since TLR agonists or inhibitors may at high doses cause cell death. Add the MTT substrate to the cell pellet, incubate at 37◦ C for 30 min and determine the OD as a measure of viability (see Notes 6 and 7). 3.3. Methods to Assess Cell Activation by Flow Cytometry
1. Resuspend cultured cells in complete DMEM medium enriched with 0.1% FCS in 24 well culture plates at 106 /mL with TLR agonists for 6 or 24 h. 2. Harvest the cells with a sterile pipette, wash gently in medium and resuspended at 106 /mL. 3. Incubate the cells with fluorochome-labelled antibodies to CD40, class II, CD80 or CD86 antibodies (usually at 1 μg/mL) for 1 h at room temperature, wash and resuspend in 200 μL culture medium. 4. Analyse the labelled and unstained cells by flow cytometry. 5. Record the percentage and absolute number of positive cells. 6. Determine the mean fluorescence of membrane-expressed antigens (see Note 8).
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3.4. Preparation of Resting and Elicited Peritoneal and BAL Macrophages from Mice
1. Lavage the peritoneal cavity by injecting with a syringe three times with 2 mL warm PBS, and gently collect the lavage cells into tubes, centrifuge and resuspend in complete DMEM medium enriched with 0.1% FCS at 105 cells in 200 μL/well in microtitre plates.
3.4.1. Resident Peritoneal Macrophages
2. Incubate the cells with the TLR agonists for 24 h.
3.4.2. Thioglycollate Activated Macrophages
1. Inject mice intraperitoneally with 3 mL thioglycollate solution.
3. Harvest the supernatant for cytokine/chemokine determination by ELISA as in Section 3.2.
2. Recover elicited macrophages after 3 days by peritoneal wash, three times with 2 mL warm PBS and resuspend in complete DMEM medium enriched with 0.1% FCS. Incubate the cells with TLR agonists for 24 h. 3. Harvest the supernatant for cytokine/chemokine determination by ELISA as in Section 3.2. 3.4.3. BAL Lavage Macrophages
1. Anesthetize the mice, open the thorax and insert a needle into the trachea. 2. Rinse with warm PBS, three times 1 mL, and collect the lavage. 3. Centrifuge to pellet the cells. 4. Resuspend the cell pellet in complete DMEM medium enriched with 0.1% FCS. 5. Incubate the cells with TLR agonists for 24 h. 6. Harvest the supernatant for cytokine/chemokine determination by ELISA as in Section 3.2.
3.5. Preparation and Culture of Human Blood-Derived Monocytes/Macrophages 3.5.1. Preparation of Human PBMC and Monocytes
The cell preparation from whole blood is performed according to Nutt et al. (25, 26). 1. Collect 30 mL whole blood from healthy volunteers in accordance with the Declaration of Helsinki, in tubes containing EDTA (5.5 mmol/L). 2. Prepare two density solutions by mixing a 60% solution of R iodixanol (Optiprep ) with RPMI/FCS medium comprising: a. Density solution of 1.084 g/mL: mix 1.5 mL of the 60% R Optiprep with 4.5 mL of RPMI/FCS medium. b. Density solution of 1.065 g/mL: mix 3 mL of the 60% R Optiprep with 13.4 mL of RPMI/FCS medium. 3. Centrifuge the whole blood for 15 min at 570×g, remove the upper plasma layer and mix the buffy coat with 0.4 volR . umes of Optiprep
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4. Load approximately 2 mL of the leukocyte-rich plasma/ R Optiprep mixture into each of the three 15 mL polypropylene centrifuge tubes, then overlay successively with 2 mL of the 1.084 g/mL density solution, 4 mL of the 1.065 g/mL and 0.5 mL of medium. 5. Centrifuge the tubes at 700×g for 15 min in a swinging bucket rotor centrifuge without application of the brake during deceleration and collect the layer below the upper interface. 6. Dilute the resultant cell suspensions with medium and count the cells. 7. Centrifuge the suspension at 400×g for 6 min and resuspend the resultant pellet in RPMI/gentamycin medium at a concentration of 106 cells/100 μL. 8. If required, purify monocytes using magnetic beads coated with anti-CD14 antibodies, as described by the manufacturer. 3.5.2. Culture and Differentiation
1. To obtain monocyte-to-macrophage differentiation, allow mononuclear cells (1 × 107 cells/well) to adhere to six-well plates for 1 h at 37◦ C in RPMI/HEPES medium. Remove non-adherent cells by washing with cold PBS and culture the remaining monocytes in macrophage/GM-CSF medium. 2. Change the medium every 2 days and culture for a total of 7 days to differentiate into macrophages.
3.5.3. Stimulation of Cytokine Production
1. Suspend cells in RPMI/gentamycin medium and adjust to 107 cells/mL. 2. Incubate with either 100 μL of RPMI/gentamycin medium (negative control) or purified LPS, Pam3Cys (10 μg/mL), heat-killed Staphylococcus epidermidis (106 /mL), MDP (10 μg/mL), or combinations of MDP and LPS, in RPMI/gentamycin medium. Other stimulants, for example by Pam3CSK4 (TLR1/2), poly-I:C (TLR3), LPS (TLR4), flagellin (TLR5) and R848 (TLR7/8), alone or in combinations of two can also be performed. 3. After 24 h, collect the supernatants and store at –70◦ C until assay. 4. Analyse cytokine levels in cell culture supernatants by ELISA. 5. Assess intracellular cytokines after adding 200 μL RPMI/ FCS medium to the adherent cells and lysing the cells by 2 cycles of freeze–thaw. 6. To investigate the role of monocytes in the production of IL-1β by stimulation of the inflammasome, stimulate
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purified monocytes for 4 h with LPS. After 4 h, centrifuge and remove the supernatants, add RPMI/ATP medium to the cells and incubate for 15 min. Harvest the supernatant by centrifugation and determine IL-1β by ELISA. 7. Determine the role of the endogenous ATP release for the stimulation of IL-1 by blocking P2X7 receptors with oxATP (300 μM) during the stimulation of cells for 4 h with LPS. 3.5.4. mRNA Determination
1. Total cellular RNA can be isolated from macrophages using the RNeasy Mini kit. Prepare cDNA from total cellular RNA (2 μg) by reverse transcription in RT medium. 2. Amplify cDNA samples in TaqMan universal PCR master mix buffer with gene expression system assay mix oligonucleotides. Each cDNA sample can be amplified in duplicate with a GeneAmp 5700 sequence detector (Applied Biosystems). 3. Calculate the relative amounts of cytokine mRNA by the comparative threshold (Ct) method, and mRNA levels are normalized against β-actin mRNA.
4. Notes 1. Sterile material is important as the culture is over 10 days, to prevent any microbial contamination. 2. Sterilin plates are critical for the growth of BMDM, as most Petri dishes do not allow the removal of the differentiated cultured macrophages. 3. Cell number is evaluated in a Malassez chamber; 15 μL of cell suspension is put in a Malassez chamber and then the number of cells is counted over 10 squares. The subsequent cell count is calculated by the following formula: C = n × dilution factor × number of square × final volume. 4. Bone marrow-derived macrophages represent a very homogenous population of cells and have an additional advantage of yielding large amounts of macrophages from single donors. However, for specific questions, macrophages from different sources may be more suitable, such as alveolar macrophages obtained by BAL lavage requiring many donor mice. 5. Differentiated BMDM may be frozen in 20% FCS and 10% DMSO by gently reducing the temperature slowly to –80◦ C and then stored in liquid nitrogen. The procedure of thawing is as for other cells rapidly, followed by gentle
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TLR2+/+ TLR4+/+ TLR2–/– TLR4+/+ TLR2+/+ TLR4–/– TLR2–/– TLR4–/–
A 14 12
TNF (ng/ml)
10 8 6 4 2 0 Medium
LPS
BLP
BCG
BCG Disp
HKBCG
BCG Lyoph
Sup BCG
B a Stimulation in wild type macrophages
c Inhibition in wild type macrophages
medium
LPS
LM
LAM
IL12p40 (ng/ml)
b Stimulation in TLR2-/- macrophages 10 8 6 4 2 0 medium
LPS
LM
LPS d IL-12P40 (ng/ml)
IL12p40 (ng/ml)
IL-12P40 (ng/ml) 10 8 6 4 2 0
10 8 6 4 2 0
10 8 6 4 2 0
LPS +
LPS +
Inhibition in TLR2-/- macrophages
LPS
LPS + LM
LPS + LAM
LAM
Fig. 9.1. (A) TNF expression of BMDM from single and double TLR2- and TLR4-deficient mice in response to different preparations of M. bovis BCG (9). (B) LM but not LAM induces IL-12/p40 expression as LPS (a, b), which is TLR2 dependent. Both LM and LAM inhibit LPS-induced IL-12/p40 expression which is TLR2 independent (c, d) (24).
centrifugation and resuspension in complete DMEM medium containing 10% foetal calf serum. 6. Cell viability is quantified by the MTT assay (23). Briefly, 50 μL of stimulated cells are incubated with 1-(4,5Dimethylthiazol-2-yl)-3,5-diphenylformazan at 2.5 mg/mL for 2–6 h at 37◦ C resulting formazan is then solubilized with 10% of SDS (Sigma, St Louis, MO) and the absorbance is read at 610 nm on a microtitre plate reader. 7. Our laboratory is interested in the role of mycobacteriainduced cell activation of BMDM. We used either several
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LPS
BLP
BCG TLR2+/+ TLR4+/+
TLR2–/– TLR4+/+
TLR2+/+ TLR4–/–
TLR2–/– TLR4–/–
100
101
102
CD40
103
0 104 10
101
102
CD40
103
104
100
101
102
103
104
CD40
Fig. 9.2. Mean fluorescent intensity of CD40 expression of BMDM from single and double TLR2- and TLR4-deficient mice in response to different preparations of M. bovis BCG as compared to BLP and LPS (bold line represent macrophages upon activation, while the thin line represents the control in the absence of stimulant) (9).
physical forms of M. bovis BCG or culture supernatant and found substantial differences in the requirement of TLR for a cytokine response. We tested the TLR2-4 receptor engagement using single- and double-deficient mice (Fig. 9.1A). Using the TLR2 and TLR4 agonistic ligands, BLP and LPS, respectively, we confirmed that the TNF response of BMDM depends strictly on the specific receptors. Live BCG-induced TNF response was only slightly reduced in TLR2-deficient BMDM. By contrast, the response to heat-killed (HK) BGG, a fully dispersed
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or lyophilized BCG as well BCG culture supernatant, was largely TLR2 dependent (9). Since BCG culture supernatant produce several active components with immunomodulatory properties, we investigated purified components derived from the mycobacterial cell wall such as LM and LAM (24) and found that LM-induced IL-12/p40 expression as LPS, while LAM had no effect. The LM effect was TLR2 dependent (Fig. 9.1B: a, b). Interestingly LAM but LM to a lesser extent had an inhibitory effect on LPS-induced IL12/p40 production, and this effect was TLR independent (Fig. 9.1B: c, d). Therefore the data suggest at a defined LM may have a dual function, that is a TLR2 agonistic function and an inhibitory effect on LPS-induced cell activation. LAM has no stimulatory effect, but is a potent inhibitor. The mechanism of LM- and LAM-mediated inhibition is not resolved yet but does not depend on TLR signalling. 8. We investigated the expression of CD40 and CD86 in BMDM from single and double TLR2- and TLR4-deficient mice in response to different preparations of M. bovis BCG (9). While LPS and BLP induced the expression of the costimulatory molecule CD40 in a TLR4- and TLR2dependent manner, BCG-induced expression of CD40 (Fig. 9.2) and CD86 (not shown) is TLR2/TLR4 independent (9, 10). Therefore, the data suggest that the expression of costimulatory molecules and adaptive immunity to mycobacteria may be TLR independent, as we showed also for virulent mycobacteria (8).
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6. Quesniaux, V., Fremond, C., Jacobs, M., et al. (2004) Toll-like receptor pathways in the immune responses to mycobacteria. Microbes Infect 6, 946–959. 7. Fremond, C. M., Togbe, D., Doz, E., et al. (2007) IL-1 receptor-mediated signal is an essential component of MyD88-dependent innate response to Mycobacterium tuberculosis infection. J Immunol 179, 1178–1189. 8. Fremond, C. M., Yeremeev, V., Nicolle, D. M., Jacobs, M., Quesniaux, V. F., Ryffel, B. (2004) Fatal Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88. J Clin Invest 114, 1790–1799. 9. Nicolle, D., Fremond, C., Pichon, X., et al. (2004) Long-term control of Mycobacterium bovis BCG infection in the absence of Toll-like receptors (TLRs): investigation
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25.
26.
vate immune cells via the TLR7 MyD88dependent signaling pathway. Nat Immunol 3, 196–200. Hemmi, H., Takeuchi, O., Kawai, T., et al. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745. Kawai, T., Adachi, O., Ogawa, T., Takeda, K., Akira, S. (1999) Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11, 115–122. Hoebe, K., Du, X., Georgel, P., et al. (2003) Identification of Lps2 as a key transducer of MyD88-independent TIR signalling. Nature 424, 743–748. Horng, T., Barton, G. M., Flavell, R. A., Medzhitov, R. (2002) The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature 420, 329–333. Ferrari, M., Fornasiero, M. C., Isetta, A. M. (1990) MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods 131, 165–172. Quesniaux, V. J., Nicolle, D. M., Torres, D., et al. (2004) Toll-like receptor 2 (TLR2)dependent-positive and TLR2-independentnegative regulation of proinflammatory cytokines by mycobacterial lipomannans. J Immunol 172, 4425–4434. Nutt, J. C., Willis, C. C., Morris, J. M., Gallery, E. D. (2004) Isolating pure populations of monocytes from the blood of pregnant women: comparison of flotation in iodixanol with elutriation. J Immunol Methods 293, 215–218. Netea, M. G., Nold-Petry, C. A., Nold, M. F., et al. (2009) Differential requirement for the activation of the inflammasome for processing and release of IL1beta in monocytes and macrophages. Blood 113, 2324–2335.
Chapter 10 In Vitro Effects of Adjuvants on B Cells Jörg Vollmer and Hanna Bellert Abstract Stimulation of B cells not only through the B cell antigen receptor (BCR) but also through Toll-like receptors (TLRs) can drive activation, proliferation, and differentiation of B cells to result in antigenspecific antibody secretion. In addition, B cells are co-stimulated by specific antigen and the presence of a TLR ligand such as for TLR9, which selectively enhances the development of antigen-specific antibodies and endows B cells with strong antigen-presenting capabilities to T cells. These effects promote antigenspecific immune responses and account for the strong adjuvant effect of TLR9 ligands. Several studies have described the activation of human or murine B cells by TLR ligands or other adjuvants. However, there are no reports summarizing the various different effects adjuvants can have on B cells, nor how to best measure these effects. Here, we will try to give an overview on the TLR expression pattern of human, primate, and murine B cells, their stimulation by TLR ligands or other adjuvants, and the outcome such as B cell proliferation and cytokine production. Key words: B cell, CpG, oligodeoxynucleotide, oligoribonucleotide, Toll-like receptor.
1. Introduction B cells are defined by the presence of surface immunoglobulins that act as specific antigen receptors (BCR) upon associating with other molecules. They constitute about 5–15% of the circulating lymphocytes in the blood. Upon stimulation through the BCR by specific antigen and followed by cooperative (cognate) T cell help via MHC class II, naïve antigen-specific B cells proliferate and differentiate into memory B cells and plasma cells (1). Memory B cells mediate secondary immune responses upon rechallenge, and the terminally differentiated plasma cells home to spleen and bone marrow and secrete high amounts of antibodies. To G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_10, © Springer Science+Business Media, LLC 2010
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maintain serological memory independent of persisting antigen, polyclonal stimuli such as microbial products and non-cognate T cell help via CD40 and cytokines may ensure sustained proliferation and differentiation of memory B cells (2). The family of Toll-like receptors (TLRs) consists of ten human TLRs that detect highly conserved components of pathogens that are not present in eukaryotic cells (3). These TLRs respond to molecular structures that are conserved in certain pathogens, but are not present or are not accessible in self-tissues. Besides the CpG motif in single-stranded DNA as a ligand for TLR9, examples of such TLR ligands include certain lipopeptides (detected by TLR2), double-stranded RNA (TLR3), endotoxins (TLR4), flagellin (TLR5), and single-stranded RNA (TLR7 and TLR8). The TLRs have a very specific pattern of cellular expression in the innate and adaptive immune system. For example, TLR9 is exclusively expressed in two human immune cell types: B cells and plasmacytoid dendritic cells (pDCs) (4). B cells harbor a very specific TLR expression (Table 10.1), with TLR1, TLR6, and TLR10 expressed on the cell surface (5–7), while TLR7 and TLR9 are found in endolysosomal compartments [4, 8]. However, the cellular patterns of TLR expression vary between different species, so that the results of TLR stimulation in one species may not be predictive of what will occur in another. For example, mice differ from humans in that they also express TLR4 in B cells (Table 10.1) [9, 10]. This makes it difficult at best to use observations with TLR ligands in murine studies to predict accurately the effects of TLR activation in humans. A large number of adjuvants have been developed which differ in their mode of action and can be broadly divided into two groups: (1) immune stimulatory molecules and (2) delivery systems which may also have immune stimulatory activity (11). From all adjuvants currently in preclinical and clinical development, synthetic CpG oligodeoxynucleotides (ODN) appear to be one of the most promising. CpG ODN induce strong antigenspecific humoral as well as cellular immune responses against a wide variety of different antigens [4, 12]. The TLR9-signaling mechanism is divided into two different signal cascades: one leading to activation of the IFN-α pathway (in pDCs) and the other leading to activation of the nuclear factor-κB (NF-κB) pathway (13–15). The first signaling protein necessary for both pathways is the adaptor protein MyD88. Subsequently, other signal transducing proteins such as members of the IL-1 receptor-associated kinase (IRAK) family, or mitogen-activated kinases (MAPK), are involved in the activation of NF-κB transcription factors and a core set of responses. CpG ODN cause upregulation of expression of costimulatory molecules and MHC class II molecules and create a microenvironment that promotes the induction of Th1biased antigen-specific adaptive immune responses (16).
Low +
CD19+ CD27–
CD19+ CD27+
Naïve B cells
Memory B cells
+ +
B220+ CD23+ IgM+
B220+ CD23+ IgG+
Naïve B cells
Memory B cells
na: not aquired.
∗ Some reports also describe low TLR2 expression [6, 7]. ∗∗ Very low expression except for splenic marginal zone B cells.
+
B220+
B cells (naïve + memory)
Mouse
+
CD19+
TLR1
B cells (naïve + memory)
Human
B cell subset
+
+
+ + +
–∗∗
–
–∗∗
–
–∗
–
–
TLR4
–∗∗
–
+
–
–∗
TLR3
–∗
TLR2
Table 10.1 TLR expression in human and murine B cells and B cell subsets
–
–
–
–
–
–
TLR5
+
+
+
+
–
+
TLR6
+
+
+
+
Low
+
TLR7
–
–
–
–
–
–
TLR8
+
+
+
+
Low
+
TLR9
na
na
na
+
Low
+
TLR10
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Human and murine B cells express a very specific pattern of TLRs (Table 10.1) [5, 7, 9]. However, this pattern differs not only between different species but also between subtypes of human and murine B cells (Table 10.1) [9, 17–20], so that care should be taken when analyzing the effects of TLR activation on B cells. The simplest method to measure activation of human B cells by TLR ligands or other stimulators is the secretion of cytokines from PBMC or the upregulation of cell surface markers by flow cytometry. However, in addition to these measurements additional methods may be used to obtain a broader profile of immune stimulatory effects mediated by B cell activators.
2. Materials 2.1. Isolation and Culture of Human Peripheral Blood Mononuclear Cells (PBMC)
1. RPMI 1640 medium: 5% heat-inactivated human AB serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, store at 4◦ C. 2. Buffy coats from healthy male and female human donors, about 100 mL. 3. Ficoll-Paque Plus store at 4◦ C. 4. 1x DPBS/Ampuwa: 10x Dulbecco’s Phosphate Buffered Saline, 1:10 diluted with Ampuwa, store at room temperature (RT). 5. CpG oligodeoxynucleotides (ODN): and oligoribonucleotides (ORN): suspended in sterile, endotoxin-free Tris– EDTA, and ORN, suspended in DNA/RNAse-free water. 6. DOTAP as delivery system for ORN.
2.2. Isolation and Culture of Human B Cells
1. CD19+ B cell isolation kit (Miltenyi Biotech, BergischGladbach, Germany), store at 4◦ C. 2. Running solution: bovine serum albumin/EDTA/PBS, store at 4◦ C. 3. Rinsing solution: EDTA/PBS. 4. 70% ethanol, store at RT. 5. FACS buffer: 3% FCS/1x PBS, store at 4◦ C. 6. Cell-Fix: 1% formaldehyde/1x PBS, store at 4◦ C. 7. Monoclonal antibody (Ab) to CD14, CD20 (BD Pharmingen, Heidelberg, Germany), BDCA-4 (Miltenyi Biotech), store at 4◦ C.
2.3. Culture of B Cell Lines
1. Ramos (Burkitt’s lymphoma) (American Type Culture Collection, ATCC, Wesel, Germany) for culture in suspension.
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2. RPMI 1640 medium: 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, and 10% heat-inactivated fetal bovine serum (FBS), store at 4◦ C. 2.4. Cytokine Secretion
1. ELISA kits IL-6 Elipair and IL-10 Elipair (Diaclone, Besancon, France), store at 4◦ C. 2. ELISA kit IP-10 DuoSet (R&D Systems, Wiesbaden, Germany), store at 4◦ C. 3. TMB Substrate Reagent Kit (BD Pharmingen), store at 4◦ C. 4. Stop solution: sulfuric acid 1 M, store at RT. 5. 1x DPBS/Ampuwa: 10x Dulbecco’s Phosphate Buffered Saline, 1:10 diluted with Ampuwa, store at RT. 6. Washing buffer 1x PBST: 10x DPBS/Ampuwa, 0.05% Tween 20.
2.5. Cell Surface Activation
1. FACS buffer: 3% FCS/1x PBS, store at 4◦ C. 2. Cell-Fix: 1% formaldehyde/1x PBS, store at 4◦ C. 3. mAb to CD19, CD80, CD86 (BD Pharmingen), store at 4◦ C.
2.6. Intracellular Cytokine Measurement
1. Brefeldin A from Penicillium brefeldianum 1 μg/mL, store at –20◦ C. 2. FACS buffer: 3% FCS/1x PBS, store at 4◦ C. 3. Cell-Fix: 1% formaldehyde/1x PBS, store at 4◦ C. 4. 1x DPBS/Ampuwa: 10x Dulbecco’s Phosphate Buffered Saline, 1:10 diluted with Ampuwa, store at RT. 5. IntraPrep Reagents 1 and 2 (Coulter, Krefeld, Germany). 6. mAb to CD19, CD14, IP-10 (BD Pharmingen), store at 4◦ C.
2.7. B Cell Proliferation
1. RPMI 1640 medium: 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, store at 4◦ C. 2. 1x DPBS/Ampuwa: 10x Dulbecco’s Phosphate Buffered Saline, 1:10 diluted with Ampuwa, store at RT. 3. CFSE 5 μM (5-(and-6-)-carboxyfluorescein diacetate succinimidyl ester. 4. FACS buffer: 3% FCS/1x PBS, store at 4◦ C. 5. Cell-Fix: 1% formaldehyde/1x PBS (36.5% formaldehyde, Sigma; 10x Dulbecco’s Phosphate Buffered Saline, BioWhittaker), store at 4◦ C. 6. mAb to CD19 (BD Pharmingen).
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2.8. Ab Secretion
1. RPMI 1640 medium: 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, store at 4◦ C. 2. Purified mouse anti-human IgG1; biotin mouse anti-human IgG1 (BD Pharmingen); human IgG1 lambda, purified myeloma protein (Sigma); Avidin-HRP (BD Pharmingen). 3. Purified mouse anti-human IgG2; biotin mouse anti-human IgG2 (BD Pharmingen); human IgG2 lambda, purified myeloma protein (Sigma); Avidin-HRP (BD Pharmingen). 4. Monoclonal anti-human IgM; anti-human IgM (peroxidase conjugated); human IgM, purified immunoglobulin (Sigma). 5. TMB Substrate Reagent Kit (BD Pharmingen), store at 4◦ C. 6. Stop solution: sulfuric acid, store at RT. 7. 1x DPBS/Ampuwa: 10x Dulbecco’s Phosphate Buffered Saline, 1:10 diluted with Ampuwa, store at RT. 8. Washing buffer 1x PBST: 10x DPBS 1:10 diluted with Ampuwa, 0.05% Tween 20, store at RT.
3. Methods B cells can be identified by the expression of cellular markers such as CD19 or CD20 for human (or primate) B cells and B220 for murine B cells. The stimulation of PBMC or purified CD19+ B cells results in a wide variety of immune effects such as the production of cytokines and chemokines (IL-6, IL-8, IL-10, and IP10), the upregulation of cell surface activation markers (CD40, CD54, CD69, CD80, CD86, and MHC class II), B cell proliferation, B cell differentiation, and Ig secretion (IgM, IgG1, IgG2), as well as their protection from apoptosis (Table 10.2) [6, 21– 26]. To obtain clear results for the stimulation of B cells by a specific adjuvant, human CD19+ B cells should be purified from PBMC and stimulated with the adjuvant to measure IL-6 or other cytokine secretion. Fewer data are available for primate B cells stimulated with TLR9 ligands (Table 10.2) [21, 27]. CD20+ primate B cells produce IL-6 and IL-8 and show enhanced cell surface activation of CD40, CD80, and MHC class II upon stimulation with CpG ODN. Similar to human B cells, primate B cells proliferate and are protected from apoptosis upon TLR9 triggering. Subtypes of human B cells are the memory B cells which express the CD27 cell surface antigen, and the naïve B cells,
+
CD19+ CD27+
Memory B cells
+
+
na
na
na
–
+
+∗ ∗ ∗
+
na
na
na
+
na
na
+∗
IL-12p70
na
na
na
+
na
na
–∗ ∗
TNFα
na
na
na
na
na
na
+
IP10
+
na
na
na
na
na
+
na
na
na
na
na
na
+
na
na
na
na
+
+
+
+
na
na
na
na
na
+
na
na
na
na
+
+
+
CD40 CD54 CD69 CD80 CD86
Cell surface activation marker
+
na
na
+
na
na
+
MHC Class II
na: not aquired.
∗ IL-12p70 secretion is observed only in the presence of T cell help (CD40L) (19). ∗∗ Low TNF-α production in human B cells upon CpG activation was reported (34). ∗∗∗ Naïve B cell activation is observed only in the presence of concomitant BCR crosslinking. ∗∗∗∗ IL-6 production upon CpG stimulation from naïve B cells can also be observed in the absence of BCR triggering (19).
B cells (naïve + memory)
CD20+
na
B220+ CD23+ IgG+
Memory B cells
Primate
na
B220+ CD23+ IgM+ na
Naïve B cells
na
na
+
B220+
B cells (naïve + memory)
na
na
+∗ ∗ ∗ ∗
CD19+ CD27–
Naïve B cells
Mouse
+
+
IL-8
CD19+
IL-6
B cells (naïve + memory)
Human
B cell subset
IL10
Cytokine and chemokine secretion
+
+
+
+
+
+∗ ∗ ∗
+
Proliferation
Table 10.2 CpG-mediated and TLR9-dependent effects on human, primate, and murine B cells and B cell subsets
na
+
+
+
+
+∗ ∗ ∗
+
na
+
+
+
+
+∗ ∗ ∗
na
+
na
na
+
na
na
+
Protection Ig secre- Differen- from apoptosis tion tiation
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which lack this cellular antigen. To investigate the activation of these B cell subsets which may become important for the determination of the stimulatory capacity of specific adjuvants, CD19+ B cells may be purified, followed by additional isolation of CD27+ memory B cells. TLR1, TLR6, TLR7, TLR9, and TLR10 expression in human memory B cells can be readily detected by RTPCR. Although TLR expression in naïve B cells appears similar, TLR1, TLR7, TLR9, and TLR10 expression is found to be very low (Table 10.1) [2, 17–20, 28]. The differential expression of the TLRs correlates with the responsiveness to TLR9 triggering, and naïve B cells respond to TLR9 stimulation, but their activation by CpG ODN only leads to the upregulation of cell surface activation markers such as CD69 and CD86 or the secretion of cytokines such as IL-6 (Table 10.2) [2, 18, 20]. In contrast, memory B cells undergo several rounds of division and differentiate to immunoglobulin (Ig)-secreting cells in response to CpG which can be measured by staining B cells with CSFE and measuring proliferation by flow cytometry or determining IgM, IgG1, or IgG2 secretion by ELISA. Naïve B cells only produce Ig and proliferate if simultaneously stimulated by CpG ODN and through the BCR (Table 10.2), which also up-regulates their TLR9 expression. BCR stimulation can be achieved by coincubation of B cells with, e.g., CpG ODN and F(ab’)2 fragments of antibody to human Ig (anti-Ig) [2, 18, 20, 28]. The differential regulation of TLR expression in memory and naïve B cells appears to prevent polyclonal activation in a primary immune response and restricts TLR9-induced Ig secretion to antigenspecific B cells. Murine differ from human B cells significantly in their response to CpG ODN (Table 10.2) [10, 29–33]. Although they produce IL-6, no IL-10 secretion upon CpG stimulation is observed. In addition, even without the concomitant activation through CD40L (19), murine B cells produce IL-12 and secrete TNF-α which is, if at all, only detected at very low levels in human B cells (34). Murine B220+ B cells show enhanced expression of MHC class II, proliferate, and differentiate in Ig-producing cells after stimulation with CpG ODN. Moreover, these effects can also be observed upon activation of both murine naïve (B220+ CD23+ IgM+ ) and memory (B220+ CD23+ IgG+ ) B cells with TLR9 ligands (9). The activation of B cells to different TLR ligands correlates to their TLR expression (Table 10.3). Whereas human B cells respond to TLR1 activation by Pam3 CSK4 or TLR7 activation by small molecule TLR7 ligands such as R-837 and R-848 or G,Urich single-stranded synthetic oligoribonucleotides (ORN) such as R-0009 (Table 10.3 and Fig. 10.1), they are not activated by TLR4, TLR6 (although expression of TLR6 on the mRNA level was observed), or TLR8 ligands [6, 17, 19, 22, 35–37], as well as
Proliferation, Ig secretion
IL-6, HLA-DR
na
–
–
–
–
Flagellin
TLR5
Proli– feration, Ig secretion
–
Source: Vollmer and Krieg (4) and Bourke et al. (5). ∗ TLR7 sensitivity is enhanced by pDC and IFN-α (17). ∗∗ See Table 10.2.
B cells
Murine
B cells
Human
Peptidoglycan Poly I:C LPS
TLR4
Pam3CSK4
TLR3
TLR2
TLR1
Table 10.3 Effects of TLR ligands on human and murine B cells G,U-rich ORN
TLR7 3M002
TLR8
na
na
IL-6, MIP-1α, MIP-1β, CD25, – CD40, CD80, CD54, CD80, proliferation, Ig CD86 secretion, apoptosis protection
R-837/ R-852A∗
TLR7
ProliProliferation, feration, Ig Ig secretion secretion
–
Zymosan/ MALP-2
TLR6
TLR9
TLR9
na
–
+∗∗
+∗∗
na
–
A,U-rich Thymosin ORN CpG ODN α
TLR8
In Vitro Effects of Adjuvants on B Cells 139
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MFI CD19+ CD80+ Cells
80
60
R-0009 Control R-0010 Dotap R-848 Media
40
20
10–8
10–7
10–6
10–5
[M]
Fig. 10.1. Stimulation of human PBMC with TLR7 ligands and small molecule ligand results in enhanced cell surface CD80 expression. Human PBMC were cultured for 48 h with ORN R-0009 (5 -UGUUUUUUCUCUUGUUUGGU-3 , all phosphorothioate (PS) modified; Biospring, Frankfurt, Germany), ORN R-0010 (5 -AUAAUUGACCUGCUUUCGCU3, PS; Biospring), control ORN (5 -AGCGAAAGCAGGUCAAUUAU-3 , PS; Biospring), or R-848 at the indicated TLR ligand concentrations in the presence (ORN) or absence (R-848) of DOTAP used as uptake enhancer. Cells were harvested and CD80 expression on CD19+ B cells measured by flow cytometry. Shown is the mean fluorescence intensity ± SEM of three blood donors.
by thymosin α, a putative TLR9 agonist previously described to stimulate cells via TLR9 (Table 10.3 and Fig. 10.2) (38). Similar to CpG ODN, the activation of human B cells with TLR1 and TLR7 ligands results in the production of cytokines, the upregulation of cell surface antigens, as well as proliferation and protection from apoptosis (Table 10.3). In contrast to human B cells and in concordance with their TLR expression pattern, murine B cells respond to TLR4 and TLR6 ligands such as LPS and MALP-2 (Table 10.3) [9, 10]. 3.1. Isolation and Culture of Human PBMC
1. Dilute buffy coats from healthy male and female human donors to 140 mL total volume by adding 1x DPBS/ Ampuwa and prepare four 50 mL-tubes with 15 mL ficoll each. Cover ficoll carefully to avoid mixing with a layer of 35 mL buffy coat and spin tubes at 400g for 30 min without brake. 2. Collect purified PBMC layer (white layer between ficoll and serum) and wash twice with 1x DPBS/Ampuwa. 3. Remove supernatant, resuspend pellet in 20 mL RPMI 1640 culture medium, and count cells.
In Vitro Effects of Adjuvants on B Cells
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400
PBMC 350
Purified B cells
IL-6 [pg/ml]
300 250 200 150 100
CPG2395
ODN1982
PolyI:C Flagellin R-848
PGN
Zymosan A
100 ng/ml
50 µg/ml
50 µg/ml
50 ng/ml
10 ng/ml
10 µg/ml
2.0 µM
1.0 µM
0.25 µM
0.063 µM
0.016 µM
2.0 µM
1.0 µM
0.25 µM
0.063 µM
0
0.016 µM
50
LPS
Med
ODN [µM]
Fig. 10.2. Stimulation of human PBMC and purified B cells by different TLR ligands. CD19+ B cells were isolated from PBMC and B cells as well as whole PBMC were cultured for 24 h with the indicated concentrations of TLR ligands (compare also Table 10.3). Supernatant was harvested and IL-6 measured by ELISA. Shown is the mean ± SEM of IL-6 secretion for two blood donors.
4. Prepare dilution series (for example, starting with 4 μM, 1:3 dilution, 7 concentrations, 50 μL/well) of ORN complexed to DOTAP (mix medium with DOTAP as indicated by the supplier, then add ORN for about 10–30 min before adding cells), or ODN, add to 96-well round-bottomed plates (see Notes 1 and 2). 5. Culture 2–5 × 106 cells/mL in 96-well round-bottomed plates in a humidified incubator at 37◦ C (see Note 3). 3.2. Isolation and Culture of B Cells
1. Purify human PBMC from buffy coat (see Section 3.1). 2. Isolate human B cells with the CD19 B cell isolation kit according to manufacture’s recommendations (Miltenyi Biotech). 3. To determine purity, stain isolated B cells with mAb solution (50 μL/well) consisting of FACS buffer and relevant CD markers: CD20 (B cells), CD14 (monocytes), and BDCA4 (plasmacytoid DC), stain for 20 min, 4◦ C in the dark, and identify B cells by flow cytometry on a FACSCalibur (Becton Dickinson) (see Note 4). For these experiments, B cells should be more than 95% pure. 4. Culture B cells (1 × 106 cells/mL, 250 μL/well) in 96-well round-bottomed plates for 24 h (see also Fig. 10.2).
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3.3. Culture of B Cell Lines
1. Human B cell lines (such as RAMOS or RPMI 8226) are cultured in suspension (see Note 5). 2. Passage the human B cell lines when approaching confluence by splitting every 3 days. 3. Seed out at a concentration between 0.5 and 1 × 106 cells/mL and culture cells in 150 cm2 flasks in a humidified incubator at 37◦ C (see also Fig. 10.3).
CD80 Expression [Mean % positive cells]
45 40 35 30 25 20 15 10 5 0 0.75
1.5 CPG 7909
0.75
1.5 ODN 1982
Med
ODN [µM]
Fig. 10.3. Stimulation of the human B cell line RAMOS by CpG ODN results in enhanced cell surface CD80 expression. RAMOS cells were stimulated for 24 h with the indicated ODN concentrations. For this experiment, the B-Class CpG ODN 7909 (PF-3512676) and a non-CpG control ODN 1982 were used [21, 43]. Cells were harvested and CD80 expression measured by flow cytometry. Shown is the percentage of CD80 positive cells.
3.4. Cytokine Secretion
1. Culture purified PBMC (5 × 106 cells/mL, 250 μL/well) in 96-well round-bottomed plates for 24 or 48 h (see Note 6). 2. Collect culture supernatants and, if not used immediately, freeze at –20◦ C until required. 3. Assess amounts of cytokines in the supernatants by using commercially available ELISA kits according to manufacturer’s recommendations (see Notes 7 and 8).
In Vitro Effects of Adjuvants on B Cells
3.5. Cell Surface Activation
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1. For detection of CD86 and CD80 expression on CD19+ B cells, culture PBMC (5 × 106 cells/mL, 250 μL/well) in 96-well round-bottomed plates for 48 h. 2. Harvest PBMC, wash once with FACS buffer, remove supernatant, resuspend pellet in 150 μL/well FACS buffer, and transfer 2 × 105 cells/well, 25 μL, to a new plate. 3. Stain cells with mAb solution (50 μL/well) consisting of FACS buffer and relevant CD markers: CD19 (B cells), CD86, and CD80 (activation marker) for 20 min, 4◦ C, in the dark. 4. Wash cells twice with FACS buffer and resuspend cells in Cell-Fix. 5. Store at 4◦ C in the dark until acquisition by flow cytometry on a flow cytometer (see also Fig. 10.1).
3.6. Intracellular Cytokine Measurement
1. For intracellular IP-10 measurement, culture PBMC (5 × 106 cells/mL, 250 μL/well) in 96-well round-bottomed plates for 18 h. 2. Add Brefeldin A solution (10 μL/well) and incubate for additional 6 h. 3. Harvest PBMC, wash once with FACS buffer, remove supernatant, resuspend pellets in 150 μL/well FACS buffer, and transfer 2 × 105 cells/well, 25 μL, to a new plate. 4. Stain cells with mAb solution (50 μL/well) consisting of FACS buffer and relevant CD markers: CD19 (B cells) and CD14 (Monocytes) for 20 min, 4◦ C, in the dark. 5. Wash PBMC twice with FACS buffer and resuspend cells in Intraprep Reagent Fixation (Reagent 1, 50 μL/well) for 15 min, 4◦ C in the dark. 6. Wash PBMC twice with 1x PBS and carefully resuspend cells in Intraprep Reagent Permeability (Reagent 2, 50 μL/well) 7. Ten minutes after starting resuspension, stain cells with mAb solution (10 μL/well) consisting of 1x DPBS/Ampuwa and mAb to IP-10 for 20 min, 4◦ C, in the dark. 8. Wash PBMC twice with FACS buffer and resuspend PBMC in Cell-Fix (100 μL/well). 9. Store at 4◦ C in the dark until acquisition by flow cytometry on a flow cytometer.
3.7. B Cell Proliferation
1. Wash purified PBMC with 1x DPBS/Ampuwa and resuspend cells in a total of 360 μL 1x PBS per 8 × 106 cells. 2. Add 40 μL CFSE per 8 × 106 cells and transfer to a new tube. Stain cells for 10 min at RT.
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3. Add 2 mL RPMI 1640 culture medium containing 10% FBS to stop reaction and wash cells twice with 1x DPBS/Ampuwa. 4. Resuspend cells in RPMI 1640 culture medium and incubate in 96-well flat-bottomed plates (2 × 106 cells/mL, 200 μL/well) for 5 days. 5. After incubation harvest PBMC, wash once with FACS buffer and stain cells with mAb solution (50 μL/well) consisting of FACS buffer and relevant CD marker: CD19 (B cells) for 20 min, 4◦ C, in the dark. 6. Wash cells twice with FACS buffer and resuspend cells in Cell-Fix (100 μL/well). 7. Store at 4◦ C in the dark until acquisition by flow cytometry. Decreased CFSE content indicates proliferating B cells (see Note 9). 3.8. Ab Secretion
1. Culture purified PBMC (5 × 106 cells/mL, 250 μL/well) in 96-well round-bottomed plates for 5 days (see Note 10). 2. Collect culture supernatant and, if not used immediately, freeze at –20◦ C until required. 3. Assess amounts of Ab in the supernatant by using in-house ELISAs developed using commercially available antibodies (BD Pharmingen and Sigma).
4. Notes 1. CpG ODN do not need to be formulated with uptake enhancers to exert their immune stimulatory activity, they work just by incubation of the primary cells or cultured cell lines with CpG ODN. This is in contrast to antisense ODN or ORN which usually require special formulation with, e.g., lipocations to facilitate uptake into cells or to down-regulate gene expression. 2. It is recommended to perform a dose–response study before deciding on one or few doses of, e.g., a single TLR9 ligand. Results may vary, and donor to donor variation may be observed, so that at least three different donors should be used in a B cell stimulation experiment. In addition, doses of adjuvants or TLR ligands to effectively stimulate B cells can differ (see also Fig. 10.1), and in these cases dose responses are highly recommended. 3. In principle, frozen PBMC or B cells can be used for these assays. However, care should be taken when the
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activation of pDCs is measured in the same assay. Plasmacytoid DCs are very sensitive to freezing and thawing and strongly reduced pDC effects are usually observed when frozen PBMC are used for TLR activation. 4. In some cases, the purity of the cells used may have been insufficient to completely exclude effects due to contaminating pDCs, which are activated even at very low concentrations. For example, CpG-induced activation of human monocytes is indirect and dependent on the presence of contaminating pDC and IFN-α. In addition, the presence of pDCs and IFN-α can enhance TLR7 expression in B cells and, therefore, sensitize B cells to respond to TLR7 ligands (17). The purity of B cell cultures can be measured with mAb to BDCA-4 and CD14 or determination of potential IFN-α production from remaining pDC in the SN to ensure lack of pDC that can influence results. 5. Activation of B cells cannot only be measured by using primary human or murine B cells. Human B cell lines such as RPMI8226 and RAMOS or murine B cell lines such as WEHI-231 were demonstrated to be responsive to TLR9 stimulation [32, 39–41]. Similar responses to CpG activation such as IL-6, IL-8, or IP-10 secretion and upregulation of the CD80 antigen on the cell surface (Fig. 10.3) are observed. The use of B cell lines also allows for automatization of B cell activation assays. 6. The duration of the culture of cells in the presence of a stimulus such as a TLR9 ligand can influence the detection of cytokines or chemokines. For example, IL-6 secretion from human B cells can be already measured earlier (starting at about 6 h upon CpG activation, with a peak around 24 h) than IL-10 or IP-10 secretion that should be measured after around 48 h of cell culture. 7. Cytokine production mediated by CpG ODN can adopt a bell-shaped nature in vitro with cytokine levels decreasing at higher doses. This may be observed for IL-6 secretion or B cell proliferation but is most pronounced for IFN-α production by pDC. Potential factors responsible for the decrease in cytokine production at higher ODN concentrations are yet to be identified. It is tempting to speculate that the strong stimulation of the NF-κB pathway leads to the induction of negative regulators and finally to the down modulation of B cell responses. 8. Different classes of CpG ODN and ORN exist depending on their sequence and secondary and tertiary structure [24, 42]. For example, A-Class CpG ODN stimulate weak TLR9-mediated NF-κB activation resulting in only moderate B cell activation, but at the same time very
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efficiently stimulate type I IFN production from pDCs. In contrast, B-Class CpG ODN induce moderate IFN-α production but strong NF-κB signaling and, therefore, B cell activation. A third class of CpG ODN (the C-Class) combines the characteristics of the A- and B-Classes and stimulates strong B cell responses but also pDC type I interferon production. 9. Stimulation of B cells also results in an increase of their size. Therefore, for flow cytometry a greater gate has to be set to allow to measure proliferation or cell surface antigen expression of all activated B cells. 10. For most B cell activation experiments human AB serum may be used, except for Ab secretion where RPMI 1640 medium supplemented with heat-inactivated human fetal bovine serum must be used.
Acknowledgements The author would like to thank Drs. Alexandra Forsbach, Marion Jurk, and Ulrike Samulowitz for experimental support and helpful scientific discussions, and Silke Fähndrich for assistance in manuscript preparation. Both authors are employees of Pfizer and may have a financial interest in the therapeutic development of TLR7 and TLR9 agonists. References 1. Rajewsky, K. (1996) Clonal selection and learning in the antibody system. Nature 381(6585), 751–758. 2. Bernasconi, N. L., Traggiai, E., Lanzavecchia, A. (2002) Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298(5601), 2199–2202. 3. Iwasaki, A., Medzhitov, R. (2004) Tolllike receptor control of the adaptive immune responses. Nat Immunol 5(10), 987–995. 4. Vollmer, J., Krieg, A. (2007) Mechanisms and therapeutic applications of immune modulatory oligodeoxynucleotide and oligoribonucleotide ligands for Toll-like receptors. Antisense Drug Technology, 2nd edn. CRC Press, Boca Raton, FL, pp. 747–772. 5. Bourke, E., Bosisio, D., Golay, J., Polentarutti, N., Mantovani, A. (2003) The Toll-like receptor repertoire of human B lymphocytes: inducible and selective
expression of TLR9 and TLR10 in normal and transformed cells. Blood 102(3), 956–963. 6. Mansson, A., Adner, M., Hockerfelt, U., Cardell, L. O. (2006) A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by PamCSK, R-837 and CpG-2006 stimulation. Immunology 118(4), 539–548. 7. Hornung, V., Rothenfusser, S., Britsch, S., Krug, A., Jahrsdorfer, B., Giese, T., Endres, S., Hartmann, G. (2002) Quantitative expression of Toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 168(9), 4531–4537. 8. Leifer, C. A., Kennedy, M. N., Mazzoni, A., Lee, C., Kruhlak, M. J., Segal, D. M. (2004) TLR9 is localized in the endoplasmic reticulum prior to stimulation. J Immunol 173(2), 1179–1183.
In Vitro Effects of Adjuvants on B Cells 9. Gururajan, M., Jacob, J., Pulendran, B. (2007) Toll-like receptor expression and responsiveness of distinct murine splenic and mucosal B-cell subsets. PLoS ONE 2(9), e863. 10. Richard, K., Pierce, S. K., Song, W. (2008) The agonists of TLR4 and 9 are sufficient to activate memory B cells to differentiate into plasma cells in vitro but not in vivo. J Immunol 181(3), 1746–1752. 11. McCluskie, M. J., Weeratna, R. D. (2001) Novel adjuvant systems. Curr Drug Targets Infect Disord 1(3), 263–271. 12. McCluskie, M. J., Krieg, A. M. (2006) Enhancement of infectious disease vaccines through TLR9-dependent recognition of CpG DNA. Curr Top Microbiol Immunol 311, 155–178. 13. Muzio, M., Natoli, G., Saccani, S., Levrero, M., Mantovani, A. (1998) The human toll signaling pathway: divergence of nuclear factor kappaB and JNK/SAPK activation upstream of tumor necrosis factor receptorassociated factor 6 (TRAF6). J Exp Med 187(12), 2097–2101. 14. Muzio, M., Polentarutti, N., Bosisio, D., Manoj Kumar, P. P., Mantovani, A. (2000) Toll-like receptor family and signalling pathway. Biochem Soc Trans 28(5), 563–566. 15. Hacker, H., Vabulas, R. M., Takeuchi, O., Hoshino, K., Akira, S., Wagner, H. (2000) Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. J Exp Med 192(4), 595–600. 16. Vollmer, J. (2006) TLR9 in health and disease. Int Rev Immunol 25(3), 155–181. 17. Berkeredjian-Ding, I. B., Wagner, M., Hornung, V., Giese, T., Schnurr, M., Endres, S., Hartmann, G. (2005) Plasmacytoid dendritic cells control TLR7 sensitivity of naive B cells via type I IFN. J Immunol 174(7), 4043–4050. 18. Huggins, J., Pellegrin, T., Felgar, R. E., Wei, C., Brown, M., Zheng, B., Milner, E. C., Bernstein, S. H., Sanz, I., Zand, M. S. (2007) CpG DNA activation and plasma-cell differentiation of CD27– naive human B cells. Blood 109(4), 1611–1619. 19. Wagner, M., Poeck, H., Jahrsdoerfer, B., Rothenfusser, S., Prell, D., Bohle, B., Tuma, E., Giese, T., Ellwart, J. W., Endres, S., Hartmann, G. (2004) IL-12p70-dependent Th1 induction by human B cells requires combined activation with CD40 ligand and CpG DNA. J Immunol 172(2), 954–963. 20. Bernasconi, N. L., Onai, N., Lanzavecchia, A. (2003) A role for Toll-like receptors in
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Chapter 11 NKT Cell Responses to Glycolipid Activation Josianne Nitcheu Tefit, Gwyn Davies, and Vincent Serra Abstract NKT cells are a distinct lineage of T lymphocytes that are usually identified by the co-expression of the semi-invariant CD1d-restricted αβ TCR and the NK1.1 allelic marker of NK lineage receptors in the C57BL/6 mice and related strains. NKT cells can be subdivided based on CD4/CD8 expression and on tissue of origin. NKT cells express significantly the TCR gene products Vα24 JαQ in humans, the homolog of mouse Vα14 Jα18, paired with Vβ11, the homolog of mouse Vβ8.2. NKT cells are most frequent in liver (up to 30% of T cells in mice and approximately 4% of hepatic T cells in human), bone marrow, and thymus and represent a smaller proportion of T cells in other tissues including spleen, lymph nodes, blood, and lung. NKT cells recognize a broad array of glycolipids in the context of CD1d presentation, and many studies have characterized a cascade of functions following in vitro and in vivo stimulation by α-GalCer, including production of high levels of immune-regulatory cytokines and bystander activation of several cell types including NK, B, T, and dendritic cells. Both in vitro and in vivo methods have been developed for the study of NKT responses to glycolipid presentation by CD1d. In practice, CD1dglycolipid-loaded tetramers would most reliably identify these cells. In vitro, splenocytes can be used to monitor cytokine release as this population contains all the cells necessary for sequestering, loading onto CD1d molecules, and presentation of glycolipids to NKT cells. Another system involves the use of NKT cell hybridoma and CD1d coated onto plastic plates to measure responses limited to NKT cells more precisely. In vivo, responses are typically measured by injecting the glycolipid into mice and monitoring plasma cytokine levels or DC maturation in the spleen. This chapter describes methods that can be used to identify NKT cells and to asses in vitro and in vivo their activation and expansion. Key words: NKT cells, glycolipid, alpha-galactosylceramide (α-GalCer), cytokines, CD1d/αGalCer tetramer.
1. Introduction Natural killer T (NKT) cells are a separate lineage of T lymphocytes that co-express receptors for T cell and natural killer (NK) cell lineages. Most NKT cells express a semi-invariant G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_11, © Springer Science+Business Media, LLC 2010
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T-cell receptor (TCR), Vα14-Jα18 paired with Vβ8.2, Vβ7, or Vβ2 in mice and Vα24-Jα18/Vβ11 in human (1–5). These cells are referred to as iNKT cells or type I NKT cells in contrast to type II NKT cells comprising the remaining NKT cells expressing noninvariant TCR (6). These cells share phenotypic and functional characteristics of T and NK cells. The phenotype of NKT cells expresses a T-cell receptor αβ (TCRαβ), the CD4 or the CD8 coreceptor or neither of them (double-negative [DN] phenotype), the NK1.1 marker, and some Ly49 receptors (7–10). Emerging evidence indicates that CD4+ and CD4– iNKT cell subsets are functionally distinct (11–13). The distribution of iNKT cells has been well studied in mice, but less well in humans. Murine iNKT cells represent approximately 0.5% of T-cell population in the blood and peripheral LN and up to 30% of T cells in the liver, and this population appears to be 10 times less frequent in humans. However, high and low expressers are found in humans and mice (14–17). Most NKT cells seem to recognize CD1d in conjunction with glycolipids, although the precise nature of these ligands is not yet clear. CD1d is a major histocompatibility complex (MHC) class I-related protein that consists of a heterodimer of a glycosylated heavy chain and β-2 micro globulin, with a binding pocket adapted for glycolipid antigens. CD1d is constitutively expressed on antigen presenting cells (APC) such as dendritic cells (DCs), macrophages, B cells, and also on Kuppfer cells and hepatocytes in the liver (18–21). All iNKT cells react strongly in vitro and in vivo with the glycolipid α-GalCer, originally isolated from the marine sponge Agela mauritianus (22–25). Because of its origin, α-GalCer seemed unlikely to be a natural antigen for NKT cells. However, despite the lack of any physiological relevance, α-GalCer has provided evidence that conserved TCR of iNKT cells evolved to recognize conserved lipids; consequently, much attention has been given to the identification of natural endogenous and exogenous ligands to which humans would be exposed. Despite controversies, the glycosphingolipid isoglobotrihexosylceramide (iGb3), one candidate family of natural endogenous ligands, was shown to activate the majority of mouse and human iNKT cells although iGb3 appeared to be a weaker agonist than α-GalCer (26–28). In addition to natural endogenous ligands, many reports have focused on the discovery of natural exogenous antigens and showed that iNKT cells could react with microbial glycolipids (29). As the identity of physiological ligands remained elusive, many advances in the function of iNKT cells have come from studies of NKT cell responses to α-GalCer. The hallmark of iNKT cell activation following TCR engagement is the rapid production of a high level of a variety of immunoregulatory cytokines, including both Th1 and Th2 cytokines, unlike conventional CD4 T cell responses that
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are usually polarized toward either Th1 or Th2 cytokine production (30). The cytokines secreted by activated NKT cells amplify the immune response resulting in bystander activation of other cell types including NK cells, B cells, conventional CD4+ T cells, CD8+ CTL cells, and DC (31–34). Because Th2 cytokines can antagonize the immunostimulatory properties of Th1 cytokines, the use of agonists that permit substantial control over the type of response generated may be important in the context of exploiting NKT cell responses to enhance adaptive cellular and humoral immunity. Many structural studies have demonstrated that CD1d glycolipid-mediated presentation to NKT cells is an important aspect of immune regulation, and structural analogs of α-GalCer have been developed in order to influence not only the magnitude of NKT cell stimulation but also the nature of the stimulation. In fact, some variants have shown decreased Th1 compared to Th2 cytokines (35). In this chapter, we describe in vitro and in vivo methods for the study of NKT cell responses to glycolipid presentation. In particular, methods are given to isolate identify, expand iNKT cells, measure CD1d glycolipid–NKT interaction, and NKT cells’ functions.
2. Materials 2.1. NKT Cell Detection In Vitro
1. Glycolipid antigen, (available upon request to the authors) α-GalCer (Kirin Breweries). 2. Antihuman antibodies: Pc5–antihuman TCR αβ (Beckman Coulter), FITC antihuman Vα24 (Beckman Coulter), PE antihuman Vα24 (Beckman Coulter), FITC antihuman Vβ11 (Beckman Coulter), PE antihuman Vβ11 (Beckman Coulter). 3. Blocking antibody: anti-mouse 0.5 mg/mL (BD Biosciences).
CD16/CD32
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4. Anti-mouse antibodies: PE-anti-mouse NK1.1 (BD Biosciences), FITC-anti-mouse TCR β (BD Biosciences). 5. FACS staining buffer: 1.7% of a 30% bovine serum albumin solution (SIGMA)/PBS (v/v), 2 mM EDTA. 6. Streptavidin, PE conjugate (Prozyme/Europa). 7. 10X Cell Fix (BD Biosciences): Dilute to 1X in distilled water before use. 8. BD falcon polystyrene round-bottomed tubes (BD Biosciences). 9. Biotinylated human or murine CD1d (available upon request to the authors).
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2.2. iNKT Cell Isolation and Culture
1. Phosphate-buffered saline (PBS). 2. Cell strainer (BD Biosciences). 3. 1 mL plastic syringe. 4. Petri dish. 5. 15 mL Falcon tubes. 6. Complete RPMI medium for culture of mouse (and NHP) mononuclear cells (MNCs): RPMI-Glutamax (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), antibiotics (100 U/mL penicillin G + 100 μg/mL streptomycin sulfate (Gibco)) and 50 mM 2mercaptoethanol (Gibco). 7. Complete RPMI AB serum medium for culture of human PBMCs: RPMI-Glutamax supplemented with 5% heatinactivated human male AB serum (Biowest), antibiotics (100 U/mL penicillin G + 100 μg/mL streptomycin sulfate (Gibco)). 8. Ammonium chloride lysing buffer (ACK buffer): 8.29 g NH4 Cl (SIGMA) + 1 g KHCO3 (SIGMA) + 37.2 mg Na2 EDTA (SIGMA) completed to 1 L of distilled H2 O to prepare a 1X solution. The pH of the 1X solution should fall within the range of pH 7.1–7.4. Store the 1X solution at 4◦ C and warm the solution to room temperature prior to use. 9. Trypan blue solution 0.4% (Sigma). 10. PE-labeled CD1d/α-GalCer tetramers (see Section 3.1.1). 11. Anti-PE microbeads (Miltenyi Biotec). 12. MACS positive selection column (Miltenyi Biotec). 13. Recombinant human IL-2 (Peprotech). 14. Recombinant mouse IL-2 (Peprotech). 15. Hank’s balanced salt solution (HBSS). 16. Percoll solution (GE Healthcare). 17. Antibodies for flow cytometry: APC-conjugated mouse anti-CD19 (BD Bioscience).
2.3. Measurement of Antigenic Potency
1. PBS 2. Recombinant soluble dimeric mouse CD1d:Ig fusion protein (BD Biosciences) or available upon request to the authors 3. DN32.D3 hybridoma (available upon request to the authors) 4. GM2a (available upon request to the authors)
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5. Mouse saposin B (available upon request to the authors) 6. Glycolipid antigen (available upon request to the authors), α-GalCer 7. ELISA kit for mouse IL-2 (BD Bioscience) 8. Complete RPMI medium for culture of mouse mononuclear cells (MNCs): RPMI-Glutamax (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), antibiotics (100 U/mL penicillin G + 100 μg/mL streptomycin sulfate (Gibco)) and 50 mM 2mercaptoethanol (Gibco) 9. BMDC (freshly isolated) 10. Thymocytes (freshly isolated) 11. CBA cytokine detection kits (BD Bioscience) 2.4. Measurement of Cytokine Release In Vitro
1. MNC from mouse spleen (see Section 3.2.1) 2. Complete RPMI medium for culture of mouse mononuclear cells (MNCs): RPMI-Glutamax (Gibco) supplemented with 10% heat-inactivated fetal bovine serum, antibiotics (100 U/mL penicillin G + 100 μg/mL streptomycin sulfate (Gibco)) and 50 mM 2-mercaptoethanol 3. Glycolipid antigen (available upon request to the authors), α-GalCer 4. ELISA kits for cytokine quantification (BD Bioscience) 5. CBA kits for cytokine detection (BD Bioscience)
2.5. Serum Cytokine Measurement
1. Mice: all strains of wild type mice 2. Glycolipid antigen, (available upon request to the authors), α-GalCer (Kirin Breweries) or available upon request to the authors 3. ELISA kits for cytokine quantification (BD Bioscience) 4. CBA kits for cytokine detection (BD Bioscience)
2.6. NKT Activation In Vivo
1. Mice: all strains of wild type mice
2.7. In Vivo Dendritic Cell Maturation Assays
1. R-PE-conjugated hamster anti-mouse CD11c (BD Biosciences)
2. Glycolipid antigen (available upon request to the authors), α-GalCer (Kirin Breweries)
2. FITC-conjugated rat anti-mouse CD8α (BD Biosciences) 3. Biotin-conjugated rat anti-mouse CD80 (BD Biosciences) 4. Biotin-conjugated rat anti-mouse CD86 (BD Biosciences) 5. Biotin-conjugated rat anti-mouse CD40 (BD Biosciences) 6. Streptavidin APC (BD Biosciences)
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3. Methods 3.1. NKT Cell Detection In Vitro
Generally, the use of CD1d/glycolipid tetramer can most reliably identify iNKT cells. Another means of identifying these cells is the use of specific monoclonal antibodies. Mouse NKT cells are usually identified by the co-expression of the αβ TCR and NK1.1 and human NKT cells by the co-expression of Vβ11 and Vα24 on the TCR αβ positive population (see Note 1).
3.1.1. Tetramer Staining of Mouse Mononuclear Cells
Importantly, CD1d/glycolipid tetramers should be prepared the day before the experiment (CD1d + glycolipid X): Here, we describe CD1d loading with α-GalCer, the most common glycolipid used for the study of iNKT cells. Unloaded CD1d tetramers are used as negative controls (see Note 2). 1. Loading of α-GalCer to CD1d: On the day before staining, mix 0.1 μL of CD1d at 2 mg/mL + 0.08 μL α-GalCer 0.2 mg/mL + 0.19 μL PBS per staining condition. To prepare empty or unloaded CD1d, mix 0.1 μL of CD1d 2 mg/mL + 0.27 μL PBS. Incubate overnight at 37◦ C. 2. PE-labeling of α-GalCer-CD1d tetramers: The following day, label CD1d/α-GalCer tetramers or empty CD1d tetramers with streptavidin-PE as follows. Mix 0.37 μL of CD1d/glycolipid tetramers (or empty CD1d tetramers) + 0.11 μL of streptavidin-PE + 1.52 μL of PBS to obtain 2 μL final volume. Tetramer is ready to use at 2 μL per 2 × 106 cells for staining (see Note 3). 3. Prepare two BD falcon polystyrene tubes with 2×106 cells per tube for CD1d/α-GalCer tetramers and empty CD1d tetramer. 4. Add 2 μL of blocking solution (anti-CD16/CD32 at 0.5 mg/mL) and 23 μL of FACS staining buffer. 5. Incubate for 10 min at 4◦ C. 6. Add 2 μL of CD1d/ α-GalCer tetramer or empty CD1d tetramer in appropriate tubes. 7. Incubate the cells for 15 min at room temperature (RT) protected from light. 8. Add 1 μL of FITC anti-TCRβ in each tube and 23 μL of FACS staining buffer (see Note 4). 9. Incubate for 30 min at RT protected from light. 10. Wash the cells twice by adding 1 mL of FACS staining buffer, centrifuge for 5 min at 350×g and discard supernatant.
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11. Resuspend cell pellet in final volume of 250 μL of cell fix 1X. The cells are now ready to be analyzed by flow cytometry.
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12. Quantify the proportion of iNKT cells on the TCR β+ CD1d/glycolipid tetramer+ population. Background consisting of TCR β+ empty-CD1d+ population is further subtracted from the positive population (Fig. 11.1).
TCR-β FITC
TCR-β FITC
Fig. 11.1. iNKT cell staining of live mouse lymphocytes using CD1d/α-GalCer-loaded tetramers. iNKT cells are the CD1d/α-GalCer+ TCRβ+ population among the gated mouse lymphocytes from the spleen. Background consisting of unloaded CD1d tetramers+ TCRβ+ is further subtracted from the positive population.
3.1.2. Tetramer Staining of Human or Nonhuman Primate (NHP) PBMC
1. The day before staining, mix 0.1 μL of CD1d 2 mg/mL + 0.08 μL α-GalCer 0.2 mg/mL + 0.19 μL PBS per staining condition. To prepare empty or unloaded CD1d, mix 0.1 μL of CD1d 2 mg/mL + 0.27 μL PBS. Incubate overnight at 37◦ C. 2. The next day, label CD1d/α-GalCer tetramers or empty CD1d tetramers with streptavidin-PE as follows. Mix 0.37 μL of CD1d/glycolipid tetramers (or empty CD1d tetramers) + 0.11 μL of streptavidin-PE + 1.52 μL of PBS to obtain 2 μL final volume. Tetramer is ready to use at 2 μL per 2 × 106 cells for staining. 3. Prepare two FACS polypropylenes tubes with 2×106 cells per tube for CD1d/ α-GalCer tetramers and empty CD1d tetramer. 4. Pellet the cells (5 min at 350×g). Add 23 μL of FACS staining buffer and 2 μL of CD1d/ α-GalCer tetramers or empty CD1d tetramers in appropriate tubes. 5. Incubate the cells for 15 min at RT protected from light. 6. Prepare antibody cocktail as follows. Mix 10 μL of FITC anti-Vβ11 (or anti-Vα24) with 10 μL of PC-5 anti-TCR αβ and 5 μL of FACS staining buffer per sample. Add 25 μL of antibody cocktail to each tube (see Note 5). 7. Incubate for 30 min at RT protected from light.
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8. Wash the cells twice by adding 1 mL of FACS staining buffer, centrifuge for 5 min at 350×g, and discard supernatant. 9. Resuspend cell pellet in final volume of 250 μL of cell fix 1X. The cells are now ready to be analyzed by flow cytometry.
M1
FSC-H
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SSC-H
10. Quantify the proportion of iNKT cells by gating on the TCR αβ+ population then analyze for both the expression of CD1d/α-GalCer tetramers and Vβ11 (or Vα24). Background consisting of Vβ11+ (or Vα24+ ) empty-CD1d+ population is further subtracted from the positive population (Fig. 11.2).
R2
TCR-β PC5
Vβ11 FITC
R3
Vβ11 FITC
Fig. 11.2. iNKT cell staining of PBMC from human samples using CD1d/α-GalCer-loaded tetramers. iNKT cells are the CD1d/α-GalCer+ Vβ11+ population gated on both the live and TCRβ+ lymphocyte population. Background consisting of unloaded CD1d tetramers+ TCRβ+ is further subtracted from the positive population.
3.1.3. Antibody Staining of Murine iNKT Cells
An alternative method for staining iNKT cells uses anti-TCR-β and anti-NK1.1 antibodies for mouse iNKT cells or anti-Vα24 and anti-Vβ11 antibodies for human iNKT cells, despite being less specific than tetramer staining. 1. Prepare BD falcon polystyrene tubes with 2×106 cells per tube. 2. Pellet the cells. Add 48 μL of FACS staining buffer and 2 μL of blocking solution (anti-CD16/CD32 at 0.5 mg/mL). 3. Incubate for 10 min at 4◦ C. 4. Wash the cells by adding 1 mL of FACS staining buffer, centrifuge for 5 min at 350×g, and discard supernatant. 5. Prepare antibody cocktail as follows: Mix 1 μL of FITC antiTCR-β (or anti-CD3) + 1 μL PE anti-NK1.1 (PK136) + 98 μL of FACS staining buffer. Add 100 μL of antibody cocktail per staining. 6. Incubate for 30 min at 4◦ C protected from light. 7. Wash the cells twice by adding 1 mL of FACS staining buffer, centrifuge for 5 min at 350×g, and discard supernatant.
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8. Resuspend cell pellet in final volume of 250 μL of cell fix 1X. The cells are now ready to be analyzed by flow cytometry.
SSC-H
PE Anti NK1.1
9. Analyze iNKT cells by gating on the TCR β+ NK1.1+ population (Fig. 11.3).
FSC-H
TCR-β FITC
Fig. 11.3. iNKT cell staining of live mouse lymphocytes using NK1.1 monoclonal antibodies. iNKT cells are the TCRβ+ NK1.1+ population among the gated mouse lymphocytes from the spleen.
3.1.4. Human iNKT Cell Detection Using Antibodies
1. Prepare FACS polypropylenes tubes with 2 × 106 cells. 2. Pellet the cells (5 min at 350×g). Prepare antibody cocktail as follows: Mix 10 μL of FITC anti-Vβ11 + 10μL of PE anti-Vα24 +10 μL of PCS anti-TCR αβ and 20 μL of FACS staining buffer. Add 50 μL of antibody cocktail per staining. 3. Incubate for 30 min at 4◦ C protected from light. 4. Wash the cells twice by adding 1mL of FACS staining buffer, centrifuge for 5 min at 350×g, and discard supernatant. 5. Resuspend cell pellet in final volume of 250 μL of cell fix 1X. The cells are now ready to be analyzed by flow cytometry. 6. Quantify the proportion of iNKT cells on the Vβ11+ Vα24+ population gated on the TCR αβ+ population.
3.2. iNKT Cell Isolation and Culture
As already mentioned, Vα14 NKT cells account for up to 30% of T cells in the liver, and approximately 5% of T lymphocytes in other organs such as the spleen. Here we present methods to isolate and expand iNKT cells from the spleen and the liver, as these organs can be easily harvested, and their iNKT cells may display different functional characteristics.
3.2.1. Mononuclear Cell (MNC) Preparations from Spleen
This method can also be applied to isolate MNC from thymus or lymph nodes (LN) (see Note 6). 1. Euthanized animals in accordance with ethic committee. 2. Cut out the spleen: Cut open the body cavity and remove the spleen using the forceps; the spleen is of the color of a kidney bean.
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3. Place the spleen into the 40 μM cell strainer. Using the plunger end of a 1-mL syringe, mash the spleen through the cell strainer into the Petri dish. 4. Rinse the cell strainer with 10 mL of PBS. Discard the strainer. Transfer the cells into 15-mL falcon tubes. 5. Spin cells at 300×g for 5 min. 6. Discard supernatant and resuspend pellet in 5 mL of ACK. 7. Gently mix by pipetting immediately after adding the ACK. 8. Incubate at RT for 10 min. 9. Add 5 mL of PBS to stop the reaction and spin cells at 300×g for 5 min. 10. Discard supernatant and resuspend pellet in 10 mL of PBS. Spin cells at 300×g for 5 min. 11. Repeat step 10, then resuspend pellet in 10 mL PBS and pass the preparation through the cell strainer (see Note 7). 12. Count viable cells in trypan blue solution to exclude dead cells. 13. Depending on the mouse age, 5–10 × 107 cells will be obtained per mouse. 3.2.2. Mononuclear Cell Preparation from the Liver
Despite high frequency of iNKT cells in the liver, the absolute number of MNCs that can be recovered from one liver is approximately 2–5 × 106 cells. Hence livers of several identical mice can be pooled to obtain high numbers of MNC. 1. Euthanize animals in accordance with ethic committee. 2. Cut out the liver: Make an abdominal liver incision to remove the liver. 3. Perfuse the liver by the portal vein: Inject ice-cold HBSS slowly into the portal vein using a 25G needle until the organ becomes pale. 4. Homogenize using a Potter–Elvehjem homogenizer and resuspend in HBSS. 5. Wash the cells once by centrifugation at 800×g for 5 min, discard supernatant. 6. Resuspend cells in a 35% Percoll solution and centrifuge at 1,400×g for 25 min at RT. The cells of interest will form a pellet. Therefore the brake should be left on. Discard supernatant. 7. Wash the pellet containing mononuclear cells by centrifugation at 800×g for 5 min, discard supernatant. 8. Resuspend pellet in 5 mL of ACK. Gently mix by pipetting immediately after adding the ACK. 9. Incubate at RT for 10 min.
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10. Add 5 mL of PBS to stop the reaction and spin cells at 300×g for 5 min. 11. Discard supernatant and resuspend pellet in 10 mL of PBS. Spin cells at 300×g for 5 min. 12. Repeat step 10, then resuspend pellet in 10 mL PBS, and pass the preparation through the cell strainer (see Note 7). 13. Count viable cells in trypan blue solution to exclude dead cells. 3.2.3. Peripheral Blood Mononuclear Cell Preparation from Human and NHP PBMC
PBMC from human or NHP are isolated from whole venous blood using standard procedures yielding approximately 1 × 106 PBMC/mL total blood containing around 0.01–0.5% iNKT cells.
3.2.4. iNKT Cell Enrichment from Mouse MNCs Using Magnetic Beads
The proportion of iNKT cells in the liver or other organs such as the spleen shows significant variations. Hence it might be necessary to further purify iNKT cells from MNCs from these organs. Positive selection using CD1d/glycolipid tetramers will achieve the highest selection by magnetic bead separation (MACS) according to the manufacturer’s instructions (Miltenyi Biotec) or FACS sorting. 1. Add 2 μL of blocking solution (anti-CD16/CD32 0.5 mg/mL) and 23 μL of FACS staining buffer per 2 × 106 cells. 2. Incubate for 10 min at 4◦ C. 3. Add 2 μL of PE-labeled CD1d/α-GalCer tetramers and 23 μL of FACS staining buffer. 4. Incubate the cells for 45 min at RT protected from light. 5. After washing, the cells are incubated with anti-PE micro beads following the manufacturer’s instructions. Enriched iNKT cells are recovered by positive selection through selection columns. 6. The purity of cells is checked by FACs and a second round of purification with enriched iNKT cells could be performed if necessary.
3.2.5. iNKT Cell Enrichment from Mouse MNCs Using FACS Sorting
1. Add 2 μL of blocking solution (anti-CD16/CD32 at 0.5 mg/mL) per 2 × 106 cells and 23 μL of FACS staining buffer 2. Incubate for 10 min at 4◦ C 3. Add 2 μL of PE-labeled CD1d/α-GalCer tetramers 4. Incubate the cells for 15 min at RT protected from light 5. Add 1 μL of FITC anti-TCRβ and 1 μL of APC-conjugated mouse-anti CD19 (BD Bioscience) and 23 μL of FACS staining buffer (see Note 4)
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6. Incubate for 30 min at RT protected from light 7. Wash the cells twice and resuspend the cells in FACS staining buffer 8. For the sorting, the gate is set to include CD1d/α-GalCer tetramer+ TCR β+ population and exclude CD19+ cells (B cells) 3.2.6. Culture of NKT Cells in Mouse MNC Preparation from the Spleen, Thymus, LN, or Liver
iNKT cells can be expanded by culturing MNC preparations for 3–7 days in the presence of rmIL-2 and specific glycolipid. 1. Following isolation of MNC, resuspend pellet with appropriate volume of complete RPMI medium to obtain final cell concentration of 2 × 106 cells/mL. 2. Plate 2 mL of cell preparation in 12-well plate. 3. Add specific glycolipid previously filtered for iNKT stimulation. In general, glycolipid is added to final concentration of 100 ng/mL (add 20 μL per well of glycolipid X at 10 μg/mL to obtain final concentration of 100 ng/mL in wells), but other concentrations of glycolipid can be used. For control wells, add complete RPMI medium. 4. Culture the cells in incubators at 37◦ C/5% CO2 . 5. The next day, 24 h following stimulation with glycolipid, add recombinant mouse IL-2 to obtain final concentration IL-2 of 100 UI/mL (add 20 μL of IL-2 at 10 UI/mL) in each well. 6. Culture the cells in incubators at 37◦ C/5% CO2 and assay iNKT cells from day 3 to day 6 following IL-2 addition by tetramer staining.
3.2.7. Culture of iNKT Cells in Human or NHP PBMC
Human iNKT cells can be expanded by culturing PBMCs preparations for 7 days in the presence rhIL-2 and the specific glycolipid. 1. When using cryo-preserved PBMC, thaw rapidly in water bath at 37◦ C in a 15-mL falcon tube containing 50 mL of cold RPMI 5% AB serum. Centrifuge at 300×g for 10 min, discard supernatant, and resuspend the pellet in 10 mL of complete RPMI AB serum. For fresh PBMCs, proceed directly to the next step. 2. Count the cells in trypan blue and adjust concentration to 1 × 106 PBMC/mL in complete RPMI-AB serum medium. 3. Plate 2 mL of PBMC suspension per well of 12-well plates. 4. Add specific glycolipid previously filtered to final concentration of 100 ng/mL (add 20 μL of glycolipid at 10 μg/mL per well to obtain final concentration of 100 ng/mL), but other concentration of glycolipid can be used (from 10 ng/mL up to 1 mg/mL). For control wells, add complete RPMI AB medium.
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5. Culture the cells in incubators at 37◦ C/5% CO2 . 6. The following day, add rh IL-2 to obtain final concentration IL-2 of 100 UI/mL (add 20 μL of IL-2 at 10 UI/mL) in each well. 7. Culture the cells in incubators at 37◦ C/5% CO2 for another 6 days, then assay iNKT cells by tetramer staining. NHP iNKT cells can be expanded by culturing PBMCS as described above, in complete RPMI-10% FCS for 10–15 days, rh IL-2 is added every 3–4 days, them iNKT cells are assayed by tetramer staining. 3.3. Measurement of Antigenic Potency
A drawback of using MNC preparation is that immune cells other than NKT cells are present; consequently responses are not limited to NKT cells and may come from recognition of antigens by other cells (e.g., recognition of LPS by monocytes). To measure CD1d-glycolipid NKT cell-restricted responses more precisely, this system involves the use of iNKT cell hybridoma or antigen presenting cells (APC) and CD1d absorbed onto plastic plates (see Note 8). To study the presentation of exogenous lipids, CD1d coated plates or glycolipid-pulsed APCs are incubated with iNKT hybridoma, and IL2 production is measured in the supernatant.
3.3.1. Measurement of Antigenic Potency Using iNKT Hybridoma
1. 100 μL of recombinant mouse CD1d protein (10 μg/mL) in PBS is coated on triplicate on 96-well microtiter plates overnight at 4◦ C. 2. The plates are washed three times with PBS. 3. 100 μL of vehicle control or glycolipid X (α-GalCer and αGalCer variants, 100 ng/mL) in PBS or glycolipid X in the presence of LTP (1 μg/well GM2a +1.7 μg/well mouse saposin B) at various pH (acidic to neutral) in PBS is added for another 48 h. 4. The plates are washed three times with PBS. 5. DN32.D3 hybridoma cells (5 × 104 cells per well) are added and incubated for a further 24 h. 6. Supernatant is then collected and assayed for IL-2 cytokine by ELISA.
3.3.2. Measurement of Antigenic Potency Using APC
1. Bone marrow-derived denditic cells (BMDC) or fresh thymocytes can be used as APCs. BMDCs are obtained using standard methods (in general from bone marrow preparations from femurs and tibia of naive mice in the presence of GMCSF). 2. Glycolipid loading: Culture BMDC or fresh thymocytes in 6-well plates and pulse with the compound of interest by adding glycolipid X at various concentrations for 4 h.
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3. Collect the BMDC or thymocytes in complete RPMI medium, count and adjust to 2 × 105 cells/mL or 5 × 105 cells/mL, respectively. 4. Co-culture: Add 100 μL of APC (2 × 104 cells/well) or fresh thymocytes (5 × 104 cells/well) to 100 μL of hybridoma DN32.D3 at 5 × 105 cells/ml (5 × 104 cells/well) in 96-well round-bottomed plates. 5. Culture the cells in incubators at 37◦ C/5% CO2 for 24 h. 6. Collect supernatant and assay IL-2 production by ELISA. 3.4. Measurement of Cytokine Release In Vitro
Cytokine can be measured in culture supernatants from MNCs cultures from the spleens 48 h following addition of specific glycolipid. Typically the Th1/Th2 bias is quantified by measuring relative amounts of Th1 cytokines such as IFN-γ, IL-2, and TNFα, and TH2 cytokines such as IL-4, IL-5 by cytometric bead array or ELISA. 1. Following isolation of cells, resuspend pellet with appropriate volume of complete RPMI medium to obtain final cell concentration of 5 × 106 cells/mL. 2. Plate 100 μL of cell preparation in round-bottomed 96-well plate. 3. Add specific glycolipid, previously filtered, for iNKT stimulation. In general, glycolipid is added to a final concentration of 100 ng/mL in 100 μL complete RPMI medium (add 100 μL/well of glycolipid X at 200 ng/mL to obtain final concentration of 100 ng/mL in wells), but other concentrations of glycolipid can be used. For control wells, add complete RPMI medium. 4. Culture the cells in incubators at 37◦ C/5% CO2 for 48 h. 5. Collect supernatants and assay for cytokine production by ELISA or CBA, or store at –20◦ C before assay.
3.5. Serum Cytokine Measurements
Direct administration of glycolipid is possible, via the subcutaneous (s.c.), intramuscular (i.m.), or intravenous route (i.v.) (see Note 9). However the effect of α-GalCer is more potent with the i.v. rather than the i.m. and s.c. routes of administration. Following administration of glycolipid (in general 1 pg to 1 μg of glycolipid is injected in mice), the serum is collected at appropriate times for cytokine measurement. Several features of activation are seen within 4–8 h of administration with a single dose of αGalCer. Systemic injection induces an early boost of IL-4 detected in the serum after around 2 h, followed by a more prolonged burst of IFN-γ (18–24 h). More sustained and efficient results have been described upon injection with of α-GalCer-pulsed DCs, particularly with respect to the production of IFN-γ (36). Detec-
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tion of serum cytokine levels can be measured using ELISA sets or mouse Th1/Th2 CBA. 3.6. NKT Activation In Vivo
Following activation, NKT cells undergo a rapid down-regulation of their TCR, followed by a massive apoptosis within 3–4 days of activation, resulting in a long lasting depletion until regeneration occurs in part from thymus precursors (37–39). Following glycolipid administration in vivo, the spleens are collected at appropriate times following injection, for MNC isolation and iNKT staining by CD1d tetramer, as described above in Sections 3.1.1 and 3.2.1. iNKT cell activation is measured by the disappearance of the iNKT cells.
3.7. In Vivo Dendritic Cell Maturation Assay
To test the in vivo efficacy of glycolipid on dendritic cells (DC) function, expression of co-stimulatory molecules such as CD40, CD80, CD86, and B7-DC is assessed on the main subsets of myeloid CD11c+ DCs in the spleen, distinguished as CD8+ and CD8– . Spleens are collected at appropriate times following glycolipid administration, for MNC isolation and staining for DC maturation markers. 1. Following isolation of MNCs cells from the spleens, resuspend pellet with appropriate volume of FACS staining buffer to obtain final cell concentration of 50 × 106 cells /mL 2. Transfer 100 μL of cell per staining, then pellet the cells 3. Prepare antibody cocktail for each specific staining as follows: 4. Antibody cocktail for CD86 staining: 2 μL PE antiCD11c + 1 μL FITC anti-CD8-α + 2 μL biotinylated anti-CD86 (0.2 μg/106 c) + 95 μL FACS staining buffer 5. Antibody cocktail for CD40 staining: 2 μL PE antiCD11c + 1 μL FITC anti-CD8-α + 5 μL biotinylated anti-CD40 (0.5 μg/106 c) + 92 μL FACS staining buffer 6. Antibody cocktail for CD80 staining: 2 μL PE antiCD11c + 1 μL FITC anti-CD8-α + 5 μL biotinylated anti-CD80 (0.5μg/106 c) + 92 μL FACS staining buffer 7. Add 100 μL of cocktail antibody as appropriate 8. Incubate for 30 min at 4◦ C 9. Add 1mL of FACS staining buffer 10. Spin cells at 300×g for 5 min, discard supernatants 11. Dilute streptavidin-APC as follows: Add 10 μL streptavidin + 90 μL FACS staining buffer. Distribute 100 μL of diluted streptavidin-APC in each tube 12. Incubate for 20 min at 4◦ C 13. Add 1 mL of FACS wash buffer
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14. Spin cells at 300×g for 5 min, discard supernatants 15. Resuspend pellet with 200 μL of Cell Fix 1X in BD falcon polystyrene tubes 16. Gate the live population on the CD11c+ positive cells, and then re-gate to show CD8-positive or CD8-negative cells expressing CD40, CD80, or CD86 activation marker
4. Notes 1. Several receptors commonly found on NK cells are also expressed on iNKT cells, chief among these is the Ctype lectin NK1.1. Subsequent reports showed that mouse NK1.1+ subsets can be divided based on CD4/CD8 expression and on tissue of origin, with approximately 60% of iNKT cells expressing CD4. A difficulty with the use of NK1.1 to identify NKT cells is that this allelic marker is only expressed in a limited number of mouse strains, primarily C57BL/6 and related strains. Humans have NKT cells with characteristics very similar to mouse NKT cells. Human NKT cells expressed the homologous TCR gene products (Vβ11, the homolog of mouse Vβ8.2, and Vα24 JαQ, the homolog of mouse Vα14 Jα18). Human NKT cells can recognize mouse CD1d and vice versa due to highly conserved specificity. 2. Because of a high degree of conserved specificity, murine CD1d/glycolipid tetramers can stain human iNKT cells and vice versa. In addition, human CD1d/glycolipid tetramers can stain iNKT cells from NHP, because of the high degree of conserved specificity between humans and rhesus macaques (40). 3. α-GalCer-loaded CD1d tetramers are commercially available (www.proimmune.com) and the staining is performed according to the manufacturer’s instructions. 4. Alternatively, FITC anti-CD3 can be used. 5. Anti-CD3 antibodies can be used in the place of anti-TCR αβ. For the staining of NHP PBMC, the use of Vα24 antibody is best suitable because of a good cross-reactivity. 6. The populations from the spleen, liver, LN, PBMC contain all of the cells necessary for sequestering glycolipids, loading them onto CD1d, and presenting them to NKT cells. 7. This step is important to remove aggregates for further steps of iNKT enrichment. 8. It has been shown that glycolipid antigens require the assistance of a lipid transfer protein (LTP) such as saposins
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A-D, GM2 activator (GM2a), and Niemann–Pick type C2 (NPC2) for efficient lipid binding and recognition of CD1d molecules by NKT cells. However, each has its own specificity and it has been shown that saposin B and GM2a has the capacity to transfer α-GalCer to CD1d (26, 41). Low pH is optimal for saposin-mediated lipid loading, consistent with their normal cellular distribution although for saposin B the functional loading is still effective at pH 7. 9. The central feature of NKT activation upon exogenous administration in vivo of NKT ligands such as α-GalCer is the reciprocal activation of NKT cells and DCs, inducing iNKT cells to up-regulate CD40L, Th1, and Th2 cytokines as well as a variety of chemokines. CD40 cross-linking induces CD40, B7.1, and B7.2 expression and IL-12 production by DCs, which in turn enhances NKT cell activation and cytokine production. The TH1/Th2 outcome of NKT activation is partially understood and may result from intrinsic properties of the glycolipid, such as the length or unsaturation of alkyl chains and composition of the polar head, as well as extrinsic variations such as solubility in water, that could modify their route of trafficking and uptake. In addition, the route of administration could modify the TH1/Th2 bias depending on the cytokine environment. References 1. Bendelac, A., Matzinger, P., Seder, R. A., Paul, W. E., Schwartz, R. H. (1992) Activation events during thymic selection. J Exp Med 175, 731–742. 2. Bendelac, A., Schwartz, R. H. (1991) CD4+ and CD8+ T cells acquire specific lymphokine secretion potentials during thymic maturation. Nature 353, 68–71. 3. Dellabona, P., Padovan, E., Casorati, G., Brockhaus, M., Lanzavecchia, A. (1994) An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J Exp Med 180, 1171–1176. 4. Hayakawa, K., Lin, B. T., Hardy, R. R. (1992) Murine thymic CD4+ T cell subsets: a subset (Thy0) that secretes diverse cytokines and overexpresses the V beta 8 T cell receptor gene family. J Exp Med 176, 269–274. 5. Porcelli, S., Yockey, C. E., Brenner, M. B., Balk, S. P. (1993) Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V
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7. 8.
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beta genes and an invariant TCR alpha chain. J Exp Med 178, 1–16. Godfrey, D. I., MacDonald, H. R., Kronenberg, M., Smyth, M. J., Van Kaer, L. (2004) NKT cells: what’s in a name?. Nat Rev Immunol 4, 231–237. Bendelac, A. (1995) Mouse NK1+ T cells. Curr Opin Immunol 7, 367–374. Bendelac, A., Rivera, M. N., Park, S. H., Roark, J. H. (1997) Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu Rev Immunol 15, 535–562. MacDonald, H. R. (1995) NK1.1+ T cell receptor-alpha/beta+ cells: new clues to their origin, specificity, and function. J Exp Med 182, 633–638. Ortaldo, J. R., Winkler-Pickett, R., Mason, A. T., Mason, L. H. (1998) The Ly49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells. J Immunol 160, 1158–1165. Gumperz, J. E., Miyake, S., Yamamura, T., Brenner, M. B. (2002) Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J Exp Med 195, 625–636.
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12. Lee, P. T., Benlagha, K., Teyton, L., Bendelac, A. (2002) Distinct functional lineages of human V(alpha)24 natural killer T cells. J Exp Med 195, 637–641. 13. Crowe, N. Y., Coquet, J. M., Berzins, S. P., Kyparissoudis, K., Keating, R., Pellicci, D. G., Hayakawa, Y., Godfrey, D. I., Smyth, M. J. (2005) Differential antitumor immunity mediated by NKT cell subsets in vivo. J Exp Med 202, 1279–1288. 14. Yoshimoto, T., Bendelac, A., Hu-Li, J., Paul, W. E. (1995) Defective IgE production by SJL mice is linked to the absence of CD4+ , NK1.1+ T cells that promptly produce interleukin 4. Proc Natl Acad Sci USA 92, 11931– 11934. 15. Gombert, J. M., Herbelin, A., TancredeBohin, E., Dy, M., Carnaud, C., Bach, J. F. (1996) Early quantitative and functional deficiency of NK1+ -like thymocytes in the NOD mouse. Eur J Immunol 26, 2989–2998. 16. Baxter, A. G., Kinder, S. J., Hammond, K. J., Scollay, R., Godfrey, D. I. (1997) Association between alphabetaTCR+ CD4-CD8- Tcell deficiency and IDDM in NOD/Lt mice. Diabetes 46, 572–582. 17. Lee, P. T., Putnam, A., Benlagha, K., Teyton, L., Gottlieb, P. A., Bendelac, A. (2002) Testing the NKT cell hypothesis of human IDDM pathogenesis. J Clin Invest 110, 793–800. 18. Brossay, L., Jullien, D., Cardell, S., Sydora, B. C., Burdin, N., Modlin, R. L., Kronenberg, M. (1997) Mouse CD1 is mainly expressed on hemopoietic-derived cells. J Immunol 159, 1216–1224. 19. Roark, J. H., Park, S. H., Jayawardena, J., Kavita, U., Shannon, M., Bendelac, A. (1998) CD1.1 expression by mouse antigenpresenting cells and marginal zone B cells. J Immunol 160, 3121–3127. 20. Bendelac, A. (1995) Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes. J Exp Med 182, 2091–2096. 21. Geissmann, F., Cameron, T. O., Sidobre, S., Manlongat, N., Kronenberg, M., Briskin, M. J., Dustin, M. L., Littman, D. R. (2005) Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids. PLoS Biol 3, e113. 22. Kobayashi, E., Motoki, K., Uchida, T., Fukushima, H., Koezuka, Y. (1995) KRN7000, a novel immunomodulator, and its antitumor activities. Oncol Res 7, 529–534. 23. Kawano, T., Cui, J., Koezuka, Y., Toura, I., Kaneko, Y., Motoki, K., Ueno, H.,
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Chapter 12 Tracking Dendritic Cells In Vivo Catherine M. Rush and James M. Brewer Abstract The reasons why certain vaccine adjuvants and/or delivery systems are more or less effective at inducing immune responses or promoting the preferential induction of particular types of response are unknown. While vaccine antigen discovery has benefited from a systematic approach, our limited understanding of the interactions of adjuvants with cells of the immune system has hampered rational adjuvant discovery and handicapped the development of new and more effective vaccines. It is well accepted that the component parts of the immune system do not work in isolation and their interactions occur in distinct and specialised micro- and macro-anatomical locations. Consequently, significant obstacles to the systematic investigation of adjuvant effects have been the complexity of the physiological environments that adjuvants interact with and the difficulty in directly investigating these interactions dynamically in vivo. Here we describe some of the immunological and microscopical techniques that have enabled the analysis of the immune cells and their interactions, in vivo, in real time. It is only by performing such detailed and fundamental studies in vivo that we can fully understand the cellular and molecular interactions that control the immune response. These types of systematic analyses of the events involved in adjuvant action are a prerequisite if we are truly to design, build and target vaccines effectively. Key words: Immunology, adjuvants, dendritic cells, imaging, in vivo, vaccines, lymph nodes.
1. Introduction Vaccine adjuvants are a diverse group ranging from particulate formulations (e.g. MF-59, liposomes, ISCOMs), inorganic minerals (e.g. aluminium compounds) to inflammatory agents (e.g. cytokines such as IL-1 and IL-12) and pathogen-derived immunomodulators (e.g. lipopolysaccharides, MPL, dsRNA, DNA containing CpG motifs) (reviewed (1)). They also produce qualitative differences in immune responses (reviewed (2)). Given G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_12, © Springer Science+Business Media, LLC 2010
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this diversity, a central mechanism to explain their activity is far from obvious. It is clear that most vaccine adjuvants do not act directly on T cells, but exert their functions indirectly via effects on antigen-presenting cells (APCs). Of the APC populations, dendritic cells (DCs) are the most effective at inducing activation and proliferation of naïve T cells in vitro and in vivo. However, to optimally activate T cells, DCs themselves require activation. Activated DCs have increased antigen presentation (Signal 1) and costimulatory (Signal 2) activities, and during this process, DCs alter chemokine receptor expression and migrate to T cell areas of lymph nodes (3, 4). At this point DCs then interact with naïve T cells and it is likely that the initial contacts between these cells are the most important determinant of programming the subsequent T cell response towards priming versus tolerance and preferential induction of T cell phenotypes (57). APC-derived factors known to influence the phenotype and magnitude of T cell responses include duration and magnitude of antigen presentation, costimulator and cytokine expression by APCs and the phenotype of the APC population. However, it is unclear what impact adjuvants have on these APC parameters and the consequences these factors have on T cell activation, differentiation and function. Critically, very few of these parameters have been defined for adjuvants in vivo. This is a major obstacle, as it is clear that the component parts of the immune system do not work in isolation and their interactions are dynamic and occur in distinct and specialised micro- and macro-anatomical locations that can only be fully determined in real time, in the physiological context, in vivo. Where and when T cells “see” antigen during the initiation, maintenance and regulation of immune responses and where they go as a consequence are critically important to our fundamental understanding of the immune system and have wideranging implications, particularly for the targeted delivery of vaccines; however, these issues remain entirely unclear. Where a T cell encounters antigen can influence the subsequent immune response in a number of ways. For example, antigen presentation in a lymph node draining a particular site imprints T cells with a recirculation program/homing address (integrins, chemokine receptors, etc.) specific for that tissue (8, 9). Furthermore, APC populations may differ in their constituents (e.g. different proportions of DC subsets, B cells, macrophages and contribution of non-professional tissue cells) and/or response to local conditions (e.g. cytokines) in different sites (e.g. tissue versus lymph node), and this can influence what type of immune response is induced and maintained (10, 11). The ability of inflammatory and particulate adjuvants to change the physical form of an antigen or influence the cellular milieu that antigen interacts with at the injection site raises the question of precisely how antigen is
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transported to the lymph node (free or APC associated), whether after initial activation T cells require subsequent antigen exposure in situ and where this occurs (injection site versus lymph node). Some of these issues were first addressed in classical studies in the 1960s and 1970s (12, 13), and more recently have been aided by the availability of trackable TcR transgenic (Tg) systems (14, 15). However, these studies were static and only examined snapshots in time, days apart. As such, they were unable to definitively demonstrate the first encounter of a T cell with an APC and their subsequent migration, interactions and function. For example, expression of activation markers on T lymphocytes in a particular location does not prove that these cells encountered antigen there as they may have been activated elsewhere and migrated. Such gaps in our knowledge partly reflect not only the paucity of animal models that track APCs and lymphocytes during the initiation of an immune response but also the fact that it has been difficult to achieve this dynamically and definitively in real time in vivo. We have adopted two approaches to address these issues. First, we have employed the Eα/Y-Ae technique developed by Itano and Jenkins (16). The Eα-GFP system represents a significant advance in our ability to track the development of immune responses as it allows identification and detection of native antigen through tracking of GFP and more significantly, identification and detection of antigen-presenting cells, through the use of the Y-Ae antibody that solely recognises MHC Class II protein loaded with Eα peptide (Fig. 12.1). Finally, the availability of Eα-specific TcR transgenic T cells from TEa mice means that identification of the T cell response to presented antigen can also be performed.
Antigen presenting cell
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Fig. 12.1. Schematic diagram of Eα-GFP and Y-Ae system for detecting cells displaying peptide–MHC complexes. When recombinant Eα-GFP protein is taken up by APCs they become green due to GFP fluorescence. Following antigen processing, the Eα peptide is loaded onto I-Ab (MHC Class II) molecules and displayed on the APC surface. The Y-Ae monoclonal Ab only recognises the Eα peptide/I-Ab complex and thus can be used to identify cells presenting Eα apeptide in vivo by immunofluorescence microscopy or flow cytometry.
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This approach has a number of advantages over conventional methods (e.g. mice expressing Tg TcRs) that we, amongst others, have used extensively in the past. In our experience, while the adoptive transfer of TcR Tg T cells has a number of very useful applications in T cell biology, it cannot be employed to definitively identify presentation of antigen in vivo. Being an antibody-based method, use of Y-Ae overcomes confounding issues of costimulation that limits indirect analysis of T cell responses, as the readout is entirely Signal 1. Furthermore this approach can be directly applied to tissue sections or freshly isolated cells to identify presentation, allowing direct phenotypic analyses of the presenting cell populations to be performed. In summary, this method overcomes the confounding issues associated with T cell readouts of presentation and is consequently more definitive, relevant and less prone to artefact. The Eα-GFP system is amenable to a number of imaging modalities, particularly immunofluorescence of tissue sections, flow cytometry and in vivo multiphoton laser scanning microscopy (MPLSM).
2. Materials 2.1. Preparation of Y-Ae Antibody
1. The Y-Ae hybridoma originally described by Rudensky et al. (17) was provided by Dr Stephen McSorley (University of Minnesota, MN) and grown serum-free prior to supernatant harvest. 2. Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 10 mM Hepes, 0.05 mM 2-mercaptoethanol, 1 mM sodium pyruvate and 1x MEM non-essential amino acids (Invitrogen). 3. Serum-free DMEM as above without 10% FCS. 4. Biotinylation kit (EZ-linkTM Sulfo-NHS-Biotin, Pierce). 5. Biotinylated Y-Ae is now commercially available (eBioscience).
2.2. Preparation and Purification of His-Tagged E α -GFP
1. LB/ampicillin broth contains LB broth (Sigma, UK) and ampicillin (Sigma, UK, 100 μg/mL) 2. LB/ampicillin agar also contains 1.5% agar (Sigma, UK) 3. rTRCHis Eα-GFP 4. E. coli DH5α (Invitrogen) 5. IPTG (Fisher Scientific) 6. Ni-NTA Superflow Columns (Qiagen)—include NPI buffers, lysozyme and Benzonase
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7. Amicon Ultra, 10 kDa Centricon columns (Amicon) 8. Detoxi-Gel columns (Pierce Cat. No. 20344) 9. Sodium deoxycholate (1%) 10. Ethanol (25%) 11. Phosphate buffered saline (PBS) 2.3. Detection of Eα Peptide–MHC Complexes in Frozen Lymph Node Sections
1. OCT embedding medium (Bayer). 2. Liquid nitrogen. 3. Phosphate buffered saline (PBS). 4. PBS containing 0.1% sodium azide and 3% H2 O2 (freshly diluted from 30% H2 O2 stock solution). This should be freshly prepared for each use. 5. TNT wash buffer: 0.1 M Tris–HCl pH 7.5, 0.15 M NaCl and 0.05% Tween 20. 6. Fc block is an anti-Fcγ receptor III/II (CD16/CD32) rat mAb prepared from the supernatant of 2.4G2 hybridoma cells with the addition of 10% mouse serum or purchased from BD Pharmingen. 7. Avidin/Biotin Blocking kit (Vector Laboratories). 8. Biotinylated Y-Ae (mouse IgG2b) as described in Section 3.1. 9. Biotinylated murine IgG2b was used as isotype control (Cambridge Biosciences). 10. Blocking reagent (BR) was from the Tyramide Signal Amplification (TSATM ) Kit # 12 (Molecular Probes/Invitrogen) and diluted in PBS to give a 1% solution. 11. TNB blocking buffer: 0.1 M Tris–HCl pH 7.5, 0.15 M NaCl and 0.5% blocking reagent from the TSATM Biotin System kit (Perkin Elmer). 12. Biotinyl tyramide and streptavidin–HRP were from the TSATM Biotin System kit (Perkin Elmer). 13. Streptavidin–Alexa Invitrogen).
Fluor
647
(Molecular
Probes/
14. Rat anti-mouse CD45R (B220) Pacific Blue (CalTag Medsystems). 15. Vectashield (Vector Laboratories). 2.4. Detection of Eα Peptide–MHC Complexes and Enhanced Detection of Eα -GFP in Tissue Sections
1. 4% PFA/30% sucrose/PBS fixation solution is prepared by dissolving 4 g paraformaldehyde in 100 mL PBS with gentle heating and stirring. Once dissolved add sucrose to approximately 30% final concentration. Prepare ahead of time and can be stored refrigerated after use.
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2. 0.5% Gly–Gly (Sigma) in PBS. 3. Rabbit anti-GFP unconjugated, IgG fraction (Molecular Probes/Invitrogen). 4. Rabbit IgG (Zymed). 5. Goat anti-Rabbit IgG–horseradish peroxidase (HRP) conjugate (New England Biolabs). 6. TSATM Kit #12 with Alexa Fluor 488 tyramide and reaction buffer (Molecular Probes/Invitrogen). Prepare reaction buffer just before use by adding 1 μL of 30% H2 O2 stock to 200 μL amplification buffer from kit and then add 1 μL of this intermediate solution to 100 μL amplification buffer. This is the reaction buffer. Add 1 μL of Alexa Fluor 488 tyramide per 100 μL of reaction buffer. 2.5. Co-localisation of pMHC Complexes and Dendritic Cell Markers
1. Biotinylated Hamster anti-CD11c (clone HL3) (BD Pharmingen). 2. Biotinylated Hamster IgG was used as isotype control (BD Pharmingen). 3. Pacific Blue tyramide (Molecular Probes/Invitrogen).
2.6. Immunofluorescence Microscopy
1. Epifluorescence or confocal microscope with ×40 and/ or ×60 objective lenses 2. Appropriate combinations of filter sets capable of distinguishing DAPI, FITC and Texas Red dyes (e.g. XF67-1; Glen Spectra, UK) 3. Image analysis software
2.7. Immunohistochemical Detection of pMHC Complexes
1. ABC-HRP reagent is prepared as follows: 2.5 mL PBS, 1 drop solution A from ABC-HRP kit (Vector), mix, 1 drop solution B from ABC-HRP kit (Vector), mix and allow to incubate for 30 min in the dark prior to use. This reagent should be prepared fresh each use. 2. DAB working solution is 2.5 mL water, 1 drop buffer, 2 drops DAB, 1 drop H2 O2 all from DAB kit (Vector). Prepare fresh each use. 3. DAB enhancing solution (Vector). 4. Gill’s Haematoxylin (Vector). 5. Acid rinse solution contains 2 mL glacial acetic acid and 98 mL distilled water and can be prepared in a coplin jar. 6. Bicarbonate solution contains 0.1 M sodium bicarbonate in distilled water and can be prepared in a coplin jar. This should be prepared fresh on each use.
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1. iRPMI is incomplete RPMI 1640 (Invitrogen) no additives. 2. Nylon Nitex mesh (Cadisch Precision Meshes, London). 3. Streptavidin-APC (BD Pharmingen). 4. FACS buffer (PBS, 2% FCS and 0.1% sodium azide).
2.9. Labelling Bone Marrow-Derived Dendritic Cells for In Vivo Imaging
1. DC medium: RPMI 1640, 10% FCS, 2 mM L -glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen) containing 10% of culture supernatant from X63 myeloma cells transfected with mouse GM-CSF cDNA (18). 2. CFSE stock solution is prepared by dilution of 50 μg lyophilised powder (Molecular Probes, Invitrogen) in cell culture grade DMSO to give 10 mM. This can be aliquoted and stored at –20◦ C protected from light. 3. Hanks Balanced Salt Solution (HBSS). 4. Complete RPMI: RPMI 1640, 10% FCS, 2 mM L -glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin. 5. Lipopolysaccharide (LPS) from Salmonella abortus (Sigma).
2.10. Two-Photon Imaging of Dendritic Cells in Lymph Nodes In Vivo
1. CO2 -independent medium (Invitrogen) 2. Vetbond glue (3 M) 3. RPMI 1640
3. Methods 3.1. Preparation of Y-Ae Antibody
1. The Y-Ae hybridoma was grown serum-free prior to supernatant harvest. 2. Crack open hybridoma vial and grow in a small flask (25 cm2 ) in complete DMEM. Grow in 5% CO2 at 37◦ C for 2–4 days. 3. Split cells 1:10 into medium flask (75 cm2 ) and continue growth until ready to split. 4. Split cells into serum-free DMEM in a large flask (225 cm2 ) and grow until cells are overgrown (medium is yellow, and cells die, this takes about 5 days). 5. Transfer contents to 50 mL tube and spin cells down, keep supernatant and freeze in aliquots, ready for column purification.
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6. Y-Ae (mouse IgG2b) is purified using Pierce columns and biotinylated using the EZ-linkTM Sulfo-NHS-Biotin kit. 7. Y-Ae (mIgG2b) was quantified using standard protein quantification assay (e.g. BCA method) and used for immunofluorescence staining and flow cytometry. Mouse IgG2b was purchased from Cambridge Biosciences and used as isotype control in all experiments at the same concentration as Y-Ae (see Note 1). 3.2. Preparation and Purification of His-Tagged E α -GFP
1. Plate pTRCHis Eα-GFP, DH5α from glycerol stock on LB/ampicillin agar and incubate overnight at 37◦ C. 2. Pick single colonies and inoculate 20 mL LB/ampicillin broth in a 50 mL tube. 3. Shake at 37◦ C for around 6 h. 4. Inoculate starter culture into flask with 1 L prewarmed LB/ampicillin broth. 5. When culture is growing in log phase (OD of 0.4–0.6 at 600 nm) add IPTG (1 mM, final concentration). 6. Shake at 37◦ C overnight. 7. Centrifuge cultures at 4,000×g, 20 min, 4◦ C. 8. Freeze cell pellets for at least 1 h, thaw and refreeze twice to optimise lysis. 9. Resuspend pellet from each 1 L culture in 10 mL NPI-10 buffer. 10. Add 1 mL of 10 mg/mL lysozyme and 3 units Benzonase for every millilitre of the original cell culture volume. 11. Incubate 30 min at room temperature. 12. Centrifuge at 15,000×g at 4◦ C for 30 min, collect green supernatant and pass through a 0.2 μm syringe filter. 13. Set up Qiagen Ni-NTA columns, remove bottom seal then top screw cap, remove storage buffer and equilibrate with at least 10 mL NPI-10 buffer. 14. Add filtered supernatant and allow to run through (column should retain GFP and turn green, flow through should be clear). 15. Wash column two times with 10 mL NPI-20 buffer. 16. Add 3 mL NPI-250 to elute the protein (most protein comes off in second and third millilitre, so collect separately). 17. Store protein at –20◦ C or proceed to step 19. 18. Wash column with filtered 0.5 M NaOH for 30 min then filtered 30% EtOH for 10 min and store for reuse, if required.
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19. Thaw aliquots of protein, add to Amicon Ultra Centricon columns, add 10 mL PBS to each and centrifuge at 4,000×g for 10 min. 20. Add 10 mL PBS and centrifuge as above (repeat four times). 21. Invert column and collect protein by centrifuging into collection cup. 22. Prepare Detoxi-Gel columns by washing with 5 mL 1% sodium deoxycholate. 23. Wash with at least 5 mL PBS. 24. Add protein sample (1 mL per column) and incubate for 60 min at room temperature (replace caps). 25. Elute protein with 1 mL PBS. 26. Quantitate protein using Pierce Micro BCA kit and store protein at –20◦ C in darkness. 27. Columns can be regenerated with 3 mL 1% sodium deoxycholate followed by 3 mL 25% ethanol then stored in ethanol at 4◦ C. 3.3. Detection of Eα Peptide–MHC Complexes in Frozen Lymph Node Sections
This method is used if demonstration of pMHC complexes is the main staining objective. See Note 2 and Section 3.4 for methods when both pMHC and GFP need to be visualised. 1. Freeze excised tissue in OCT and liquid nitrogen, store at –70◦ C. 2. Cut sections at 6–8 μm. Allow to dry for few hours at room temperature. Can freeze slides after this at –20◦ C. 3. Bring slides to room temperature in acetone 10 min. Allow to air dry. Mark areas to be stained with wax pen. Allow this to dry. Draw around sections on the back of the slide using a fine marker. 4. Keep slides in a humidified chamber from now on and use only enough of each reagent to cover sections. 5. Rehydrate sections in PBS for 5–10 min. 6. Incubate at RT in 0.1% sodium azide/3% H2 O2 in PBS three times for 10 min. Make this up fresh every time. 7. Incubate in Fc block containing 10% mouse serum for 10 min. 8. Wash twice in PBS for 3 min each. 9. Add Avidin blocking reagent, 4 drops in 1 mL TNB for 15 min. 10. Wash for 5 min in TNT. 11. Add Biotin blocking reagent, 4 drops in 1 mL TNB for 15 min.
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12. Wash for 5 min in TNT. 13. Add biotinylated Y-Ae diluted 1/250 in 1% blocking reagent (or mIgG2b diluted to same concentration). Incubate 30 min (see Note 1). 14. Wash three times for 5 min each in TNT. 15. Add streptavidin–HRP, diluted 1/100 in 1% BR (30 min). 16. Wash three times for 5 min in TNT. 17. Add biotinyl tyramide diluted 1/50 (20 μL aliquot + 980 μL amplification diluent, 10 min, 100 μL per section). 18. Wash three times for 5 min in TNT. 19. Add Streptavidin-AF647 (Molecular Probes) diluted 1/500 and Pacific Blue anti-CD45R (B220) diluted 1/100 in TNB if B cell follicle staining is desired (30 min, 100 μL/section). 20. Wash three times for 5 min in TNT. 21. Dry briefly and mount in Vectashield without DAPI. 3.4. Detection of Eα Peptide–MHC Complexes and Enhanced Detection of Eα -GFP in Tissue Sections
This method allows for sensitive detection of GFP protein and pMHC complexes on the same section (see Notes 2 and 3). 1. Incubate excised lymph nodes for 60 min in 4% PFA/30% sucrose solution at 4◦ C. 2. Quench lymph nodes by removing to 0.5% Gly–Gly in PBS for 10 min. 3. Rinse in PBS and blot excess liquid prior to embedding in OCT and snap freezing in liquid nitrogen. 4. Complete Steps 2–21 as described in Section 3.3. 5. Quench any residual HRP with 0.1% sodium azide/3% H2 O2 (three times for 10 min). 6. Wash three times for 5 min in TNT. 7. Incubate each section in 50 μL 1% BR for 15 min. 8. Incubate in rabbit anti-GFP (diluted 1/500; i.e. 4 μg/mL) in 1% BR or rabbit IgG (4 μg/mL). 9. Wash three times for 5 min in TNT wash buffer. 10. Add goat anti-rabbit IgG–HRP diluted 1/500 in 1% BR (30 min). 11. Wash three times for 5 min in TNT. 12. Add Alexa Fluor 488 tyramide diluted 1/100 in reaction buffer for 10 min and Pacific Blue anti-CD45R (B220) diluted 1/100 in TNB (30 min, 100 μL per section) if staining of B cell follicles is desired (see Note 3). 13. Wash three times for 5 min in TNT. 14. Dry briefly and mount in Vectashield.
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This method allows for co-localisation of pMHC complexes and dendritic cell markers on the same section. 1. Follow Steps 1–18 in Section 3.3 for staining of Eα peptide–MHC complexes. 2. Quench any residual HRP with 0.1% sodium azide/3% H2 O2 (three times for 10 min). 3. Wash three times for 5 min in TNT. 4. Add neat Avidin blocking reagent for 15 min. 5. Wash for 5 min in TNT. 6. Add neat Biotin blocking reagent for 15 min. 7. Wash for 5 min in TNT. 8. Incubate each section in 50 μL 1% BR for 15 min, flick off residual buffer. 9. Incubate in biotinylated hamster anti-CD11c (diluted 1/200; i.e. 2.5 μg/mL) in 1% BR or biotinylated Hamster IgG (2.5 μg/mL). 10. Wash three times for 5 min in TNT wash buffer. 11. Add streptavidin–HRP diluted 1/100 in 1% BR (30 min). 12. Wash three times for 5 min in TNT. 13. Add Pacific Blue tyramide diluted 1/100 in reaction buffer for 10 min and anti-CD45R (B220) FITC diluted 1/100 in TNB (30 min, 100 μL per section) if staining of B cell follicles is desired (see Note 4 for alternative colour combinations). 14. Wash three times for 5 min in TNT. 15. Dry briefly and mount in Vectashield.
3.6. Immunofluorescence Microscopy
3.7. Immunohistochemical Detection of pMHC Complexes
Immunofluorescence staining was analysed on an Olympus BX51 microscope, equipped with ×40 and ×60 objective lenses. In our studies, individual fluorochrome excitation was achieved by appropriate filter sets (XF1006 (400DF15); XF1042 (485DF15); XF1045 (560DF15); XF1027 (640DF20)) and emitted light passed through a quadruple bandpass dichroic (XF2046) and individual emission filters XF3078 (465AF30); XF3017 (530DF30); XF3019 (605DF50); XF3076 (695AF55) (all filters from Glen Spectra, UK). Fluorescence images in each channel were captured using a connected OrcaER CCD camera (Hammamatsu) and regulated via Openlab version 3.0.9 digital imaging program (Improvision, Warwick, UK). 1. Complete Steps 1–14 as escribed in Section 3.3. 2. Prepare ABC-HRP and incubate in dark for 30 min.
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3. Wash tissue sections three times in TNT (5 min each) and once in PBS (5 min). 4. Incubate in ABC-HRP for 30 min at RT. 5. Wash samples three times in TNT (5 min each), then add DAB, incubate for 10–30 min. 6. Rinse in distilled water for 5 min, then add DAB enhancing solution for 30s. 7. Rinse in distilled water and counterstain in Gill’s Haematoxylin for 2 min. 8. Rinse in tap water and blot. 9. Give slides 10 quick dips in acid rinse solution followed by 10 quick dips in tap water and blot. 10. Incubate in bicarbonate solution for 1 min, then give 10 quick dips in tap water and blot. 11. Dehydrate, clear and mount. 12. Observe using standard upright light microscope. 3.8. Detection of pMHC Complexes by Flow Cytometry
1. Excise lymph nodes and place into ice-cold iRPMI. 2. Remove lymph node into 60 mm petri dish with 3 mL iRPMI added and macerate lymph node through nylon mesh using a syringe plunger. 3. Collect cells and make single-cell suspension by passing cells through a fresh piece of nylon mesh into a 5 mL FACS tube. Rinse petri dish with 1 mL RPMI to remove as many cells as possible. 4. Pellet cells by centrifugation at 450×g for 5 min at 4◦ C, resuspend in residual liquid, add 1 mL iRPMI and count cells using a haemocytometer. 5. Remove approximately 107 cells for staining, centrifuge at 450×g for 5 min at 4◦ C, resuspend in 100 μL Fc Block and incubate at 4◦ C for 10 min. 6. Divide sample into two tubes for Y-Ae and isotype control (murine IgG2b) staining. 7. Dilute biotinylated Y-Ae and biotinylated mIgG2b to approximately 10 μg/mL and stain for 30 min at 4◦ C. 8. Add 4 mL FACS buffer, centrifuge 450×g for 5 min at 4◦ C and resuspend in residual liquid. 9. Add 50 μL streptavidin-APC diluted 1/200 to tubes and incubate for 20 min at 4◦ C. 10. Add 4 mL FACS buffer, centrifuge 450×g for 5 min at 4◦ C and resuspend in 200 μL FACS flow.
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11. Acquire on flow cytometer and analyse FITC (FL1) and APC (FL4) channels (for BD FACSCalibur) for Eα-GFP and Y-Ae fluorescence, respectively. 12. Figure 12.2 shows examples of flow cytometry analysis of lymph nodes taken from mice injected 24 h prior with Eα-GFP/LPS and control mice (see Note 5).
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Y-Ae Fig. 12.2. Flow cytometric detection of Eα-GFP and pMHC complexes in lymph nodes following Eα-GFP protein injection. Hundred micrograms of Eα-GFP protein plus 1 μg LPS was injected into the neck scruff and 24 h later draining cervical and brachial lymph nodes were collected and stained as described in Section 3.8. Control mice were injected with LPS only. PE-conjugated anti-CD11c was included at Step 9 of protocol to demonstrate dendritic cells displaying pMHC complexes. Top panela shows typical plots for Eα-GFP and Y-Ae and bottom panel shows plots of Y-Ae and CD11c.
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3.9. Labelling Bone Marrow-Derived Dendritic Cells for In Vivo Imaging
1. Collect femurs and tibias of mice and harvest bone marrow by flushing bone with iRPMI. 2. Culture cell suspensions in petri dishes in DC media. 3. Fresh medium was added to the cell cultures every 3 days. 4. On day 6 add 1 μg/mL LPS to each well. 5. On day 7 pulse with 1 mg/mL antigen for 90 min at 37◦ C if required. 6. Harvest cells from petri dish into sterile 50 mL Falcon tube and centrifuge at 450×g for 5 min. 7. Collect supernatant and re-centrifuge to collect as many cells as possible. 8. Resuspend pooled cell pellets in at least 20 mL sterile incomplete RPMI. 9. Count cells and wash once in 20 mL HBSS (room temperature). 10. Resuspend washed cells in HBSS at room temperature to give 5 × 107 cells/mL. 11. Add 0.8 μL of CFSE per millilitre cells (8 μM final concentration), invert gently and incubate in 37◦ C incubator for 10–12 min, inverting gently twice during incubation period. 12. Add fivefold to tenfold excess volume of complete RPMI (room temperature) and centrifuge 300×g for 5 min at room temperature. 13. Wash cells twice more in approximately 20 mL HBSS at room temperature per wash. 14. Resuspend in incomplete RPMI to give at least 5 × 106 dendritic cells in 50 μL for adoptive transfer. 15. Inject into hind footpad of syngeneic recipients. 16. Image popliteal lymph node from 8–24 h.
3.10. Two-Photon Imaging of Dendritic Cells in Lymph Nodes In Vivo
This protocol is for imaging lymph nodes using the Radiance (Bio-Rad) multiphoton excitation laser scanning system with a solid-state, tuneable titanium: sapphire laser system (5W Chameleon, Coherent Laser Group) as the laser source. 1. Excise lymph node into CO2 -independent medium at room temperature. 2. Attach LN onto a plastic coverslip with veterinary glue (Vetbond, 3 M). 3. Adhere plastic coverslip with vacuum grease to the bottom of the imaging chamber continuously supplied with warmed
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(36.5◦ C) and gassed (95% O2 and 5% CO2 ) RPMI before and throughout the period of microscopy. 4. For standard conditions used for our imaging system see Note 6.
4. Notes 1. Concentration of Y-Ae antibody to use should be determined by the user. We have found that concentrations in the range 2–20 microgram (Mg)/mL are optimal depending on the batch and biotinylation efficiency. 2. Section 3.3 is used when detection of pMHC complexes is the primary focus rather than Eα-GFP detection. GFP is highly water soluble and we and others (19) have found that immobilisation of GFP in mouse tissues by fixation prior to freezing preserves fluorescence. To visualise intrinsic Eα-GFP in Y-Ae stained sections, we have found that the following protocol preserves GFP fluorescence: after excision of lymph nodes or other tissues, prefix tissues for 60 min in 4% PFA/30% sucrose at 4◦ C, quench in 0.5% Gly–Gly in PBS 10 min (to neutralise formaldehyde), then rinse in PBS prior to snap freezing in OCT. Steps 2–21 of Section 3.3 can then be followed. Sucrose is optional, but the tissue morphology is better if 10–30% sucrose is included. We also use a second protocol (Section 3.4) incorporating a GFP-specific antibody to further increase Eα-GFP detection sensitivity. This amplification protocol is useful to definitively confirm GFP presence, particularly when the protein is only present in low amounts. 3. Anti-GFP can be put on FITC channel using Alexa Fluor 488 tyramide (i.e. same as intrinsic GFP fluorescence) as in Section 3.4 to free up other channels for counterstains. However, because we often found background autofluorescence in lymphoid tissue using the FITC filter set (excitation 488 nm, emission 520 nm), the combination of antiGFP, anti-rabbit-HRP and Alexa Fluor 647 tyramide was sometimes used as confirmation of Eα-GFP presence in tissue. Tissue autofluorescence is minimal at these longer wavelengths. 4. In Section 3.5 we have used the Pacific Blue tyramide to visualise CD11c+ dendritic cells and B220-FITC to show B cell follicles. If Eα-GFP also needs to be visualised then the B cell follicle stain can be omitted. Reagents can always be substituted depending on available microscope filter combinations.
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5. In Section 3.8 we have used flow cytometry to demonstrate Eα-GFP+ and Y-Ae+ cells following Eα-GFP injection. Further phenotypic characterisation of these cells using the dendritic cell-specific antibody CD11c (Fig. 12.2) confirms that most cells displaying surface pMHC (i.e. Y-Ae+ cells) are also CD11c+ and therefore dendritic cells. Other phenotypic and activation markers can also be used. 6. The scan head was aligned through an upright microscope (E600-FN; Nikon). The objective lens used for all imaging investigations was the CFi-60 Fluo-W 40X/0.8 water-dipping objective lens (Nikon). The sample was illuminated with 780–830 nm, ∼210 fs pulse duration and 76 MHz repetition frequency. The emission spectrum was separated with a 550 nm dichroic mirror (Chroma Technologies). Each imaged volume consisted of between 11 and 18 planes 2.55 μm apart. Volumes were acquired every 18–38s. References 1. Pashine, A., Valiante, N. M., Ulmer, J. B. (2005) Targeting the innate immune response with improved vaccine adjuvants. Nat Med 11, 63–68. 2. Brewer, J. M., Pollock, K. G. J. (2004) Adjuvant induced Th2 and Th1 dominated immune responses in vaccination, in (Kaufmann, S. H. E., ed.) Novel Vaccination Strategies. Wiley VCH, Weinheim, pp 51–73. 3. Catron, D. M., Itano, A. A., Pape, K. A., Mueller, D. L., Jenkins, M. K. (2004) Visualizing the first 50 hr of the primary immune response to a soluble antigen. Immunity 21, 341. 4. Germain, R. N., Jenkins, M. K. (2004) In vivo antigen presentation. Curr Opin Immunol 16, 120–125. 5. Dustin, M. L. (2004) Stop and go traffic to tune T cell responses. Immunity 21, 305–314. 6. Zinselmeyer, B. H., Dempster, J., Gurney, A. M., Wokosin, D., Miller, M., Ho, H., Millington, O. R., Smith, K. M., Rush, C. M., Parker, I., Cahalan, M., Brewer, J. M., Garside, P. (2005) In situ characterization of CD4+ T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance. J Exp Med 201, 1815–1823. 7. Shakhar, G., Lindquist, R. L., Skokos, D., Dudziak, D., Huang, J. H., Nussenzweig, M. C., Dustin, M. L. (2005) Stable T cell-dendritic cell interactions precede the
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development of both tolerance and immunity in vivo. Nat Immunol 6, 707–714. Campbell, D. J., Butcher, E. C. (2002) Rapid acquisition of tissue-specific homing phenotypes by CD4(+) T cells activated in cutaneous or mucosal lymphoid tissues. J Exp Med 195, 135–141. Campbell, D. J., Kim, C. H., Butcher, E. C. (2003) Chemokines in the systemic organization of immunity. Immunol Rev 195, 58–71. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B., Palucka, K. (2000) Immunobiology of dendritic cells. Ann Rev Immunol 18, 767–811. Iezzi, G., Scheidegger, D., Lanzavecchia, A. (2001) Migration and Function of Antigenprimed Nonpolarized T Lymphocytes In Vivo. J Exp Med 193, 987–994. Rannie, G. H., Ford, W. L. (1978) Recirculation of lymphocytes: its role in implementing immune responses in the skin. Lymphology 11, 193–201. Gowans, J. L. (1959) The recirculation of lymphocytes from blood to lymph in the rat. J Physiol 146, 54–69. Klonowski, K. D., Williams, K. J., Marzo, A. L., Blair, D. A., Lingenheld, E. G., Lefrancois, L. (2004) Dynamics of blood-borne CD8 memory T cell migration in vivo. Immunity 20, 551–562. Catron, D. M., Rusch, L. K., Hataye, J., Itano, A. A., Jenkins, M. K. (2006) CD4+ T cells that enter the draining
Tracking Dendritic Cells In Vivo lymph nodes after antigen injection participate in the primary response and become central-memory cells. J Exp Med 203, 1045–1054. 16. Itano, A. A., Jenkins, M. K. (2003) Antigen presentation to naive CD4 T cells in the lymph node. Nat Immunol 4, 733–739. 17. Rudensky, A., Rath, S., Preston-Hurlburt, P., Murphy, D. B., Janeway, C. A., Jr. (1991) On the complexity of self. Nature 353, 660–662.
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18. Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N., Schuler, G. (1999) An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Meth 223, 77–92. 19. Kusser, K. L., Randall, T. D. (2003) Simultaneous detection of EGFP and cell surface markers by fluorescence microscopy in lymphoid tissues. J Histochem Cytochem 51, 5–14.
Chapter 13 Adjuvant Effects on Antibody Titre Barry Walker and Ian Feavers Abstract Antibody titre is a measure of the presence and amount of antibodies specific to an antigen that are present in the blood. In particular, the titre of an antibody sample is a measure of the antibody concentration determined under a defined set of conditions, with the antibody concentration being commonly established by enzyme-linked immunosorbent assay, also called ELISA, or a variation of this technology. Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used to detect the presence of an antibody or an antigen in a sample. The ELISA/EIA approach is an extremely robust technology and readily amenable to validation and quality control if adherence to ISO quality standards is required. This chapter describes in detail a specific ELISA protocol which has potential generic application. Key words: Enzyme-linked immunosorbent assay, ELISA, EIA, antibody, titre, adjuvant.
1. Introduction An adjuvant is an agent that may stimulate the immune system and increase the response to a vaccine or an antigen, without having any specific antigenic effect in itself. The word “adjuvant” comes from the Latin word adjuvare, meaning to help or aid. Adjuvants can be described as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens. In the case of antibody production, adjuvants in combination with antigen can have a wide range of effects, ranging from increasing the amount of antigen-specific antibody being produced, modulating specificity and focussing the specific antibody classes being produced (1, 2). G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_13, © Springer Science+Business Media, LLC 2010
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The reason for using adjuvants may vary from the simple requirement to produce a specific antibody-based reagent for use in assays or as standards through R&D studies of possible vaccine formulations for further development. It is clear therefore that the technology employed and approaches used in the measurement and characterisation of the antibody responses elicited by an antigen adjuvant combination is an important fundamental aspect of immune studies. Antibody titre is a measure of the presence and amount of antibodies specific to an antigen that are present in the blood. In particular, the titre of an antibody sample is a measure of the antibody concentration determined under a defined set of conditions, with the antibody concentration being commonly established by ELISA. 1.1. Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used to detect the presence of an antibody or an antigen in a sample. The ELISA/EIA approach is an extremely robust technology and readily amenable to validation and quality control if adherence to ISO quality regulations is required. This discussion will use the term ELISA throughout. The ELISA has long been used as a diagnostic tool in medicine, research immunology and plant pathology, as well as a quality control check in various industries (3–5). In simple terms, the ELISA consists of an unknown (but sufficient) amount of antigen affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a suitable substrate is added for the enzyme linked to the antibody to convert to some detectable signal. There are many variations of the ELISA technology (direct, indirect, sandwich, competition and inhibition are the main variants) but for the purposes of detecting and characterising antibodies present in serum, the basic direct ELISA is the most robust approach. Where the required outcome is an estimate or a measurement of the amount of antibody specific for the antigen, then a constant but sufficient antigen concentration is added to all wells and the serum in question is serially diluted, with an additional step using a secondary labelled antibody against the primary antibody (human, mouse, etc.) being used. The signal developing step remains the same. This is the approach summarised in Fig. 13.1. The results can be quantified by generation of standard curves using a serum of known activity (to provide quantitative data) or may be defined as the reciprocal dilution giving an optical density (OD) reading at the specified absorption wavelength (nm) of 1. The pre-vaccinated serum of the animal represents the negative
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Summary of method 1. Coat 96 Well plates with antigen at (eg 0.5 µg/ml) or at appropriate dilutions in 100 µl. 2. Incubate o/n 4°C 3. Wash (PBS x3) 4. Block 1% skim milk powder in PBS for 3h ambient temperature 5. Wash x3 PBS/Tween 6. Add serum and controls sera 100 µl at 1/1000 in blocker 7. Incubate 4°C o/n 8. Wash x6 PBS/Tween 9. Add conjugate (for example anti-human IgG-HRP) 100 µl 1/5000 in blocker ; 3h ambient 10. Wash x6 PBS/Tween 11. Add OPD/H2O2 100µl ; 20 min ambient or 37°C Read 492 nm or other suitable absorbance frequency.
Fig. 13.1. Generic scheme for detecting serum antibody levels against a specific antigen.
control. An alternative is to report the serum titre as that reciprocal dilution where the OD is equal to that of the negative control. Performing an ELISA to detect antigen-specific antibodies in serum, where the objective is to determine the potency, antigenicity or immunogenicity of a vaccination/adjuvant combination, involves the serum bleeds from the study and at least one antibody with specificity for the species being used (or the subclass if more detailed information is required). For example, in assessing immunogenicity studies in mice, an anti-mouse antibody labelled with a suitable enzyme or a developing system would be used [for example, goat anti-mouse IgG (F(ab )2), peroxidase conjugated]. For a more specific assessment of the nature of the immune response generated, it may be appropriate to select a suitable conjugated antibody directed specifically to one class or subclass of antibodies (IgG1, IgA, etc.). With improvements in technology and biochemistry, the range of conjugated antibodies has broadened and there is now the option of using a range of secondary antibodies each independently conjugated to different fluorescent labels, allowing the possibility of multiple assay outcomes where sample access is limiting. Note that there are notable technical complications in using multiple fluorophores in single-assay systems. 1.2. Developing Technologies
Alternatives to ELISA are now becoming available and are significantly more sophisticated in both the technology and the approach and, as a consequence, the information that can be provided. One of the most powerful new technologies is based on the phenomenon of plasmon resonance exemplified by proprietary technology of the Biacore© Device. The Biacore is one of a number of devices based on the same technology that are now available. However, the complexity of the requirements for using this approach is such that a separate section would be appropriate and the authors refer the reader to a number of useful overviews that may assist in deciding to invest in this approach (6–10).
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1.3. Specific Antigen ELISA
Below we describe a specific ELISA to determine serum antibody activity against malaria-specific antigens. Although the protocol is specific for malaria antigens, the approach is generic and much of the detail can be readily transferred as required. The protocol describes the single-point determination of total IgG antibodies specific for four recombinant Plasmodium falciparum antigens and total IgE in serum. This procedure takes 3 days in all to complete: coating on day 1, blocking and serum/plasma addition on day 2 and treatment with conjugate and development on day 3. Controls and standards/references are important if reliable and consistent results are to be obtained. A positive and negative serum control (control sera) is imperative and in the case where quantitative data is required, a strongly positive serum of sufficient volume to last the study period should be aliquoted and stored safely. It can be assigned an arbitrary unitage or activity and a standard curve established using this reference serum. The positive serum, if used independently and separately from the reference serum, can act as an assay performance indicator, when used in every assay. Monitoring and trend analysis of the cumulative data from the values ascribed to the positive control sample can provide early indications of reagent degradation/contamination and allow for rapid recovery from such situations. This type of assay performance monitoring is mandatory in quality controlled situations and can be very useful in research laboratories where the assays are performed regularly. It must be appreciated that this approach will require the use of a reference standard curve and the designation of a quantitative value for the control samples in every assay. In the case of the protocol described below, a reference serum for determination of each of the (a) antigen-specific antibody concentration and (b) total IgE concentration has been prepared from a high-activity sample of sufficient volume for a large number of assays. The specific volumes and concentrations for each antigen have been optimised for the specific conditions being used in this protocol. When establishing these assays for the first time, preliminary optimisation of the volumes, concentrations and conditions is critical to ensuring appropriate specificity and sensitivity of the assay in the laboratory.
2. Materials 1. Ninety-six-well ELISA plates (e.g., Immulon 4; Dynatech). 2. Deep Well 96-well polypropylene plates 500 μL for dilutions with sealing mats: these can be washed and re-used.
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3. Plastic containers (2 L) for washing plates (or automated plate washer if available). 4. Pipettes: 5–50 μL × eight channel; 20–200 μL × 12 channel, single channel 10 μL, 1 mL. 5. Pipette tips, 200 μL and 1 mL. 6. Pipetting troughs. 7. Plate reader. 8. Large plastic self-sealing food bags for incubations. 9. Antigens: for this Malaria ELISA, AMA-1, MSP-2, MSP119 , CSP (NANP16 ) have been used, sourced from NIBSC and malaria vaccine developers. 10. Capture anti-IgE MAb (detects all human IgE) for determination of total IgE concentration. 11. Rabbit anti-human–IgG–HRP conjugate for total IgG (Dako). 12. (If undertaking subclass determinations) Sheep antisubclass–HRP conjugates (the binding site): anti-IgG1– HRP AP06; anti-IgG2–HRP AP07; anti-IgG3–HRP AP08; anti-IgG4–HRP AP09. 13. (If undertaking subclass determinations) Myeloma IgG subclass references (κ) (the binding site): BP078 (IgG1), BP080 (IgG2), BP082 (IgG3), BP084 (IgG4) (all 1 mg/mL); purified human IgG (Sigma)—make up 1 mg/mL in PBS with 0.2% NaN3. 14. Control samples—positive control plasma and negative plasma pool. The positive sample can be used as a reference (see above) for generating a standard curve for quantitation or as a simple positive control for assay performance (obtained via research collaborators, NIBSC). 15. Phosphate buffered saline (PBS) 10×, pH 7.2. 16. Horseradish peroxidase-conjugated rabbit anti-human IgG (Dako). 17. HRP-conjugated rabbit anti-IgE (Dako). 18. H2 SO4 (2M)—note: concentrated H2 SO4 is hazardous, check local safety requirements. 19. Coating buffer: 1.59 g Na2 CO3 + 2.93 g NaHCO3 /L in distilled water, pH 9.4–9.6. Can be kept for up to 1 month at 4◦ C. 20. Buffer A: 0.1 M citric acid [9.6 g anhydrous in 500 mL distilled water (10.5 g of monohydrate)]. Store at 4◦ C. 21. Buffer B: 0.2 M phosphate (14.2 g Na2 HPO4 in 500 mL water). Exclude light by wrapping bottle completely in foil and store at ambient temperature.
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22. OPD substrate solution: Add 6 mL buffer A plus 6.4 mL buffer B to 12.5 mL distilled water. Check 5
3. Methods 3.1. Plate Washing
In the absence of an automated plate washer, manual washing is achieved by any number of practical methods, as long as the plates are thoroughly and repeatedly exposed to the washing buffers. Some laboratories use wash bottles containing the wash buffers to flood the plates, others operate as below. 1. Set out three wash containers, half-fill each with PBS-T. 2. To wash a plate, tip the plate/well contents into the sink or the waste container, plunge the plate into container 1 of PBS-T, remove and tip the well contents out into the sink or the waste container. Repeat in container 2 of PBS-T and finally in container 3 of PBS-T. 3. Tip out the final PBS-T, drain and pat the inverted plate dry on a pad of tissue/blotting paper (see Note 2). Note: After coating plates or for the blocking, wash as above as a single cycle. However, after serum or conjugate addition, wash as above twice.
3.2. ELISA 3.2.1. ELISA Plate Antigen Coating
1. Mark out and individually label ELISA plates according to the specific protocol. A template for the plate layout is shown in Fig. 13.2. 2. For determination of overall antigen-specific antibody levels, to each well of a 96-well ELISA plate, add 50 μL of the appropriate antigen (0.5 μg/mL in coating buffer for AMA-1, MSP-2 and MSP-119 ; 1 μg/mL for NANP16 ) (see Note 3). 3. For determination of the levels of specific IgE, add the capture antibody (anti-IgE MAb 1 μg/mL).
Adjuvant Effects on Antibody Titre Reference Dilutions (or use 2 wells for positive control
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4. Tap the plate gently to distribute the antigen solution evenly over the flat base of the well, place plates stacked together with a cover plate on top in a plastic food bag, seal and incubate coated plates overnight at 4◦ C. 5. Wash as noted above with PBS–0.05% Tween 20 (PBS/T) (single cycle) (see Note 2). 6. Add 200 μL of blocking solution (2% skimmed milk in PBS/T). 7. Incubate for 3 h at ambient temperature. 3.2.2. Preparation of Sera for ELISA
The serum samples, test samples, negative, positive controls (control serum) and serum for standard curve (reference—if standard curve being prepared), are diluted as described below into the Deep Well plates. These prepared samples are then transferred across to the corresponding ELISA plate for assay. 1. Remove control, test samples and reference serum aliquots from freezer and allow thawing. Prepare the control or reference and sample primary dilutions in the Deep Well plates for subsequent transfer to the assay plate as described below. 2. If preparing a standard curve from a reference serum sample, make up reference serum dilutions, in a series of sequential 1:5 dilutions in the Deep Well plate as follows: Pipette 200 μL blocking buffer into the Deep Well plate wells for the standard curve, then pipette 50 μL of the reference
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serum into the first well and mix well by pipetting. Serially transfer 50 μL to the next well to make a fivefold dilution series (the final 50 μL can be left in the last well or discarded). 3. Where using the IgE subtype references for analysing subtype concentrations, prepare each of the reference subtype antibodies in a similar fashion. 4. If only using positive and negative controls, rather than the standard curve, these can be prepared in the relevant wells of the Deep Well plate by adding 1 μL each of the positive and negative controls to 200 μL blocking buffer. 5. For the test samples, firstly pipette 190 μL blocking buffer into the appropriate wells of the Deep Well plate. 6. Pipette 10 μL test sample sera (in duplicate) into appropriate wells in the Deep Well plate. 3.2.3. Transfer to ELISA Plate for Assay
1. To each ELISA plate (one ELISA plate for each antigen), add blocking buffer to the different plates as follows: AMA-1, 90 μL/well; MSP-2 and MSP-119 , 40 μL/well; CSP, no addition; IgE, 40 μL/well (see Note 4). 2. Using a multi-channel pipette, add 10 μL of each sample serum from the corresponding wells of the Deep Well plate into the appropriate well of the 96-well assay plate, using fresh tips each row. 3. This gives the optimised dilution for each sample added to the 96-well plate and the controls (where appropriate) and a fivefold dilution series for the positive standard. 4. Cover and seal with the sealing mat, press every stopper carefully into place and mix the plate by inversion five times. 5. Incubate overnight at 4◦ C.
3.2.4. Developing ELISA Assay
1. Wash plates six times in PBS/T, pat dry, inverted, on tissue. Then for the antigen plates, add 50 μL/well horseradish peroxidase-conjugated rabbit anti-human IgG diluted 1/5000 in PBS/T. For the IgE plate, add 50 μL of anti-IgE–HRP. Incubate for 3 h at room temperature. 2. Wash six times with PBS/T, pat dry. Add 100 μL/well of OPD substrate solution. Leave at room temperature for 10–15 min (20–30 min for CSP) for the assay to develop. 3. Stop the reaction by adding 25 μL/well 2 M H2 SO4 . Read plates as soon as possible at 492 nm (see Notes 5 and 6).
3.3. Data Analysis
When using the reference sample and standard curve approach, the data can usually be exported from the ELISA reader as a tabdelimited text file, or where proprietary software is being used,
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the plate template can be directly linked to the data, simplifying analysis and data output. It is relatively straightforward to use modern spreadsheet programmes (e.g. Excel) and prepare suitable template files, with embedded data import macros, that will simplify routine importing and analysis of the text file output from most ELISA readers. The assay format reported below is designed with a reference sample standard curve, allowing quantitative reporting of the test sample value to be derived (see Note 7). In both automated and manual analysis of test sample values from this type of assay format, the principle of the operation is the same. 1. Where manual analysis is required, plot the absorbance values and reference sera concentrations, and then linearise the absorbance/concentration curve (e.g. plot absorbance against log dilution). 2. Read the values for the test samples from this curve using the mean duplicate value of the test sample absorbances. Dilution compensation of the sample preparation will need to be taken into account when finalising the concentration values so derived (see Note 8).
4. Notes 1. The most common problems with ELISA relate to the substrate. Check you have used Na2 HPO4 to make up buffer B. You may have used NaH2 PO4 by mistake. Was the Na2 HPO4 anhydrous? If not, correct the weight of Na2 HPO4 for the extra waters of crystallisation (buffer B is 0.2 M). Check the OPD solution with a piece of narrow range pH paper, pH 2–7. The pH should not be lower than 5.0. If it is 3.0 or lower, then you have probably used NaH2 PO4 rather than Na2 HPO4 . Check (crudely) that the OPD is OK by adding 100 μL of OPD solution (including H2 O2 ) to 100 μL of the diluted anti-human-HRP. This should go brown very quickly (very noticeable within 2 min)—this is only a rough test, and if the pH is wrong, it may still seem to work. Also check the H2 O2 . This is very stable but can be entirely decomposed if the smallest amount of catalyst (blood/serum, iron compounds, etc.) is introduced. Test by taking 100 μL into a 1.5-mL Eppendorf tube. Cautiously add 1 small crystal of FeSO4 (ferrous sulphate). The solution will react violently, going brown, boiling or getting very hot. If it does not, then the peroxide has decomposed.
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2. After coating plates or for the blocking, wash as described but as a single cycle. However, after serum or conjugate addition, wash as described but twice. 3. For individual antigens the concentration for plate coating will have to be established by preliminary experiments. 4. The volumes and final concentrations of sera will vary between each antigen plate (see Section 1). The volumes and concentrations have been previously optimised for each antigen combination. 5. If the ELISA is not functioning and the substrate solution is correct (see Note 1), check the following: a. The pH of the PBS should be 7.2 (use pH paper). If it is very high or very low, you have used one of the wrong salt forms of sodium phosphate. b. The antigen concentration—do a coating titration of antigen followed with the positive control serum (at 1:1000), then conjugate. The antigen may have degraded and you may have to replace it or use more for coating. c. The conjugate—mix leftover conjugate with OPD as above; if all seems well, then check by carrying out a rough ELISA comparing 1/5000 with 1/1000 and 1/200 conjugate. There should be almost no difference. If there is, the conjugate needs re-titrating: coat a plate with Ag as usual, wash, block and add 1/1000 control serum to each coated well, incubate for 3 h at ambient temperature, wash and add conjugate as a 3× dilution series from 1/100–100 μL blocker in all but the first row of wells, then add 150 μL of 1/100 conjugate and serially transfer 50 μL. Develop as usual. Interpretation is the same as for antigen—take the dilution corresponding to just less than the maximum OD; this is the most economical one to use. d. Run ELISA with the last known combination of things that worked (if you can). e. If the problem is a high background, check what happens if you use serum but no antigen—do you still get a reading even if all you have done is blocked the well? 6. It is inevitable that batches of reagents will need replacement during the course of a study and there are two approaches that can be used to manage the potential disruption in assay performance that may result. Assay behaviour can be significantly affected as new batches of reagent-specific detection reagents, monoclonal antibody preparations (even commercially obtained) and assay developing substrates can vary in behaviour. To mitigate this, a structured stock monitoring
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and replacement trigger protocol can be established where there is sufficient material available to ensure that at least three assays can be run with the original and replacement reagents included to ensure operation of the assay within specification. It is evident here that the use of control and references is critical to this process. Where replacement of critical reagents occurs and assessment of assay performance in parallel is not possible, a series of assay optimisation protocols are recommended where the new reagents are reviewed at varying concentrations against established assay reagent ensuring that the optimal conditions within the assay are determined and recorded. Reference to historic performance data on reference and controls will assist in determining the best combinations for the assay protocol. 7. Where simple titre is being recorded as an outcome, the assay format will need to be adjusted so that each of the test samples have been serially diluted (usually in a 1:1 series) and then the inverse dilution value where the absorbance equals the negative control or where it reaches a predetermined absorbance value can then be reported (for example, a titre of 1:16 means that the dilution of the test sample at 1:16 is the closest to the absorbance of the negative control or the predetermined value). The criteria for reporting this value must be clearly described and recorded in the analysis and other documentation deriving from the assay. 8. The use of spreadsheet software will require the inclusion of these operations into the internal processes used but can make curve fitting and test sample value interpolation/readout semi-automated and potentially relatively error proof.
Acknowledgements The help of Paul Risley and Patrick Corran finalising the details of this document is gratefully acknowledged. References 1. Hunter, R. L. (2002) Overview of vaccine adjuvants: present and future. Vaccine 20(Suppl 3), S7–S12. 2. Edelman, R. (2002) The development and use of vaccine adjuvants. Mol Biotechnol 21, 129–148. 3. Warnes, A., Fooks, A. R., Stephenson, J. R. (2004) Design and preparation of recombinant antigens as diagnostic reagents in solid-
phase immunosorbent assays. Methods Mol Med 94, 373–391. 4. Voller, A., Bidwell, D., Huldt, G., Engvall, E. (1974) A microplate method of enzymelinked immunosorbent assay and its application to malaria. Bull World Health Organ 51, 209–211. 5. Wright, P. F., Nilsson, E., Van Rooij, E. M., Lelenta, M., Jeggo, M. H. (1993)
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Standardisation and validation of enzymelinked immunosorbent assay techniques for the detection of antibody in infectious disease diagnosis. Rev Sci Tech 12, 435–450. 6. Hoa, X. D., Kirk, A. G., Tabrizian, M. (2007) Towards integrated and sensitive surface plasmon resonance biosensors: a review of recent progress. Biosens Bioelectron 23, 151–160. 7. Phillips, K. S., Cheng, Q. (2007) Recent advances in surface plasmon resonance based techniques for bioanalysis. Anal Bioanal Chem 387, 1831–1840.
8. Wiltschi, B., Knoll, W., Sinner, E. K. (2006) Binding assays with artificial tethered membranes using surface plasmon resonance. Methods 39, 134–146. 9. Pattnaik, P. (2005) Surface plasmon resonance: applications in understanding receptor-ligand interaction. Appl Biochem Biotechnol 126, 79–92. 10. Rich, R. L., Myszka, D. G. (2000) Survey of the 1999 surface plasmon resonance biosensor literature. J Mol Recognit 13, 388–407.
Chapter 14 Functional Antibody Assays Ian Feavers and Barry Walker Abstract Functional antibody assays can broadly be divided into three categories: neutralisation, serum bactericidal antibody (SBA) and opsonophagocytic assays (OPA). These biological assays are generally more complex than antibody-binding counterparts. They invariably involve multiple biological components, many of which are difficult or impossible to standardise. The aim of this chapter is to provide working examples of these assays and highlight the key issues to be addressed to ensure they are able to provide reliable data. Key words: Bacterial neutralisation, serum bactericidal antibody (SBA), opsonophagocytic assays (OPA), adjuvant, functional assays.
1. Introduction Most currently licensed vaccines work by inducing antibodies that interfere with microbial invasion of the host, kill the pathogen or neutralise the effects of microbial toxin. During the evaluation of novel vaccine formulations, the measurement of an antibody response usually provides the first evidence that the formulation has the potential to offer protection. Although antibody titres frequently serve as very reliable correlates of protection, only the ability of antibodies to mediate immune mechanisms that are known to protect the host can be taken as a surrogate, or substitute, for clinical protection. It is not simply the quantity of antibody; it is also their functional quality that is important for the prevention and clearance of infectious disease. The issues surrounding the use of correlates of protection in the evaluation of vaccines have been the subject of recent reviews (1, 2). G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_14, © Springer Science+Business Media, LLC 2010
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As the role of adjuvant is to modulate the nature (e.g. Ig classes and subclasses and antibody avidity) as well as the size of the antibody response, the measurement of a relevant functional antibody response is of particular importance when examining the protective potential of vaccine formulations containing novel adjuvants. Increasingly new vaccines are licensed on the basis of serological surrogates of protection rather than direct evidence of protection in placebo-controlled, blinded clinical studies. This is often because of the low incidence of the target disease or ethical problems associated with carrying out challenge studies in humans. Similarly new formulations or combinations, designed to replace existing vaccines, are typically evaluated against a background of low disease burden where it would also be unethical to withhold vaccine from a placebo group. Recent examples of vaccines licensed in the absence of formal efficacy trials include group C meningococcal conjugate vaccines, the 10-valent pneumococcal conjugate SynflorixTM and a number of new paediatric combination vaccines. Functional antibody assays can broadly be divided into three categories: neutralisation, serum bactericidal antibody (SBA) and opsonophagocytic assays (OPA). These biological assays are generally more complex than antibody-binding counterparts. They invariably involve multiple biological components, many of which are difficult or impossible to standardise. The aim of this chapter is to provide working examples of these assays and highlight the key issues that have to be addressed if they are to provide reliable data. 1.1. Toxin Neutralisation
Historically the potency of toxoid vaccines and antitoxins was determined by the administration of the vaccine to an appropriate animal species followed by a challenge with the corresponding toxin (3, 4). An improved understanding of the biochemical basis for the action of bacterial toxins offers the prospect of the replacement of animal challenge models with cell-based or biochemical assays (5–7). Irrespective of whether an animal model or an in vitro neutralisation assay is employed, the principle underlying these assays is the same. The antibodies elicited by vaccination are tested for their ability to block the activity of the toxin, either by binding to an active site on the toxin or blocking a domain recognised by its receptor on susceptible cells. Using the following example of a neutralisation assay, the potency of a diphtheria vaccine can be determined from the titre of anti-diphtheria toxin (DT) antibodies required to neutralise the metabolic inhibition of vero cells by DT (8). The endpoint can be determined by the microscopic examination of the cells or visually by the change in colour of the cell culture medium. A variation on this method (not shown) uses the neutralisation of cytotoxicity as the endpoint of the assay. In this version of the assay thiazolyl blue MTT,
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which is reduced to a black formazan product by viable cells, is used to indicate when the toxin has been neutralised. The use of this toxin neutralisation assay for the evaluation of diphtheria vaccine potency in a Guinea pig model is detailed in the European Pharmacopoeia (3). Vero cells are susceptible to a number of bacterial toxins including the Shiga toxin (ST) of Shigella dysenteriae and certain pathogenic isolates of Escherichia coli and enterotoxin from Clostridium perfringens. Variations of the vero cell assay have been used to explore the neutralising potential of their antitoxins (9–12). 1.2. Serum Bactericidal Antibody
Serum bactericidal antibody (SBA) assays measure the level of antibody in serum required to kill bacteria by the classical pathway of complement activation. In essence, the assay involves treating a suspension of bacteria with dilutions of an antiserum that has been heat treated to inactivate endogenous complement followed by the addition of an exogenous source of complement. After a prescribed period of incubation, the viability of the bacterial suspension is assessed at each serum dilution and the bactericidal antibody titre is usually expressed as the dilution of antiserum that kills a defined proportion of bacteria. Bacterial viability at the end of the assay may be determined directly by a viable counting method or colorimetrically by the metabolic reduction of a tetrazolium dye. The SBA assay is complex as it utilises a number of biological materials, some of which are ill defined and difficult to standardise. Arguably the least well-defined biological component used in the assay is the bacterium, so the choice of strain warrants particularly careful consideration during the development of an assay. Bacteria may be susceptible to complement-mediated killing via the alternative and lectin activation pathways, which do not require antibody. Clearly, the SBA assay cannot be used with bacteria that are too sensitive to complement in the absence of antibody, so this should be tested in advance and controlled in the assay. Conversely, some bacteria that cause invasive infections have evolved mechanisms to evade complement-mediated killing in the host and may be too complement resistant to be useful in this assay. These mechanisms include, for example, the production of bacterial proteins that interfere with the control of the complement cascade (13, 14) or the overproduction of capsular polysaccharide (15). Besides the variable nature of bacterial resistance to the complement, the antigens themselves are frequently variable in both their structure and level of expression. Bacteria exposed to the immune response of the host are usually under immunoselective pressure that is reflected in the diversity of their antigens (16). The expression of antigen-encoding genes is also often tightly regulated by environmental factors or is phase variable (17). Consequently the
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choice of culture medium and the extent to which the organism has been passaged or colony purified may have an impact on the assay (18). The other biological reagent that requires particular consideration during the development of an SBA assay is complement. A cursory review of the literature on SBA assays reveals that sera from a wide range of animal species have been used as the source of complement in different versions of this assay. Given the propensity of invasive pathogens to evolve mechanisms of resistance to complement-mediated lysis, there is a potent argument in favour of using complement from the host of the particular disease in question, as this should provide a more accurate reflection of immune protection. For example, the SBA titre has long been accepted as the surrogate of protection against meningitis and septicaemia caused by Neisseria meningitidis, the meningococcus (19). Meningococci only colonise humans, occasionally causing invasive disease; they have no other environmental or animal niche. Human serum is, therefore, the preferred complement source for this assay. However, as humans are frequently carriers of meningococci, it usually contains endogenous antibodies to meningococcal antigens. The antigenic targets for these antibodies vary from one person to the next depending on the exposure of the individual to different meningococcal strains. It is therefore essential to screen potential complement sources for their lack of endogenous antibodies to the bacterial isolate that is to be used in the assay. In some cases, unwanted endogenous antibody activity may be diluted to a level at which it does not interfere with the result of the assay, while retaining complement activity, and SBA assay protocols differ in the extent to which the complement source is diluted. Another approach is to use serum from agammaglobulinaemic individuals; however, they are rare and invariably receive immunoglobulin replacement therapy, making this a scarce resource that is unlikely to be sufficient for the analysis of sera from large vaccine trials. To ensure an adequate supply of complement, free from endogenous antibodies, an alternative approach is to use serum from a different species that has been demonstrated to be free of cross-reactive bactericidal antibodies. During the recent clinical evaluation of conjugate vaccines against group C meningococcal disease, baby rabbit complement was used in SBA assays (20). This proved to be satisfactory for the measurement of bactericidal antibodies in both adult and infant sera. In this assay, the protective titre was higher than the surrogate of protection previously defined using human complement, which is consistent with meningococci possessing mechanisms (e.g. complement factor H-binding protein) that specifically enhance their resistance to the bactericidal activity of human complement.
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When designing an SBA assay, some thought should be given to the choice of endpoints and the analysis of the resulting data. Although beyond the scope of this chapter, statistical considerations in the design and analysis of bioassays have been thoroughly reviewed elsewhere (21). In general, functional antibody assay data are expressed as titres. So in the case of the SBA assay, results are normally expressed as the reciprocal of the serum dilution at which a defined percentage of the bacteria are killed. Clearly, the choice of percentage killing is critical to the definition of the endpoint. Similarly, the result will be affected by whether the endpoint dilution is determined by interpolation between dilutions or taken as the nearest dilution above or below the chosen level of killing. These choices ultimately have to be made on the basis of their relevance to the experiment. If further standardisation of the assay were required, the result could be expressed in arbitrary units relative to a suitable reference serum. Serum bactericidal antibody assays have been used in the evaluation of functional antibody responses to a diverse list of bacteria, often in the context of vaccine development. These bacteria include type b and non-typeable Haemophilus influenzae, Salmonella species, Vibrio cholerae, Borrelia burgdorferi (Lyme disease) and Pseudomonas aeruginosa. As a result of interest in the development of meningococcal vaccines, SBA assays for the evaluation of anti-meningococcal antibodies are predominant in the recent scientific literature. 1.3. Opsonophagocytic Killing
The clearance of and recovery from many bacterial infections is thought to be mediated by opsonophagocytic activity by host immune cells (2). For these infections, the level of serum antibodies that opsonise the bacteria for phagocytosis is expected to correlate with protection. Opsonophagocytic antibody (OPA) assays measure the amount of serum antibody required to opsonise bacteria in the presence of complement and thereby facilitate phagocytosis. In the assay a bacterial suspension is treated with dilutions of the test serum, then complement is added followed by a suspension of phagocytic cells. After a period of incubation, the degree of opsonophagocytosis is determined from the number of bacteria either killed or taken up by the phagocytes. In the killing assay, the viable count is determined by inoculating the assay mixture on to agar plates of suitable bacterial culture medium and is typically reported as the reciprocal dilution of serum that kills a defined percentage of bacteria in the suspension. As with the SBA assay, 50% killing is usually taken as the endpoint because this part of the killing curve varies the least. In an uptake assay, the number of fluorescently labelled bacteria taken up by phagocytes is measured by flow cytometry. The killing assay has been multiplexed to measure the OPA titres of serum against several bacterial strains in the same assay (22). In this approach the strains are resistant to
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a different antibiotic and these antibiotics are incorporated into a set of strain-specific selective media used to determine the viable counts separately for each strain. As with the SBA assay, care should be taken over the choice of bacterial strains and growth conditions. Ideally, if the assay is to be biologically relevant, the strains should be epidemiologically representative and express the target antigen at normal levels. The culture conditions should be chosen to ensure that the target antigen is expressed at an appropriate level and repeated passaging should be avoided. Human serum is frequently used as the complement source in OPA assays, although some assays use serum from laboratory animals. Endogenous antibodies in the complement source may interfere with the results, if not removed by adsorption. Therefore, during the development of the assay, the complement should be screened to ensure it does not kill the chosen strains in the absence of antibodies and effector cells. This should also be routinely controlled in the assay. There are a number of important issues to consider when choosing the phagocytic effector cells for the assay (23). Peripheral blood lymphocytes (PBLs) from volunteer donors are frequently used in the research laboratory; however, many consider the use of cell lines as phagocytes to be more convenient and reproducible, especially when large numbers of serum samples have to be analysed. Promyelocytic leukaemia cell lines, when subjected to certain chemical conditions, differentiate into neutrophil-like cells. N,N-dimethylformamide (DMF) and alltrans-retinoic acid (ATRA) are the most commonly used inducers of differentiation (24). Depending on the method of induction, differences are observed in cell viability, markers of apoptosis and the expression of cell surface receptors. The process of differentiation can be followed by monitoring the expression of cell surface markers. Despite the availability of many such cell lines, only HL-60 and NB-4 cells have been used extensively in OPA assays. In choosing a cell line for the assay, it is important to consider characteristics such as cell viability and proliferation, as well as the expression of specific receptors that enable the phagocytic cells to recognise the Fc portions of bound antibody and the C3b and iC3b complement components deposited on the bacterial surface. For the evaluation of vaccine-induced serum opsonophagocytic activity, the key immunoglobulin receptors of interest are Fcγ type II and III receptors. Both receptors can bind human IgG1 and IgG3 subclasses, but only the FcγRIIa receptor binds IgG2. Complement receptors for C3b (CR1, CD35) and iC3b (CR3, CD11b) (CR4, CD11c) play an important role in the opsonophagocytosis of encapsulated bacteria (25). Increased expression of CD11b has been identified as a marker of differentiation for effector cells for use in the assay (24).
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Serum OPA levels have been used to evaluate the immune responses to disease and vaccination for numerous bacterial species that include the following: group A and B streptococci, staphylococci, pathogenic Neisseria, Pseudomonas aeruginosa, Bordetella pertussis and pneumococci.
2. Materials 2.1. Toxin Neutralisation
1. Vero cells (ATCC) 2. Growth medium: Dulbecco’s modification of Eagle’s medium (DMEM), supplemented with 10% heat-inactivated foetal bovine serum (FBS) 3. Maintenance medium: Dulbecco’s modification of Eagle’s medium (DMEM), supplemented with 2% heatinactivated foetal bovine serum (FBS) and Dulbecco’s PBS 4. Trypsin/EDTA: 0.25% (w/v) Trypsin-0.53 mM EDTA composition 5. Assay medium: modified medium 199 (Invitrogen) supplemented with 2 mM L-glutamine, 2% FBS and 2% penicillin/streptomycin solution 6. Serum from immunised animals and negative controls 7. Diphtheria toxin (NIBSC)
2.2. Serum Bactericidal Antibody
1. Serum samples from immunised or control animals 2. Target bacterium 3. Complement: from rabbit serum (Pelfreeze) 4. Bacterium-specific monoclonal antibody (mAb) reference serum 5. Bactericidal buffer: 0.5% (w/v) bovine serum albumin, in Gey’s balanced salt solution, filter sterilised through 2 μm filter 6. Microtitre plates 7. Plates for cfu determination
2.3. Opsonophagocytic Killing
1. HL60 cells (ATCC) 2. RPMI 1640 medium (Invitrogen)
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3. CM2 medium: RPMI 1640 medium containing 1% Lglutamine and 20% foetal calf serum but lacking phenol red (CM2) 4. CM2/DMF medium: RPMI 1640 medium containing 1% L-glutamine and 20% foetal calf serum but lacking phenol red (CM2) and containing 100 mM N,Ndimethylformamide (DMF) 5. Glycerol (sterile) 6. Opsono buffer: Hank’s balance salt solution containing 0.1% gelatine 7. Serum samples from immunised or control animals 8. Baby rabbit serum (Pelfreeze) 9. Hank’s balance salt solution 10. Microtitre plates
3. Methods 3.1. Toxin Neutralisation
1. Vero cells are cultured in growth medium at 37◦ C, in an atmosphere of 5% CO2 and 90% relative humidity. After 2–3 days of growth, the growth medium is replaced with maintenance medium. Once a confluent monolayer is obtained, the culture supernatant is discarded and the cell layer washed gently with modified Dulbecco’s phosphate-buffered saline. The cells are stripped from the flask with trypsin/EDTA and resuspended at approximately 1 × 105 cells/mL in assay medium. 2. Place 25 μL of assay medium in each well except those of column 1. Place 25 μL of positive control serum in wells A1, A2 and A11. Place 25 μL of test serum samples in wells B–G of columns 1, 2 and 11. Place 25 μL of negative control serum in row H of columns 1, 2 and 11. Make twofold serial dilutions across the plate (from column 2 up to column 10 for rows A–G and up to column 8 for row H). Discard 25 μL from the wells in column 10 in rows A–G and from well H8. 3. Prepare a 50 IU/mL solution of diphtheria toxin in saline and dilute it 50-fold in assay medium to obtain a working solution of 1.0 IU/mL. Add 25 μL of this working solution to wells A12 and B12 (toxin control). Make 25 μL twofold serial dilutions from well B12–H12, discarding 25 μL from well H12. Add 25 μL of assay medium to wells B12–H12. Then, place 25 μL of the working dilution of
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toxin (1.0 IU/mL) in each well of rows A–H, from column 1–10, except in wells H9 and H10 (cells only, without serum and without toxin). 4. Cover the plates with lids or sealer and shake gently. Incubate the plates for at least 2 h at 37◦ C in a humidified environment containing 5% CO2 . 5. Add 200 μL of cell suspension containing 1 × 105 cells/mL to all the wells. Cover the plates and continue the incubation at 37◦ C for 5 days. Check for microbial contamination by microscopic examination. 6. A yellow colour indicates the presence of viable cells and is recorded as negative while red indicates dead cells and is recorded as positive. The results can be confirmed by microscopic examination for viable and dead cells. 7. The potency of the antiserum samples to neutralise toxin is obtained by comparing the last wells of the standard and test preparations showing complete neutralisation of the toxin (see Note 1). 3.2. Serum Bactericidal Antibody
Variations of the following method have been widely used in the evaluation of meningococcal vaccine candidates but can easily be adapted for use with other organisms. Bactericidal buffer is used for all dilutions. The assay is carried out in the wells of a microtitre plate. 1. Heat inactivate complement in the serum samples for 30 min at 56◦ C. 2. Incubate a culture of the target bacterium until it reaches about mid-logarithmic phase of growth. Harvest the cells by centrifugation and resuspend them in 5 mL of bactericidal buffer. Measure the absorbance of the cell suspension at a wavelength of 600 nm and adjust to an OD600 of 0.1. This typically corresponds to approximately 2 × 108 organisms per mL. As the assay requires 8 × 104 organisms per mL dilute the suspension sequentially, first 1/10 and then 1/250 (see Note 2). 3. Complement should be stored in aliquots at –70◦ C. Slowly thaw an aliquot at RT and keep on ice until required. Immediately before the assay, heat inactivate 200 μL of complement for 30 min at 56◦ C for use in the control wells. 4. Make serial dilutions (20 μL) of serum or mAb in rows across columns 1–9 of a microtitre plate. Twenty microlitres of the starting dilution of sera (i.e. the same serum concentration used in column 1) is also put into column 12 which serves as a complement-independent control. Twenty microlitres of bactericidal buffer is put into columns 10 and
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11 which will serve as complement control (CC) and viable count (VC) wells, respectively. 5. Pipette 10 μL of the bacterial suspension into each well and tap the plate gently to ensure that the bacteria have mixed with the serum sample. 6. Add 10 μL of heat-inactivated complement to columns 11 and 12. Add 10 μL of active complement to columns 1–10. Tap the plate gently on the bench to ensure that complement, bacteria and serum are mixed. 7. Immediately plate out 10 μL from each well in column 11 to determine the viable count at the start of the assay (i.e. T0 count). Then cover the microtitre plate with sticky film and incubate for 1 h at 37◦ C. Plate out 2 × 10 μL from each well in each column to determine the viable count. Although not essential, some laboratories use electronic colony counters. 8. T60 counts from column 11 are used as the viable count in the absence of active complement, i.e. 100% survival or 0% killing (see Note 3). The % killing at a given dilution of serum is calculated from the following equation: % killing = 100 −
3.3. Opsonophagocytic Killing
cfu at dilution × 100 cfu at T60
The following summary protocol is based on an opsonophagocytic assay developed at the CDC to evaluate immune responses to Streptococcus pneumoniae (26). Details of this and a multiplex version of the assay are available through the website of the Bacterial Respiratory Pathogen Reference Laboratory at the University of Alabama (http://www.vaccine.uab.edu/). 1. Preparation of HL-60 cells: During the period that assays are required, maintain three cultures of undifferentiated cells: two for differentiation and one for further expansion (see Note 4). The cells are maintained in RPMI 1640 medium containing 1% L-glutamine and 20% foetal calf serum but lacking phenol red (CM2). Prior to differentiation, perform a viable count and calculate the volume of undifferentiated cell culture required to induce 200 mL of differentiated cells at 2×105 cells/mL. Harvest by centrifugation, resuspend the cells to 200 mL in CM2 containing 100 mM N,N-dimethylformamide and incubate for 5 days at 37◦ C in 5% CO2 . By this time 90–95% of cells should be differentiated and ready to use in the assay. The cell density should be in the range 6-8×105 cells/mL. 2. Preparation of bacterial cells: Dilute an overnight bacterial culture 1:10 and incubate to mid-logarithmic growth (OD600 = ∼0.5), add 15 mL of sterile glycerol to each
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100 mL culture, dispense in aliquots, flash freeze and store frozen at –70◦ C until required. Viable counts should be performed to determine the cell density before and after the freezing. 3. Thaw an aliquot of bacterial cells and serially dilute in opsono buffer to 103 cfu per 20 μL. Keep on ice until ready to use. 4. Treat test serum samples for 30 min at 56◦ C to heat inactivate complement. 5. Add 10 μL of opsono buffer to each well of a 96-well plate except for row A. 6. Add 20 μL of test serum to the wells of row A. Wells A9– A12 are reserved for QC sera. 7. Serially dilute test serum in twofold steps (rows A–H). Wells H9–H12 should be reserved for the complement control. 8. Add 20 μL of bacterial suspension to each well (i.e. 103 cfu/well) and incubate at 37◦ C for 15 min, 5% CO2 . 9. Add 10 μL of complement (in this assay baby rabbit serum). The baby rabbit serum is kept frozen at –70◦ C in 1 mL aliquots until ready to use. One hundred microlitres are heat inactivated at 56◦ C for 30 min as a control. 10. Wash the differentiated cells once in Hanks buffer and resuspend in opsono buffer at room temperature. Add 40 μL of differentiated HL-60 cells to each well (4 × 105 cells/well) and incubate at 37◦ C for 45 min, shake horizontally (∼220 rpm). 11. Stop the reaction and bacterial growth by cooling the plate on ice for 1 min. Then plate 5 μL from each well onto suitable medium for the determination of viable count. Incubate at 37◦ C overnight and count the colonies. A viable count of the initial number of bacteria added at time zero (T0 ) to each well is included per run (see Note 5). 12. The phagocytic titre is usually expressed as the reciprocal of the serum dilution with at least 50% killing when compared to the average growth in the complement control wells (H9–H12) (see Note 6).
4. Notes 1. For calculations of potency, it should be remembered that the endpoint may fall between dilutions.
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2. To minimise the risk of generating a potentially infectious aerosol, meningococci are often grown on the surface of solid culture medium for about 4 h and then resuspended in 5 mL of bactericidal buffer. 3. T0 (column 11), the complement control (column 10, killing by complement alone, in the absence of antibody) and complement-independent control CIC (killing by serum alone in the absence of complement, column 12) should all be as low as possible. In the case of the meningococcal assay, these values have to be less than 30% for the assay to be considered valid. 4. The ability of HL-60 cells to differentiate satisfactorily for this assay varies between sources. Experience suggests it is best to purchase undifferentiated cells from the American Type Culture Collection (ATCC) at passage 20. 5. The complement control counts and T0 counts are expected to be within 20% of each other. 6. Occasionally sera with high titres may need to be retested at higher initial dilutions. References 1. Qin, L., Gilbert, P. B., Corey, L., McElrath, M. J., Self, S. G. (2007) A framework for assessing immunological correlates of protection in vaccine trials. J Infect Dis 196, 1304–1312. 2. Plotkin, S. A. (2008) Vaccines: correlates of vaccine-induced immunity. Clin Infect Dis 47, 401–409. 3. Council of Europe. (2008) Assay of diphtheria vaccine (adsorbed), Chapter 2.7.6. Ph Eur, 6th edn. 4. Council of Europe. (2008) Assay of tetanus vaccine (adsorbed), Chapter 2.7.8. Ph Eur, 6th edn. 5. Gommer, A. M. (1996) Vero cell assay validation of an alternative to the Ph. Eur. diphtheria potency tests. Dev Biol Stand 86, 217– 224. 6. Winsnes, R., Sesardic, D., Daas, A., Rigsby, P. (2002) A Vero cell method for potency testing of diphtheria vaccines. Dev Biol (Basel) 111, 141–148. 7. Rao, K. N., Kumaran, D., Binz, T., Swaminathan, S. (2005) Structural analysis of the catalytic domain of tetanus neurotox. Toxicon 45, 929–939. 8. Hoy, C. S., Sesardic, D. (1994) In vitro assays for detection of diphtheria toxin. Toxicology In Vitro 8, 693–695.
9. Cornick, N. A., Jelacic, S., Ciol, M. A., Tarr, P. I. (2002) Escherichia coli O157:H7 infections: discordance between filterable fecal shiga toxin and disease outcome. J Infect Dis 186, 57–63. 10. Mendes-Ledesma, M. R., Rocha, L. B., Bueris, V., Krause, G., Beutin, L., Franzolin, M. R., Trabulsi, L. R., Elias, W. P., Piazza, R. M. (2008) Production and characterization of rabbit polyclonal sera against Shiga toxins Stx1 and Stx2 for detection of Shiga toxin-producing Escherichia coli. Microbiol Immunol 52, 484–491. 11. Pirro, F., Wieler, L. H., Failing, K., Bauerfeind, R., Baljer, G. (1995) Neutralizing antibodies against Shiga-like toxins from Escherichia coli in colostra and sera of cattle. Vet Microbiol 43, 131–141. 12. Mahony, D. E., Gilliatt, E., Dawson, S., Stockdale, E., Lee, S. H. (1989) Vero cell assay for rapid detection of Clostridium perfringens enterotoxin. Appl Environ Microbiol 55, 2141–2143. 13. Biedzka-Sarek, M., Jarva, H., Hyytiainen, H., Meri, S., Skurnik, M. (2008) Characterization of complement factor H binding to Yersinia enterocolitica serotype O:3. Infect Immun 76, 4100–4109.
Functional Antibody Assays 14. Madico, G., Welsch, J. A., Lewis, L. A., McNaughton, A., Perlman, D. H., Costello, C. E., Ngampasutadol, J., Vogel, U., Granoff, D. M., Ram, S. (2006) The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance. J Immunol 177, 501–510. 15. Uria, M. J., Zhang, Q., Li, Y., Chan, A., Exley, R. M., Gollan, B., Chan, H., Feavers, I., Yarwood, A., Abad, R., Borrow, R., Fleck, R. A., Mulloy, B., Vazquez, J. A., Tang, C. M. (2008) A generic mechanism in Neisseria meningitidis for enhanced resistance against bactericidal antibodies. J Exp Med 205, 1423–1434. 16. Lipsitch, M., O’Hagan, J. J. (2007) Patterns of antigenic diversity and the mechanisms that maintain them. J R Soc Interface 4, 787–802. 17. van der Woude, M. W., Baumler, A. J. (2004) Phase and antigenic variation in bacteria. Clin Microbiol Rev 17, 581-611, table. 18. Borrow, R., Aaberge, I. S., Santos, G. F., Eudey, T. L., Oster, P., Glennie, A., Findlow, J., Hoiby, E. A., Rosenqvist, E., Balmer, P., Martin, D. (2005) Interlaboratory standardization of the measurement of serum bactericidal activity by using human complement against meningococcal serogroup b, strain 44/76-SL, before and after vaccination with the Norwegian MenBvac outer membrane vesicle vaccine. Clin Diagn Lab Immunol 12, 970–976. 19. Goldschneider, I., Gotschlich, E. C., Artenstein, M. S. (1969) Human immunity to the meningococcus. I. The role of humoral antibodies. J Exp Med 129, 1307–1326.
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20. Andrews, N., Borrow, R., Miller, E. (2003) Validation of serological correlate of protection for meningococcal C conjugate vaccine by using efficacy estimates from postlicensure surveillance in England. Clin Diagn Lab Immunol 10, 780–786. 21. Plikaytis, B. D., Carlone, G. M. (2005) Statistical considerations for vaccine immunogenicity trials. Part 1: Introduction and bioassay design and analysis. Vaccine 23, 1596–1605. 22. Nahm, M. H., Briles, D. E., Yu, X. (2000) Development of a multi-specificity opsonophagocytic killing assay. Vaccine 18, 2768–2771. 23. Fleck, R. A., Romero-Steiner, S., Nahm, M. H. (2005) Use of HL-60 cell line to measure opsonic capacity of pneumococcal antibodies. Clin Diagn Lab Immunol 12, 19–27. 24. Fleck, R. A., Athwal, H., Bygraves, J. A., Hockley, D. J., Feavers, I. M., Stacey, G. N. (2003) Optimization of nb-4 and hl-60 differentiation for use in opsonophagocytosis assays. In Vitro Cell Dev Biol Anim 39, 235–242. 25. Gordon, D. L., Johnson, G. M., Hostetter, M. K. (1986) Ligand-receptor interactions in the phagocytosis of virulent Streptococcus pneumoniae by polymorphonuclear leukocytes. J Infect Dis 154, 619–626. 26. Romero-Steiner, S., Libutti, D., Pais, L. B., Dykes, J., Anderson, P., Whitin, J. C., Keyserling, H. L., Carlone, G. M. (1997) Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells. Clin Diagn Lab Immunol 4, 415–422.
Chapter 15 Determining Adjuvant Activity on T-Cell Function In Vivo: Th Cells Thomas Lindenstrøm, Peter Andersen, and Else Marie Agger Abstract Adjuvants constitute a critical component in vaccine development in terms of both stimulating and directing immune responses of a suitable profile to promote protection against a diverse range of disease targets. In the past, the field of adjuvant research was mainly dominated by empirical testing and serendipity. However, there is a strong need to develop new generations of adjuvants based on rational design, as well as a requirement to characterise and comprehend their mechanism(s) of action. Adjuvant development can be characterised as an iterative process where potential candidates are repeatedly tested in vitro and in vivo for immunogenicity and optimised in terms of formulation and delivery. Novel lead candidates of adjuvants with a suitable immunological profile relative to specific disease targets are subsequently selected and evaluated in efficacy studies. A central aspect in such a development and selection process is to determine the adjuvant activity on T-cell function in vivo. Expanding our knowledge on these mechanisms will improve our chance of developing new successful vaccines designed to target specific diseases. Key words: Adjuvants, T-cell polarisation, antigen recall, cytokine profile, T-cell frequency, T-cell proliferation, multifunctional T cells.
1. Introduction Innate immunity has been revitalised since the discovery of the Toll-like receptors more than a decade ago (1, 2). The ensuing years of research have thus greatly improved our understanding of how the innate immune system perceives pathogens and tissue injury/stressors through pattern recognition receptors (PRRs) (3). These basic findings have clearly invigorated the field of adjuvant research, not only in terms of developing new adjuvants but also in understanding their mode of action (4–9). It is becoming G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_15, © Springer Science+Business Media, LLC 2010
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increasingly clear that innate immunity not only initiates adaptive responses but also plays a central role for shaping and modulating the subsequent adaptive responses by eliciting cytokines that influence the polarisation of T-helper cells into Th1, Th2 and Th17 T-cell subsets (10, 11) (Fig. 15.1). In vitro assays can be employed to determine adjuvant activity on T cells. This can be tremendously helpful in comprehending their mode of action and additionally gives clues to how they might promote differentiation and polarisation of T-helper cells. Still, such studies do not take into account the in vivo complexity or consider factors such as those related to route of administration and immunisation schedules. For characterising adjuvant activity on T-cell composition/polarisation, magnitude and quality of the induced T-cell responses as well as the ability for memory formation, in vivo assays thus remain one of the ultimate read-outs. The current section describes a number of techniques that will guide in characterising these aspects of T-cell function following vaccination with adjuvanted vaccines. The following part will thus describe the major steps involved in characterising the adjuvant activity on T-cell function in vivo from the initial isolation of cells, subsequent recall stimulation of vaccine-induced T cells to a thorough characterisation of the response in terms of T-cell polarisation by cytokine ELISAs, cytometric bead analysis (CBA) for multiplex analysis of secreted cytokines and for assessing the frequency of antigen-specific T cells by ELISPOT and intracellular flow cytometry assays. Finally, functional aspects of the response will be dealt with by assays measuring the proliferative potential of the
Fig. 15.1. The type of imprinting signal provided by the adjuvant itself or through released damage/danger-associated molecular pattern molecules highly influences the subsequent polarisation of T-helper cells. In the presence of IL-12, naïve T cells differentiate towards a Th1 lineage by inducing IFN-γ secretion, which mediates STAT-1 signalling and activation of the Th1 transcription factor Tbet. IL-4 promotes Th2 induction by signalling through STAT-6 leading to induction of the GATA3 transcription factor. Finally, under influence of IL-6 and TGF-β, the lineage-determining transcription factor RORγt is activated leading to Th17 responses.
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vaccine-induced response by the CFSE dilution method and for the quality of the response in terms of measuring the degree of T-cell multifunctionality (capacity of cytokine co-expression).
2. Materials 2.1. Isolation of PBMCs
1. 0.9% NaCl; sterile. 2. Lympholyte (Cedarlane CL5120), sterile. 3. RPMI medium: RPMI-1640 supplemented with 5 × 10–5 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco). 4. Foetal calf serum (FCS) (Biochrome AG).
2.2. Isolation of Cells from Spleen and Lymph Nodes
1. Cell strainer (BD Biosciences) or sterile metal grid. 2. RPMI medium: RPMI-1640 supplemented with 5 × 10–5 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco). 3. Foetal calf serum (FCS) (Biochrome AG).
2.3. Cell Culture and Antigen Recall
1. U-bottom 96-well tissue culture-treated microtitre plates (NUNC). 2. RPMI medium: RPMI-1640 supplemented with 5 × 10–5 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco). 3. Foetal calf serum (FCS) (Biochrome AG). 4. Relevant recall antigens (proteins, peptides).
2.4. Conventional Capture-ELISAs—A: IFN-γ ELISA; B: IL-17A ELISA
1. F-bottom 96-well Maxisorp microtitre plates (NUNC). 2. Coating buffer (carbonate buffer): 0.03 M Na2 CO3 , 0.056 M NaHCO3 pH 9.6 in milliQ-water + phenol red 1 mL/L. 3. Capture antibody (see Note 1) (a) For IFN-γ ELISA: purified rat anti-mouse IFN-γ (clone R4-6A2; BD Pharmingen) diluted in coating buffer to a final concentration of 1 μg/mL. (b) For IL-17A ELISA: purified rat anti-mouse IL-17A (clone: TC11-18H10.1; BioLegend) diluted in coating buffer to a final concentration of 1 μg/mL.
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4. Blocking buffer: 2% skimmed milk powder in PBS. 5. Wash buffer: 0.2% Tween20 in PBS. 6. Assay buffer: 2 or 1% bovine serum albumin (BSA) in PBS. 7. ELISA standards (a) For IFN-γ ELISA: IFN-γ standard (BD Pharmingen). (b) For IL-17A ELISA: IL-17A recombinant standard (BioLegend). 8. FCS (Biochrome AG). 9. Biotinylated detection antibody (see Note 1). (a) Biotinylated rat anti-mouse IFN-γ (clone XMG1.2, BD Pharmingen) diluted in 1% assay buffer to a final concentration of 0.1 μg/mL. (b) Biotinylated rat anti-mouse IL-17A (clone: TC118H4; BioLegend) diluted in 1% assay buffer to a final concentration of 0.25 μg/mL. 10. Horseradish peroxidase-conjugated streptavidin (SA-HRP) (Zymed) diluted in 1% assay buffer to a final concentration of 0.25 μg/mL. 11. Substrate: TMB Plus ready-to-use substrate (Kem-EnTec). 12. Stop solution: 0.2 M H2 SO4 . 2.5. Cytometric Bead Analysis—Th1/Th2 Cytokine CBA
1. Mouse Th1/Th2 Cytokine CBA Kit (BD Biosciences) (see Note 2) containing (a) Recombinant mouse Th1/Th2 cytokine standards. (b) Assay diluent. (c) Capture Beads A1–A5 (A1: IL-2; A2: IL-4; A3: IL-5; A4: IFN-γ; A5: TNF-α). (d) PE detection reagent. (e) Wash buffer. (f) Cytometer Setup Beads. (g) FITC and PE positive control detector.
2.6. ELISPOT—A: IFN-γ ELISPOT; B: IL-5 ELISPOT
1. MAHA S45 10 (cellulose ester) ELISPOT plates (Millipore). 2. Sterile PBS.
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3. Capture antibody (see Note 1) (a) For IFN-γ ELISPOT: rat anti-mouse IFN-γ (clone R4-6A2; BD Pharmingen) diluted in sterile PBS to a final concentration of 4 μg/mL. (b) For IL-5 ELISPOT: rat anti-mouse/human IL-5 (clone TRFK5; BD Pharmingen) diluted in sterile PBS to a final concentration of 2 μg/mL. 4. RPMI medium: RPMI-1640 supplemented with 5 × 105 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco). 5. Foetal calf serum (FCS) (Biochrome AG). 6. Blocking buffer: 10% FCS in RPMI medium. 7. Wash buffer: 0.05% Tween20 in PBS. 8. Biotinylated detection antibody (see Note 1) (a) For IFN-γ ELISPOT: biotinylated rat anti-mouse IFN-γ (clone XMG1.2, BD Pharmingen) diluted in PBS to a final concentration of 1.25 μg/mL. (b) For IL-5 ELISPOT: biotinylated rat anti-mouse IL-5 (clone TRFK4; BD Pharmingen) diluted in PBS to a final concentration of 1 μg/mL. 9. Alkaline phosphatase-conjugated streptavidin (SA-AP) (Jackson ImmunoResearch) diluted in PBS to a final concentration (a) For IFN-γ ELISPOT: SA-AP 0.5 μg/mL. (b) For IL-5 ELISPOT: SA-AP 2 μg/mL. 10. Substrate: BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; Sigma-Aldrich). 2.7. Proliferation: CFSE Dilution Assay
1. U-bottom 96-well tissue culture-treated microtitre plates (NUNC). 2. RPMI medium: RPMI-1640 supplemented with 5 × 10−5 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco). 3. Foetal calf serum (FCS) (Biochrome AG). 4. Relevant recall antigens (proteins, peptides). 5. Carboxyfluorescein diacetate, succinimidyl ester (CFDASE/CFSE; Molecular Probes) diluted to a stock of 20 mM in 100% DMSO. Aliquot and keep in the dark (–20◦ C).
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6. FACS buffer: PBS + 1% FCS. For longer term storage at 4◦ C supplement with 0.05–0.1% NaN3 . 2.8. Intracellular FACS Staining for Multifunctional T Cells
1. ThermostatPlus heatblock (Eppendorf). 2. V-bottom 96-well cell culture-treated microtitre plates (Corning). 3. RPMI medium: RPMI-1640 supplemented with 5 × 10–5 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco). 4. Foetal calf serum (FCS) (Biochrome AG). 5. Co-stimulatory antibodies: anti-murine CD28 (clone 37.51) and anti-murine CD49d (clone 9C10(MFR4.B)) (both BD Pharmingen) (see Note 3). 6. Brefeldin A 5 mg/mL in EtOH (Sigma-Aldrich). 7. Monensin/“GolgiStop” (BD Pharmingen). 8. FACS buffer: PBS + 1% FCS. 9. Conjugated antibodies for surface markers and cytokines: anti-CD4:APC-Cy7 (clone GK1.5), anti-CD8:PerCpCy5.5 (clone 53-6.7), anti-CD44:FITC (clone IM7), anti-IFN-γ:PE-Cy7 (clone XMG1.2), anti-TNF-α: PE (MP6-XT22) and anti-IL-2:APC (clone JES6-5h4) (all BD Pharmingen, except anti- IFN-γ:PE-Cy7: eBiosciences). 10. Fluorophore-conjugated anti-CD4 (clone GK1.5) or anti-CD8 (clone 53-6.7) for compensation (conjugates: FITC, PE, PerCp-Cy5.5; PE-Cy7, APC, APC-Cy7)— alternatively use compensation beads (e.g. CompBead; BD Pharmingen). 11. Cytofix/Cytoperm kit (BD Pharmingen).
3. Methods 3.1. Isolation of PBMCs
1. The lympholyte solution has to be at room temperature (rt) before usage. Remember to shake well. Work aseptically. Add 5 mL lympholyte solution to a 15 mL Falcon tube. 2. Dilute the blood from an immunized animal (approximately 500–750 μL) with an equal volume of 0.9% NaCl before adding to the tube containing lympholyte. Carefully layer the diluted blood over the lympholyte solution.
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3. Centrifuge 20 min at 800 × g without brake. 4. Carefully harvest the cells at the interface layer with a Pasteur pipette and transfer to a new centrifuge tube. Fill it up with RPMI medium and pellet the cells by centrifugation at 800 × g for 5 min. 5. Remove the supernatant, and wash the cells one more time in RPMI medium. Remember to resuspend the cells prior to washing (see Note 4). 6. After the last spin, resuspend the cells in 1 mL RPMI medium with 10% FCS. Count cells and adjust to the desired concentration. 3.2. Isolation of Cells from Spleen and Lymph Nodes
1. Work aseptically. Grind the spleen/lymph node through a cell strainer or metal grid to obtain a single cell suspension. The cells are washed twice in RPMI medium as above (see Note 4) and resuspended in RPMI medium with 10% FCS. Count cells and adjust to appropriate cell concentration.
3.3. Cell Culture and Antigen Recall
1. Use sterile reagents and work aseptically. Dilute the antigens for stimulation in RPMI medium + 10% FCS at the double concentration relative to the desired final concentration (final concentration in the range of 0.2–5 μg/mL; see Note 5). Add antigens at 100 μL/well, though avoid using the outermost wells. Remember to include negative (RPMI medium + 10% FCS) and positive (Con A 5 μg/mL) control wells. Fill outermost wells with 200 μL PBS to avoid evaporation during incubation (see Note 6). 2. Cell suspensions, e.g. PBMCs, spleen or lymph node cells, are adjusted to 2 × 106 cells/mL in RPMI medium + 10% FCS and added at 100 μL/well, leaving a total of 200 μL/well (2 × 105 cells/well). 3. Incubate the plates (covered with lid) in an incubator at 37◦ C; 5% CO2 for 72 h (see Note 7) and subsequently collect supernatants (150–175 μL/well), for cytokine-specific ELISAs and/or CBA. If not used immediately, store at –20◦ C.
3.4. Conventional Capture-ELISAs—A: IFN-γ; B: IL-17A
1. Coat plates with 100 μL/well of capture antibody diluted in carbonate buffer (final concentration 1 μg/mL). Incubate plates overnight at 4◦ C. (a) For IFN-γ ELISAs, use anti-mouse IFN-γ (clone R46A2) as capture antibody. (b) For IL-17A ELISAs, use anti-mouse IL-17A (clone: TC11-18H10.1) as capture antibody.
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2. The following day, discard the coating buffer and add 200 μL/well of 2% milk powder in PBS. Incubate for 1.5 h at room temperature. Subsequently wash plates 3x in wash buffer. 3. Standards and samples: If necessary dilute samples in 2% assay buffer (see Note 8) and add 100 μL/well. Samples should be run in duplicate or triplicate. (a) For IFN-γ ELISA: Dilute the IFN-γ standard to a topstandard of 5,000 pg/mL. Perform six 2-fold serial dilutions in PBS + 10% FCS (5,000, 2,500, 1,250, 625, 312, 156, 78 pg/mL + blank). Each standard is added at the volume of 50 μL to each well. Subsequently add 50 μL of 2% assay buffer to the standard wells, leaving a total volume of 100 μL/well. Each plate should contain a column of standard concentrations. (b) For IL-17A ELISA: Dilute the IL-17A standard to a top-standard of 4,000 pg/mL. Perform six 2-fold serial dilutions in PBS + 10% FCS (4,000, 2,000, 1,000, 500, 250, 125, 62.5 pg/mL + blank). Each standard is added at the volume of 50 μL to each well. Subsequently add 50 μL of 2% assay buffer to the standard wells, leaving a total volume of 100 μL/well. Each plate should contain a column of standard concentrations. 4. Incubate plates for 2 h at rt. Wash plates 3x in wash buffer. 5. Detection step: Incubate with biotinylated detection antibody for 1 h at rt. Subsequently wash plates 3x in wash buffer (a) For IFN-γ ELISA: Add 100 μL/well of biotinconjugated anti-mouse IFN-γ (0.1 μg/mL). (b) For IL-17A ELISA: Add 100 μL/well of biotinconjugated anti-mouse IL-17A (0.25 μg/mL). 6. Following the washing steps, add 100 μL/well HRPStreptavidin (0.25 μg/mL) and incubate for 30 min at rt. Wash plates 5x in wash buffer. 7. Develop plates by adding 100 μL/well of TMB substrate. Incubate in the dark for 5–15 min at rt. 8. Stop reaction with 100 μL/well 0.2 M H2 SO4 . Read absorbance at 450 nm (see Note 9). 9. Make a standard curve (log OD(450 nm) against log cytokine concentration (pg/mL)) and determine the unknown cytokine concentrations. Remember dilution factors if samples were diluted and take into account the 1:2 dilutions of standards (3A and 3B).
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1. Reconstitute a vial of the lyophilised recombinant cytokine standards in 0.2 mL of Assay Diluent (bulk standard (10x); 50 ng/mL). 2. Dilute the cytokine standards 1:10 by mixing 25 μL bulk standard (10x) with 225 μL Assay Diluent. Perform eight 2-fold serial dilutions in Assay Diluent (75 + 75 μL Assay Diluent; hence 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256). 3. Determine the total number of samples, including standards (nine dilutions) and blank (Assay Diluent). Make the Capture Bead mix: Vortex the Capture Bead suspensions and aliquot 2.4 μL (see Note 10) of each Capture Bead for each assay tube to be analysed into a single tube (“Mixed Beads”) and vortex vigorously. 4. If necessary, dilute samples in Assay Diluent (standard range 20–5,000 pg/mL) (see Note 8). Add 10 μL standard/blank/unknown samples to each well. 5. Vortex the “Mixed Beads” and add 10 μL to each of the wells. Finally, add 10 μL PE detection reagent to each well. 6. Briefly spin plate to collect reagents and samples at the well bottom and then carefully resuspend using a multipipette to ensure mixing. 7. Incubate plate in the dark for 2 h at rt. 8. After incubation, add 140 μL wash buffer to each well and centrifuge the plate 5 min at 300 × g. Carefully aspirate and discard the supernatant, avoid touching the bead pellet. 9. Resuspend in 150 μL wash buffer and transfer to cluster tubes. 10. Before acquiring samples, prepare the Cytometer Setup Beads: Vortex Setup Beads and add 50 μL to three separate tubes (A, B and C). Add 50 μL of FITC Positive Control Detector to tube B and 50 μL of PE Positive Control Detector to tube C. Mix well. 11. Incubate tubes (A–C) in the dark for 30 min. Add 450 μL wash buffer to tube A and 400 μL to tube B and C. 12. Analyse samples on a flow cytometer the same day as performing the staining procedure. Set up instrument using the Setup Beads for adjusting the PMT levels and carry out compensation. Collect at least 1,800 events/beads per sample. 13. Make standard curves for each cytokine and determine the cytokine concentrations for the unknown samples based on
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the fluorescent intensity value of each circulating analyte relative to their standards. Remember any dilutions factors. 3.6. ELISPOT—A: IFN-γ; B: IL-5
1. Use sterile reagents and work aseptically. Coat ELISPOT plates with 50 μL/well of capture antibody diluted in PBS. Cover with tinfoil and incubate overnight at rt (a) For IFN-γ ELISPOTs, use anti-mouse IFN-γ (clone R4-6A2) as capture antibody (4 μg/mL). (b) For IL-5 ELISPOTs, use anti-mouse IL-5 (clone: TRFK5) as capture antibody (2 μg/mL). 2. The following day, discard the capture antibody and wash plates 5x with sterile PBS (100 μL/well). Subsequently block plates in blocking buffer (RPMI medium + 10% FCS) 100 μL/well for 1.5 h at 37◦ C in a CO2 incubator. 3. Dilute the antigens for stimulation in RPMI medium + 10% FCS at the double concentration relative to the desired final concentration (final concentration in the range of 0.2– 5 μg/mL; see Note 5). Remove the blocking solution by flicking the plates and add the diluted antigens at a volume of 100 μL/well. Include negative controls (RPMI medium + 10% FCS). 4. Cell suspensions, e.g. PBMCs, spleen or lymph node cells, are adjusted to 2 × 106 cells/mL (see Note 11) and added 100 μL/well, leaving a total volume of 200 μL/well (2 × 105 cells/well). 5. Incubate the plates (covered with lid) in an incubator at 37◦ C; 5% CO2 for 48 h (see Note 12). 6. From this point, aseptic working conditions are no longer needed. Wash the plates thoroughly 5x with Wash Buffer (0.05% Tween20 in PBS). 7. Incubate with biotinylated detection antibody for 2 h at rt. Subsequently flick plates and wash 5x with wash buffer. (a) For IFN-γ ELISPOT: Add 100 μL/well of biotinconjugated anti-mouse IFN-γ (final concentration 1.25 μg/mL). (b) For IL-5 ELISPOT: Add 100 μL/well of biotinconjugated anti-mouse IL-5 (final concentration 1 μg/mL). 8. Following the washing steps, add 100 μL/well APStreptavidin (a) For IFN-γ ELISPOT: SA-AP 0.5 μg/mL; incubate for 45 min at rt. (b) For IL-5 ELISPOT: SA-AP 2 μg/mL; incubate for 1 h 15 min at rt.
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9. Flick plates and wash 5x in wash buffer. Carefully disassemble the bottom plate and rinse under running water. Reassemble the plate, taking care not to destroy the cellulose-ester-bottomed wells. 10. Dissolve BCIP/NBT substrate tablets in double-distilled dH2 O, filter through a 0.2 μm filter and develop plates in the dark for 10–20 min. 11. Stop the reaction by washing plates under running water. 12. Allow plates to dry at rt and keep in the dark until reading the plates. 13. Enumerate spots using an ELISPOT reader or manually using a stereomicroscope. Calculate the frequency of spot forming units (SFU) by expressing the number of SFUs per 106 cells. 3.7. Proliferation: CFSE Dilution Assay
1. Use sterile reagents and work aseptically. Resuspend cells, e.g. PBMCs, lymph node or splenic cells in 1 mL RPMI medium without FCS (max. cell concentration: 2×107 cells/mL). 2. Predilute the CFSE stock (20 mM) 1:100 in RPMI medium without FCS. Add 25 μL/mL cells to a final concentration of 5 μM CFSE (see Note 13). Mix well to ensure uniform staining and incubate in the dark for 10 min at 37◦ C. 3. Stop staining and bind surplus CFSE by adding 5 volumes of ice-cold RPMI medium + 10% FCS and put on ice for 5 min. 4. Spin cells at 800 × g for 3 min at 4◦ C and repeat washing three times in ice-cold RPMI medium + 10% FCS. Finally resuspend in RPMI medium + 10% FCS at a cell concentration of 2 × 106 cells/mL and stimulate with appropriate antigen(s), including positive and negative controls, as described in Section 3.3. Incubate at 37◦ C; 5% CO2 (2 × 105 cells/well) for 3–5 days (see Note 13). 5. Harvest cells and transfer to cluster tubes. Set up instrument and acquire samples—if cell are stained for surface markers, remember to compensate (see Note 14). Refer to Fig. 15.2 for data analysis and calculate the percentage of original cells dividing, the precursor frequency and the proliferative index.
3.8. Intracellular FACS Staining for Multifunctional T Cells
1. Adjust cell suspensions, e.g. PBMCs, spleen or lymph node cells, to 1–2 × 107 cells/mL and add 100 μL/well. Add cells to 7–8 extra wells for compensation purposes (6 wells) and for unstained controls (1–2 wells)—otherwise use compensation beads.
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Fig. 15.2. Schematic outline showing the principles of the CFSE dilution assay. (a) the CFSE fluorescent signal is partitioned evenly among daughter cells at each division round. (b) Use a flow-software (e.g. FlowJo) to analyse the obtained CFSE diagram using a proliferation algorithm and identify each generation (shaded grey). Alternatively, manually identify generations, gate around these and obtain the number of events within each. The precursor frequency can be identified as depicted by calculating the cohort total. The proliferative index can be a useful parameter and denotes the sum of the cells in all generations divided by the calculated number of precursors. For detailed information on the background of the assay and the calculations, see Lyons and Parish (12).
2. Dilute the antigens for stimulation in RPMI medium + 10% FCS at the double concentration relative to the desired final concentration (final concentration in the range of 1–5 μg/mL; see Note 5) and add at a volume of 100 μL/well leaving a total volume of 200 μL/well. Likewise add 100 μL/well of RPMI medium + 10% FCS to unstimulated controls as well as unstained/compensation controls. 3. Add/well anti-CD28 and anti-CD49d antibodies (final concentration each 1 μg/mL). 4. Incubate plate 1 h at 37◦ C in a CO2 -incubator (see Note 15). 5. Dilute BFA (5 mg/mL) 25x in RPMI medium + 10% FCS to a concentration of 200 μg/mL. Add 10 μL/well media containing Brefeldin A (final concentration 10 μg/mL) and 0.7 μL/well of Monensin/GolgiStop. Mix well using a multichannel pipette. 6. Incubate for 5–6 h in the ThermostatPlus heatblock (see Note 16). Programme it to cool down to 4◦ C after the 5– 6 h incubation at 37◦ C. Alternatively, incubate at 37◦ C in a CO2 -incubator for 5–6 h and subsequently transfer the plate to 4◦ C for the next day. 7. Spin plate at 1,000 × g for 3 min at 4◦ C. Discard the supernatant. 8. Wash plate 1x in 200 μL FACS buffer. Spin plate at 1,000 × g for 3 min at 4◦ C. Discard the supernatant.
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9. Stain cells for surface markers in 50 μL/well. Calculate the volume needed (add extra for pipetting errors) and make a surface stain mix by diluting antibodies for surface markers (CD4:APC-Cy7; CD8:PerCp-Cy5.5 and CD44:FITC) 1:200 in FACS buffer (final concentration of each antibody: 1 μg/mL). Vortex surface stain mix and add 50 μL/well. Do not add surface stain mix to compensation wells. Add 50 μL/well single colour antibodies (anti-CD4 or anti-CD8) at 1 μg/mL for each fluorescence channel (FITC, PE, PerCp-Cy5.5, PE-Cy7, APC and APC-Cy7: 6 wells) and FACS buffer for the negative unstained control. Incubate in the cold and dark for 30 min. 10. Add 150 μL FACS buffer to each well. Spin at 1,000 × g for 3 min at 4◦ C. Discard the supernatant. Repeat the wash in FACS buffer 200 μL/well. Spin plate at 1,000 × g for 3 min at 4◦ C. Discard the supernatant. 11. Fix cells by adding 100 μL Cytofix/Cytoperm/well. Mix by pipetting. Incubate in the cold and dark for 30 min. 12. Spin plate at 1,000 × g for 3 min at 4◦ C. Discard the supernatant. Wash plates with Perm Wash Buffer, 200 μL/well. Spin at 1,000 × g for 3 min at 4◦ C. Discard the supernatant. 13. Intracellular stain; 50 μL/well: Make an intracellular stain mix by diluting antibodies for cytokines (IFN-γ-PE:Cy7; TNF-α:PE; IL-2:APC) 1:200 in Perm wash buffer (final concentration of each antibody: 1 μg/mL). Vortex and add 50 μL/well, except to compensation wells. Add Perm wash buffer 50 μL to each of these. Incubate the plate for 30 min in the cold and dark. 14. Add 150 μL Perm wash buffer to each well. Spin plate at 1,000 × g for 3 min at 4◦ C. Discard the supernatant. Wash plates with Perm wash buffer, 200 μL/well. Let the plate stand for 2–3 min, then spin plate at 1,000 × g for 3 min at 4◦ C. Discard the supernatant. 15. Finally resuspend cells in 200 μL FACS buffer and transfer to cluster tubes. 16. Set up instrument by adjusting the PMT levels and perform compensation using either unstained and single stain tubes or compensation beads. 17. When running samples: Acquire as many cells as possible— preferentially no less than 200,000 events within the lymphocyte gate. Gate as in Fig. 15.3 by making IFNγ, TNF-α and IL-2 +ve gates within the CD4 and CD8
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T cells followed by creating Boolean combination gates to asses the frequency of each cytokine co-expressing subset and their share of the total responding T-cell population.
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4. Notes 1. We have found these capture–detection antibody pairs to work excellently for ELISAs and ELISPOT assays. Numerous competitive reagents are available from other commercial sources. 2. Cytometric Bead Analysis is a multiplexing assay in which a series of soluble analytes are detected and quantified simultaneously in a single tube by virtue of catching particles with discrete fluorescent intensities. Numerous competitive reagents are available from commercial sources with slightly different setups—e.g. Bio-Plex (23-plex, Bio-Rad), Flowcytomix (10-plex; Bender MedSystems) and CBA (BD Biosciences). Here we describe a fixed Th1/Th2-cytokine CBA—but in most cases, the “Flex Set” can be customised for specific needs. 3. The employment of anti-CD28 and anti-CD49d costimulation is an optional step. It was originally introduced for optimising intracellular staining techniques (14, 15). It increases Ag-specific responses thereby revealing the full functional Ag-specific response and thus increases the sensitivity resulting in better signal–noise ratios. In our experiences it results in a 2- to 4-fold enhancement in the frequency of cytokine-secreting CD4+ T cells following Ag-specific stimulation without affecting background levels. However, controls are essential to ensure that background levels/non-specific staining do not increase. 4. Resuspend cells thoroughly by flicking tubes over a rack. 5. Antigen concentrations for stimulation need to be established according to the specific experimental settings. In most cases, a final concentration in the range of 0.2– 5 μg/mL can be employed. 6. The usage of outermost wells is avoided, and these are filled with 200 μL PBS to prevent evaporation of supernatant from sample wells. Otherwise, this could impact the concentration of cytokines to be subsequently measured. 7. Incubation times can vary according to the specific experimental settings. In most cases, 48–72 h incubation will be adequate. 8. Experimental samples need to be diluted to be within the linear range of O.D. values obtained by the cytokine standards.
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9. If microplate reader can perform wavelength correction, set to subtract readings at 540 or 570 nm from the readings at 450 nm. This will correct for optical imperfections in the plate(s). 10. The supplier recommends the use of 10 μL of each Capture Bead for each assay tube. In our experience this can be down-scaled 1:5 without compromising read-outs. The use of 2.4 μL is suggested to ensure adequate volume for pipetting errors. 11. Cell concentrations for ELISPOT assays should be adjusted according to the specific experimental settings to ensure clear resolution of spots. Initially, a titration experiment should be performed. 12. Incubation times should be adjusted according to the specific experimental settings; in most cases 24–48 h would be ideal. During incubation, it is imperative to avoid any disturbances of the sedimented cells to obtain distinct and well-demarcated spots. 13. The concentrations of CFSE to be used depend on the duration of analysis/stimulation. For experiments examining division up to a week (<7 days), use 5–10 μM for staining 2×107 cells/mL or less. Duration of stimulation varies, but in most cases, 3–7 days would be a good starting point. 14. CFSE is detected in the Fl-1 channel (FITC). There is a substantial overlap of fluorescein emission into the PEchannel, especially when CFSE staining is bright. This can make compensation of CFSE challenging and indeed very tricky. Although it can be done, avoid using the PE-channel, if possible. Make sure to have an unstimulated control for determining the undivided peak and to compensate for CFSE-bleeding into adjacent channels. The unstimulated control must be incubated for the same duration as the stimulated cells. Also include non-CFSElabelled cells to determine the degree of autofluorescence of divided cells, as this will aid in determining the number of divisions resolvable. 15. Incubating 1 h before addition of BFA works well when stimulating with both protein and peptide antigens. However, stimulation time needs to be established according to the specific experimental settings. 16. The use of a thermostat heatblock is dispensable, but eases the workflow greatly.
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Acknowledgements Linda Christensen and Maria Nørtoft Sørensen are acknowledged for their excellent technical assistance. We are indebted to all colleagues, present and former, at the Department of Infectious Disease Immunology for input to the described protocols. References 1. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M., Hoffmann, J. A. (1996) The dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973–983. 2. Medzhitov, R., PrestonHurlburt, P., Janeway, C. A. (1997) A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, 394–397. 3. Hargreaves, D. C., Medzhitov, R. (2005) Innate sensors of microbial infection. J Clin Immunol 25, 503–510. 4. Garcon, N., Chomez, P., Van Mechelen, M. (2007) GlaxoSmithKline Adjuvant systems in vaccines: concepts, achievements and perspectives. Expert Rev Vaccines 6, 723–739. 5. Mosca, F., Tritto, E., Muzzi, A., Monaci, E., Bagnoli, F., Iavarone, C., et al. (2008) Molecular and cellular signatures of human vaccine adjuvants. Proc Natl Acad Sci USA 105, 10501–10506. 6. Seubert, A., Monaci, E., Pizza, M., O’Hagan, D. T., Wack, A. (2008) The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells. J Immunol 180, 5402–5412. 7. Schellack, C., Prinz, K., Egyed, A., Fritz, J. H., Wittmann, B., Ginzler, M., et al. (2006) IC31, a novel adjuvant signaling via TLR9, induces potent cellular and humoral immune responses. Vaccine 24, 5461–5472. 8. Hornung, V., Bauernfeind, F., Halle, A., Samstad, E. O., Kono, H., Rock, K. L., et al. (2008) Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 9, 847–856.
9. Korsholm, K. S., Agger, E. M., Foged, C., Christensen, D., Dietrich, J., Andersen, C. S., et al. (2007) The adjuvant mechanism of cationic dimethyldioctadecylammonium liposomes. Immunology 121, 216–226. 10. Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A., Coffman, R. L. (1986) Two types of murine helper T cell clone. 1. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 136, 2348–2357. 11. Harrington, L. E., Hatton, R. D., Mangan, P. R., Turner, H., Murphy, T. L., Murphy, K. M., et al. (2005) Interleukin 17producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 6, 1123–1132. 12. Lyons, A. B., Parish, C. R. (1994) Determination of lymphocyte division by flow-cytometry. J Immunol Methods 171, 131–137. 13. Seder, R. A., Darrah, P. A., Roederer, M. (2008) T-cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol 8, 247–258. 14. Gauduin, M. C., Kaur, A., Ahmad, S., Yilma, T., Lifson, J. D., Johnson, R. P. (2004) Optimization of intracellular cytokine staining for the quantitation of antigen-specific CD4+ T cell responses in rhesus macaques. J Immunol Methods 288, 61–79. 15. Horton, H., Thomas, E. P., Stucky, J. A., Frank, I., Moodie, Z., Huang, Y. D., et al. (2007) Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. J Immunol Methods 323, 39–54.
Chapter 16 Quantitative Multiparameter Assays to Measure the Effect of Adjuvants on Human Antigen-Specific CD8 T-Cell Responses Laurent Derré∗ , Camilla Jandus∗ , Petra Baumgaertner, Vilmos Posevitz, Estelle Devêvre, Pedro Romero, and Daniel E. Speiser Abstract Large numbers and functionally competent T cells are required to protect from diseases for which antibody-based vaccines have consistently failed (1), which is the case for many chronic viral infections and solid tumors. Therefore, therapeutic vaccines aim at the induction of strong antigen-specific T-cell responses. Novel adjuvants have considerably improved the capacity of synthetic vaccines to activate T cells, but more research is necessary to identify optimal compositions of potent vaccine formulations. Consequently, there is a great need to develop accurate methods for the efficient identification of antigenspecific T cells and the assessment of their functional characteristics directly ex vivo. In this regard, hundreds of clinical vaccination trials have been implemented during the last 15 years, and monitoring techniques become more and more standardized. Key words: direct ex vivo monitoring, CD8 T lymphocytes, staining, cytokines, proliferation, adjuvants.
1. Introduction Considerable efforts have been directed during the last years toward the development of new, defined antigen-based cancer vaccines (2, 3). A T-cell vaccine is composed of at least three major components, i.e., antigen(s), adjuvant(s), and a delivery system. Vaccine optimization requires careful step-by-step development, whereby each component needs to be investigated with respect to its capacity to induce protective immune responses (4). ∗ These
authors contributed equally to this work.
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Antigens need to recruit high-avidity T cells capable of efficient recognition of antigen-bearing cells. Adjuvants are required to activate dendritic cells that optimally promote and sustain T-cell activation. Finally, delivery systems may be optimized such that they provide the vaccine components in a targeted fashion and during prolonged time to lymphoid tissues where immune responses are generated. Induction of specific antitumor T-cell responses has been observed and monitored using several techniques (5). However, one of the current major limitations for a valid measurement of naturally acquired as well as vaccineinduced tumor antigen-specific T-cell responses remains the difficulty of ex vivo detection of specific T cells using accurate and reproducible methods. In this regard, one major breakthrough has been the development of fluorescent peptide-MHC-I multimers (6). This technique allows direct identification, enumeration, and phenotyping, as well as isolation of antigen-specific T cells. Additional approaches can be applied for the direct ex vivo assessment of effector functions of antigen-specific T cells and include the use of ELISPOT assays for enumeration of cytokine-secreting cells (7) or intracellular cytokine staining of cell suspensions (8). The latter further enables the direct visualization and quantification of single cytokine+ cells, with the concomitant phenotyping in multiparameter, multicolor flow cytometry format. Our current monitoring strategy is therefore based on the use of a combination of standardized monitoring methods consisting in the direct ex vivo quantification of specific T cells together with the assessment of their functionality, in order to gather a comprehensive picture of the specific T-cell response in the patients, avoiding the bias introduced by the in vitro cell expansion (9, 10). In this regard, the initial handling of patients’ samples, both peripheral blood as well as tissue material, is of paramount importance to the accuracy and reproducibility of quantitative and direct T-cell assays (11). Thus, only high-quality processing and preservation of the sampled material will enable reproducible analyses and will allow to draw conclusions on the efficacy of newly developed cancer vaccines. This chapter represents a detailed description of materials, methods, and notes of laboratory techniques, with the aim to promote common procedures leading to standardization of assays, ultimately allowing wide application and improved understanding of the role of CD8 T cells in infection, cancer, autoimmune disease, and transplantation. Methods for the isolation and cryopreservation of human CD8 T cells from peripheral blood as well as from lymph nodes and tumor tissues are described first. Subsequently, we show ex vivo multimer and intracellular cytokine staining. Finally, we discuss limiting dilution analysis, ELISPOT assay, and carboxyfluorescein succinimidyl ester proliferation assays.
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2. Materials 2.1. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Blood
1. Heparinized human blood. 2. 50 mL tubes (Falcon, Becton Dickinson). 3. LymphoprepTM (Axis-Shield PoC AS), store at 4◦ C. 4. PBS store at room temperature. 5. RPMI 1640+GlutamaxTM -I medium (Gibco), store at 4◦ C. 6. 10 and 2 mL sterile pipettes (Falcon, Becton Dickinson).
2.2. Isolation of Human Lymphocytes from Tumor-Infiltrated Lymph Nodes (TILNs) or Primary Tumor Material (TILs)
1. Pair of sterile scissors. 2. Petri dishes. 3. Autoclavable metallic “tea strainer.” 4. Ethanol 70% in water. 5. 50 mL tubes (Falcon, Becton Dickinson). 6. 50 mL sterile syringe. 7. 10 mL sterile pipette. 8. CTL culture medium: RPMI 1640+GlutamaxTM -I supplemented with 8% human serum (recommended is the use of pooled A+ healthy donors’ serum), 1% penicillin/streptomycin (Amimed), L-glutamine (1.5 mM, Gibco), L-asparagine (0.24 mM, Sigma,), L-arginine (0.55 mM, Sigma), 1% HEPES buffer (Amimed), 1% sodium pyruvate (100 mM, Gibco), and 0.1% 2-mercaptoethanol (5 × 10–2 M 100 mL, Sigma-Aldrich). Store at 4◦ C. 9. Tumor cell culture medium: RPMI 1640+GlutamaxTM -I supplemented with 10% FCS, 1% penicillin/streptomycin (Amimed), 1% L-glutamine (200 mM, Amimed), 1% HEPES buffer (1, Amimed), 1% nonessential amino acids (Gibco), 1% sodium pyruvate (100 mM, Gibco). Store at 4◦ C. R , Roche) and 10. Cytokines: 150 UI/mL of IL-2 (Proleukine 10 ng/mL of IL-7 (R&D). Store stock dilutions at –20◦ C.
11. 6-well plates (Corning Incorporated, Costar). 2.3. Cryopreservation of Human Cells
1. Round-bottom 1.8 mL Nunc CryoTubesTM (Nunc). 2. Freezing solution: pure FCS supplemented with 20% dimethyl sulfoxide (DMSO). Make fresh as required. 3. RPMI 1640+GlutamaxTM -I medium (Gibco), store at 4◦ C.
2.4. Thawing of Human Cells
1. Water bath at 37◦ C. 2. RPMI 1640+GlutamaxTM -I medium (Gibco), store at 4◦ C.
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3. DNAse I (Sigma) reconstituted at 10 mg/mL in buffer according to manufacturer’s instructions. Store at –20◦ C. Prepare aliquots at 1 mg/mL by diluting the stock in RPMI 1640 medium. Store aliquots at 4◦ C. 4. 15 mL tubes (Sarstedt). 2.5. Ex Vivo Multimer Staining and Phenotyping of Human PBMCs or TILNs and Limiting Dilution Analysis (LDA)
1. Staining buffer: PBS containing 0.2% BSA (Merck Fraction V for biochemical use) and 5 mM EDTA. Store at 4◦ C. 2. CD8 microbeads (Miltenyi Biotec). Store at 4◦ C. 3. MiniMACSTM MS columns (Miltenyi Biotec). 4. MiniMACSTM separation unit (Miltenyi Biotec). 5. 15 mL tubes (Sarstedt). 6. 4 mL V-bottom tubes (Greiner). 7. 4 mL U-bottom tubes (Falcon). 8. HLA-A2/peptide multimer-PE (internal production facility). Avoid exposure to light, store at 4◦ C in humid chamber. 9. PBS. 10. Monoclonal antibodies: our standard format successfully used for ex vivo monitoring of tumor antigen-specific CD8 T cells consists in the use of CD8-PercP (Becton Dickinson), CCR7-PECy7 (Becton Dickinson), CD45RAECD (Beckman Coulter), CD28-APC (Becton Dickinson), CD27-APC-Alexa780 (ebioscience), CD127-Pacific Blue (ebioscience), PD1– FITC (Becton Dickinson). Avoid exposure to light, store at 4◦ C. R Fixable Aqua Dead Cell Stain Kit (Invit11. LIVE/DEAD rogen).
12. RPMI 1640+GlutamaxTM -I medium (Gibco), store at 4◦ C. R , Roche). 13. Cytokine: 150 UI/mL of IL-2 (Proleukine ◦ Store stock dilutions at 4 C.
14. Synthetic peptides of the specificity of interest (internal production facility). Store stock dilutions at –20◦ C. 15. CTL medium (see Section 2.2). 16. Round-bottom 96-well plates (Sarstedt). 2.6. ELISPOT Assay for Detection of IFN-γ-Producing Human CD8 T Cells
1. CTL culture medium (see Section 2.2). 2. 24-well plates (Corning Incorporated, Costar). 3. IFN-γ ELISPOT PVDF—Enzymatic kit (Diaclone) containing antihuman-IFN-γ capture and detection antibodies, streptavidin alkaline phosphatase conjugate, and readyto-use BCIP/NBT substrate.
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4. Microtiter plates MultiscreenTM -HA (Millipore). 5. PBS containing 5% FCS. 6. 15 mL tubes (Sarstedt). 7. Phytohemagglutinin-L (PHA) (Remel). Store stock dilutions at –20◦ C. 8. Synthetic peptides of the specificity of interest (internal production facility). Store stock dilutions at –20◦ C. 9. Tween-20. Store at room temperature. R 5000 (BioSys GmbH, 10. Automatic plate reader: Bioreader Karben-Frankfurt Germany).
2.7. Intracellular Cytokine Staining
1. CTL culture medium (see Section 2.2). 2. 24-well plates (Corning Incorporated, Costar). 3. Synthetic peptides of the specificity of interest (internal production facility). Store stock dilutions at –20◦ C. 4. Phorbol 12-myristate 13-acetate (PMA) (Sigma), ionomycin (Sigma). Store stock dilutions at –20◦ C. 5. Brefeldin A (Sigma). Store stock dilutions at –20◦ C. 6. 4 mL V-bottom tubes (Greiner). 7. Staining buffer: PBS containing 0.2% BSA (Merck Fraction V for biochemical use), and 5 mM EDTA (Gibco). Store at 4◦ C. 8. Fixing buffer: PBS supplemented with 1% formaldehyde (Fluka), 2% glucose (Fluka), and 5 nM sodium azide (Merck). Store at 4◦ C. 9. Saponin (Sigma). Store stock dilutions at –20◦ C. 10. 4 mL U-bottom tubes (Falcon). 11. Monoclonal antibodies: INFγ-PECy7 (Becton Dickinson), TNFα-Alexa700 (Becton Dickinson), IL-2-APC (Becton Dickinson), CD107a-FITC (Becton Dickinson).
2.8. Carboxyfluorescein Succinimidyl Ester (CFSE) Proliferation Assay
1. CFSE (Molecular Probes/Invitrogen). Store stock dilution at –80◦ C. 2. Labeling buffer: PBS containing 2% FCS. Store at 4◦ C. 3. Washing buffer: PBS containing 5% FCS. Store at 4◦ C. 4. 24-well plates (Costar). 5. CTL medium (see Section 2.2). 6. Synthetic peptides of the specificity of interest (internal production facility). Store stock dilutions at –20◦ C. R , Roche). Store stock dilu7. 150 UI/mL of IL-2 (Proleukine ◦ tions at 4 C.
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3. Methods 3.1. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Blood
1. Dilute freshly collected heparinized human peripheral blood in 1:1 or 1:2 ratio with PBS or RPMI 1640+GlutamaxTM -I medium in 50 mL tubes (see Note 1). 2. Distribute, using a 10 mL sterile pipette, 15 mL of LymphoprepTM (at room temperature) in 50 mL tubes and carefully overlay 35 mL of the diluted blood on the LymphoprepTM layer, creating a sharp blood– LymphoprepTM interface (see Note 2). Cap the tube in order to prevent accidental contamination. 3. Centrifuge the tube at 800 × g for 20 min without brake, in a swing-out rotor, at room temperature (see Note 3). Centrifugation will result in sedimentation of erythrocytes and polymorphonuclear leukocytes to the bottom of the tube; thus, peripheral blood mononuclear cells will form a distinct white layer at the LymphoprepTM /medium interface, with the appearance of an opalescent ring. 4. Carefully harvest the PBMC layer, together with about half of the LymphoprepTM solution laying directly below the cells, using a 2 mL sterile pipette. Transfer the cells into a new 50 mL tube. Fill up the tube with PBS or RPMI 1640+GlutamaxTM -I, mix well, and centrifuge for 5 min, at 600 × g, with break, at room temperature. 5. Discard the supernatant, add PBS or RPMI 1640+GlutamaxTM -I, then centrifuge the PBMCs at 200 × g for 10 min at room temperature without brake (see Note 4). 6. Wash the cells once more in PBS 1640+GlutamaxTM -I (5 min at 600 × g).
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7. Resuspend the cell pellet in RPMI 1640+GlutamaxTM -I and count them before use. 3.2. Isolation of Human Lymphocytes from Tumor-Infiltrated Lymph Nodes (TILNs) or Primary Tumor Material (TILs)
1. Put 3 mL of RPMI 1640+GlutamaxTM -I in a Petri dish together with the TILNs or the tumor sample (see Note 1). 2. Cut the tissue sample into small pieces using sterile scissors (see Note 5). 3. Transfer into a sterile strainer and grind with a syringe plunger above the Petri dish (see Note 6). Rinse the strainer with additional 5 mL of RPMI 1640+GlutamaxTM -I. 4. Collect with a sterile 10 mL pipette the dissociated tissue together with the medium from the Petri dish, into a 50 mL tube. Centrifuge for 5 min at 600 × g.
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5. Discard the supernatant and resuspend the pellet in 10–20 mL of RPMI 1640+GlutamaxTM -I. Mix gently using a 10 mL pipette and count both the lymphocytes and the tumor cells. 6. Put part of the cells in culture, in 6-well plates (1–2 × 106 cells/2 mL/well), either with tumor cell culture medium in order to derive a tumor cell line (see Note 7) or with CTL culture medium supplemented with IL-2 (150 UI/mL) and IL-7 (10 ng/mL) in order to derive TILNs lines (see Note 8). Place the plates in an incubator at 37◦ C, with 5% CO2 and add fresh medium every 3–4 days. 7. Freeze remaining cells. 3.3. Cryopreservation of Human Cells
1. Prepare the freezing solution and put it on ice. 2. Label cryotubes and put them on ice. 3. Count cells, wash them with pure medium, and resuspend them in 500 μL of ice-cold culture medium per vial to be frozen. Distribute 500 μL of cell suspension into cryotubes on ice (see Note 9). 4. Add 500 μL of ice-cold freezing solution dropwise on the cell suspension in each vial. Final DMSO concentration will be 10% per vial and final total volume will be of 1 mL (see Note 10). 5. Close the cryotubes and mix the suspension gently. 6. Immediately transfer the vials into –80◦ C precooled polystyrene box (see Note 11). 7. Cryotubes can be transferred to liquid nitrogen 24 h after freezing at the earliest.
3.4. Thawing of Human Cells
1. Heat water bath at 37◦ C and keep RPMI 1640+GlutamaxTM -I medium at room temperature. 2. Distribute 10 mL of culture medium into 15 mL tubes, add 100 μL of DNAse I to give a final concentration of 10 μg/mL. 3. Thaw frozen cell vials (no more than two frozen cell vials at a time) in the water bath until a small piece of frozen medium is still left. 4. Immediately transfer the cells dropwise into the tubes containing the RPMI 1640+GlutamaxTM -I medium and the DNAse I. It is critical to dispense the cell suspension dropwise as the changes in osmotic pressure may be important and only a gradual adjustment may preserve cell viability and ensure high recovery after thawing. Centrifuge for 5 min at 600 × g.
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5. Resuspend the cell pellet in RPMI 1640+GlutamaxTM -I medium and count the cells using trypan blue. 3.5. Ex Vivo Multimer Staining and Phenotyping of Human PBMCs and TILNs and Limiting Dilution Analysis (LDA)
3.5.1. Ex Vivo Multimer Staining and Phenotyping of Human PBMCs and TILNs
The use of peptide MHC-I multimers allows direct identification and, when combined with other markers, the phenotyping of single antigen-specific T cells in ex vivo samples by flow cytometry. The current limit of detection of specific cell populations using MHC-I/peptide multimers is set at 0.01%, rendering impossible the direct monitoring of populations at lower frequencies. In order to do that, a limiting dilution analysis (LDA) (12, 13) that uses multimers to count individual microcultures can be applied and gives as result an estimation of the precursor frequency of the cells of the specificity of interest. Both methods will be described hereafter. 1. Thaw, wash, and count PBMCs or TILNs following the methods explained in Section 3.4. 2. Resuspend PBMCs/TILNs (up to 10 × 106 ) in 85 μL icecold staining buffer. Add 15 μL of CD8 microbeads (see Note 12). Incubate for 20 min at 4◦ C. 3. During this incubation period, hang the magnet on its support and place the column on the magnet. Equilibrate the column with 0.5 mL of ice-cold staining buffer. 4. After the incubation period, add 5 mL of ice-cold staining buffer to the cell suspension and centrifuge for 5 min at 600 × g. 5. Resuspend the cell pellet in 0.5 mL of ice-cold staining buffer. Place a 15 mL tube under the column to collect the CD8-negative fraction. Apply the sample into the column, and let the negative fraction pass through. Then rinse the column three times with 0.5 mL ice-cold staining buffer. 6. When all the staining buffer has passed through, remove the column from the magnet and place it on a 4 mL V-bottom tube. Pipette 2 mL of ice-cold staining buffer onto the column and firmly flush out the positive fraction by using the plunger supplied with the column. 7. Count the cells from the CD8-positive fraction. Spin down and resuspend up to 106 cells/tube in 50 μL of ice-cold staining buffer containing the optimal concentration of PElabeled HLA-A2/peptide multimer (see Note 13). Sample is then incubated for 45 min at 4◦ C in the dark (see Note 14). 8. Add the appropriate amounts of antibody conjugates and incubate for 20 min at 4◦ C (see Note 15) in the dark. 9. Wash the cell with 1 mL of ice-cold PBS buffer (see Note 16). In the meantime, prepare Aqua solution according to the manufacturer’s instructions. Remove the
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supernatant and add 200 μL of Aqua solution on pelleted cells. Incubate for 30 min at 4◦ C in the dark. 10. Wash the cells with 1 mL of ice-cold PBS buffer. After removal of the supernatant, cell pellet (up to 106 cells) is resuspended in 300 μL of ice-cold staining buffer and transferred into 4 mL U-bottom tube. Samples are analyzed on a flow cytometer (LSR II, using DIVA software, Becton Dickinson, San Diego, CA). In order to have a reliable picture of the multimer-positive CD8 T-cell population, we recommend to acquire at least 3–5 × 105 total events (see Note 17) (Fig. 16.1). Multimer gate: CM
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Fig. 16.1. Representative example of Melan-AMART-1 -specific CD8 T cells monitoring from a vaccinated melanoma patient using HLA-A2/Melan-AMART-1 multimers (leftmost dot plot). Naïve, central memory, effector memory, and effector subsets are represented according to CD45RA and CCR7 expression (middle panels) from total CD8 or multimer-specific populations. CD28 expression in each subset is depicted in the right panels.
3.5.2. Limiting Dilution Analysis (LDA)
1. Purify CD8-positive cells and collect the CD8-negative fraction as described in Section 3.5.1 from Steps 1–6. 2. Count both cell fractions. 3. Pipette 100 μL of CTL culture medium into 50 wells in a round-bottom 96-well plate (five times 10 wells in each row). Add 100 μL of the CD8 T-cell suspension at a 1 × 106 cells into the first 10 wells. Proceed with a serial dilution of the CD8 T cells by pipetting 100 μL from the first 10 wells (remaining volume 100 μL, containing 50 × 103
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CD8-positive cells) into the following 10 wells in the second row, mix well by pipetting up and down five consecutive times, and so on. Finally 10 replicates of five cell doses will be obtained (50 × 103 ; 25 × 103 ; 12.5 × 103 ; 6.25 × 103 ; 3.125 × 103 cells). 4. Irradiate the CD8-negative fraction at 3,000 rad and resuspend cells at a concentration of 2 × 106 cells/mL in CTL culture medium. Pipette 50 μL of this suspension to all the 50 wells (each well gets a constant number of 1 × 105 “feeder” cells). 5. Add to each well 50 μL of CTL culture medium containing the peptide of interest at a final concentration of 2 μM. Incubate for 14 days at 37◦ C. 6. At day 2, replace 100 μL of CTL culture medium with medium containing IL-2 at a final concentration of 150 IU/mL. Change medium every 2–3 days, split the cells if necessary. 7. At day 14, each well is stained individually with multimers and CD8 antibody, according to the methods described in the Section 3.5.1 from point 7–10. 8. For each cell dose, the number of multimer-negative wells out of the 10 replicates is counted and the percentage of negative wells versus the dose of CD8 T cells is plotted in a graph. If the dots are aligned within a confidence interval of 95% the results follow a single-hit curve and the single-hit Poisson analysis can be applied, which says that 37% of negative wells correspond to the estimated precursor frequency (see Note 18) (Fig. 16.2). 3.6. ELISPOT Assay for Detection of IFN-γ-Producing Human CD8 T Cells
1. Thaw, wash, and count PBMCs following the methods explained in Section 3.4. PBMCs are then kept overnight in CTL culture medium (see Note 19) in a 24-well plate (2 × 106 cells/well, in 2 mL of CTL culture medium). 2. Anti-IFN-γ capture antibody (IFN-γ ELISPOT PVDF— Enzymatic kit) is coated on ELISPOT plates. In detail, antibody is diluted in PBS (10 μL antibody in 1 mL of PBS, for a final antibody concentration of 10 μg/mL) and 100 μL are distributed into each well. Plates are then incubated overnight at 4◦ C. 3. The capture antibody solution is removed from wells and plates are washed six times with 200 μL of PBS/well (see Note 20). 4. Antibodies’ free sites are blocked by adding 200 μL of PBS supplemented with 5% FCS into each well, followed by an incubation of 1 h at 37◦ C.
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10 replicates for each CD8 T cell dose
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Fig. 16.2. Limiting dilution analysis of antigen-specific CD8 T cells. (a) Representative diagram of a 96-well plate loaded with 10 replicates of each CD8 T-cell dose. Black dots indicate multimer positive wells (CD8+ multimer+ > 1%), defined according to multimer staining on each individual well as shown in (b). (c) Estimation of the precursor frequency of antigen-specific CD8 T cells based on the plotted fraction of multimer-negative wells (Y-axis) versus the different CD8 T-cell doses (X-axis). The dashed line represents the calculated fitting line with the 95% CI shown by the two continuous lines. Precursor frequency is then calculated using the single-hit Poisson distribution analysis, which says that the CD8 T-cell dose required for 37% of negative wells corresponds to the estimated precursor frequency.
5. During this incubation, PBMCs are collected in a 15 mL tube, counted, and washed once. Each condition (i.e., tested peptides, negative and positive controls) will be performed in triplicates and 5 × 105 PBMCs will be used per condition. PBMCs are therefore resuspended in CTL
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culture medium in order to have 1.67 × 105 cells per 180 μL of CTL culture medium (corresponding to 5 × 105 cells per triplicates). 6. After the incubation period, remove the blocking solution from the plates and add 180 μL of medium containing PBMCs into each well. 7. Peptides of interest are added to the desired wells at the final concentration of 10 μg/mL. Thus 20 μL of a stock solution at 100 μg/mL are added to the PBMCs. For the negative control, add 20 μL of CTL culture medium alone or an irrelevant peptide (at 100 μg/mL). For the positive control, add 20 μL of PHA at 10 μg/mL (final concentration 1 μg/mL). Plates are incubated at least for 20 h at 37◦ C. 8. Plates are washed three times with 200 μL/well of PBS supplemented with 0.05% of Tween-20 and three additional times with 200 μL of pure PBS per well. 9. Dilute antihuman IFN-γ detection antibody (IFN-γ ELISPOT PVDF—Enzymatic kit) by adding 10 μL of antibody into 1 mL of PBS. 100 μL of diluted antibody is thereafter distributed in each well. Plates are wrapped with aluminum paper and incubated at room temperature for 2 h. 10. Plates are washed three times with 200 μL/well of PBS 0.05% Tween-20 and then three times with 200 μL/well of pure PBS. 11. Dilute 1 μL of streptavidin alkaline phosphatase (IFN-γ ELISPOT PVDF—Enzymatic kit) into 1 mL of PBS and add 100 μL to each well. Plates are incubated for 1 h at room temperature. 12. Wash the plates three times with PBS 0.05% Tween-20 and three times with pure PBS. 13. Add 100 μL of substrate solution/well (ready-to-use BCIP/NBT substrate from IFN-γ ELISPOT PVDF— Enzymatic kit) (see Note 21). 14. Plates are incubated in the darkness at room temperature until distinct dark spots develop and entirely fill the positive control wells (around 15 min) (see Note 22). 15. Stop the color development by rinsing plates three times with tap water. 16. Remove the base of the plates and dry them overnight at room temperature or for 2 h at 40◦ C. Then count the spots with the automatic reader (Fig. 16.3a).
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(a)
(b)
Fig. 16.3. Direct ex vivo analysis of Melan-AMART-1 -specific T-cell function. (a) PBMCs from patient LAU 1015 taken after four vaccine injections were tested ex vivo in IFN-γ ELSIPOT assays without peptide (left panel), Melan-AMART-1 natural and analog peptides (respectively EAAGIGILTV and ELAGIGILTV, middle panels), and PHA (right panel). The number of spots is indicated at the bottom right of each well. For the PHA condition, since the spots are numerous and confluent, counting of spots is not accurate which is actually not necessary as long as results reflect a valid positive control. (b) Intracellular staining of PBMCs from patient LAU 1164 after 4 h of stimulation without peptide (top panels), with MelanAMART-1 analog (middle panels) or PMA/ionomycin (bottom panels). In each condition, IFN-γ, TNF-α, IL-2, and CD107a expression are represented from A2/Melan-AMART-1 multimer-positive cells.
3.7. Intracellular Cytokine Staining
1. Thaw, wash, and count PBMCs following the methods explained in Section 3.4. 2. Perform a multimer staining as described in Section 3.1 (see Note 23).
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3. Resuspend cells in culture medium in order to have 2 × 106 cells/mL. Distribute 1 mL of cell suspension/well in 24-well plates. 4. Add 1 mL of culture medium containing either specific or irrelevant peptides at 2–20 μg/mL, together with Brefeldin A at 10 μg/mL. As positive control, use PMA/ionomycin at a final concentration of 1 and 0.5 μg/mL, respectively. Incubate for 4–6 h at 37◦ C (see Note 24). 5. After incubation, collect the cells of each condition in a 4 mL V-bottom tube. Perform a multimer staining as described in Section 3.5.1, and if desired additional extracellular stainings can be performed followed by staining with Aqua solution in order to discriminate dead cells from living cells, as describe in Section 3.5.1. 6. Wash the cells with 1 mL staining buffer/tube and resuspend the pellet in 300 μL of fixing buffer. Mix gently and incubate for 20 min at room temperature, in the dark. 7. Wash with 1 mL/tube of staining buffer (see Note 25). 8. Prepare a mix of fluorescent antibody specific for cytokines, diluted in staining buffer supplemented with 0.1% of Saponin. Incubate for 30 min at room temperature in the dark (see Note 26). 9. Wash samples with 1 mL of staining buffer and resuspend them in 200–300 μL of staining buffer/tube. Transfer cells into 4 mL-U-bottom tube and analyze on a flow cytometer (Fig. 16.3b). 3.8. Carboxyfluorescein Succinimidyl Ester (CFSE) Proliferation Assay
1. Thaw, wash, and count PBMCs following the methods explained in Section 3.4. Resuspend them at a maximal concentration of 20 × 106 cells/mL in labeling buffer, at room temperature. 2. Prepare CFSE dilution in pure PBS in order to have a CFSE concentration of 2–4 μM. 3. Add the CFSE dilution on the cell suspension (1:1 v:v) (see Note 27), mix gently, and incubate for 4–5 min at room temperature. Do not exceed this labeling time. 4. Stop labeling by adding 10-fold volume of ice-cold washing buffer. Wash twice by centrifugation at 600 × g using washing buffer. 5. Count the cells and resuspend them in order to have 2 × 106 cells/mL of CTL culture medium. Distribute 1 mL/well in a 24-well plate. 6. Dilute the cognate and irrelevant peptides at 1–10 μg/mL in CTL culture medium supplemented with 150 UI
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IL-2/mL. Add 1 mL of peptide dilution on the CFSElabeled cells. Incubate at 37◦ C. 7. Measure cell proliferation by flow cytometric analysis after 3– 14 days upon stimulation (see Note 28). In addition, multimer as well as extracellular and/or intracellular staining can be performed in combination with the CFSE proliferation assay, on the day of analysis. A caveat is that profound TCR downregulation may follow 1 day after stimulation with the cognate antigen, which is antigen dose dependent. Cell surface expression of TCR is restored to the pre-stimulation levels gradually over the first week of in vitro stimulation.
4. Notes 1. Always wear gloves when handling human tissues and perform PBMCs isolation in a sterile environment, under a clean laminar flow hood. 2. High-quality separation of PBMCs after density gradient centrifugation depends upon a sharp interface between lymphocytes and the separation solution after the layering step. This step can be achieved by a slow overlay of the diluted peripheral blood onto the LymphoprepTM solution using a 10 mL pipette. The second method involves inclining the two tubes (one containing the Lymphoprep solution and the other the blood) in an upside down “V” position with the rims firmly touching each other, such that the blood can be carefully and continuously poured onto the solution along the side of the tube. Avoid any shaking of the tubes to avoid mixing of the two solutions and progressively straighten the receiving tube as it becomes filled. 3. If the blood is stored for more than 2 h, increase the centrifugation time to 30 min. 4. This centrifugation step at lower speed allows elimination of contaminating platelets. 5. All the instruments used for tissue dissociation should be sterilized either by autoclaving them or by using 70% alcohol. If the tumor tissue is hard and compact, perform a collagenase digestion. Transfer small pieces of tumor into a tube containing tumor cell culture medium with 0.1% collagenase type I (Sigma), 0.02% DNase I (Sigma) and incubate at 37◦ C. Mix regularly by inverting the tube. Depending on the sample, the digestion may take incubation times ranging from 2 h up to overnight. After few hours,
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check the digestion extent by pipetting and counting viable cells in suspension. If enough cells have been released from the tissue sample, let large undigested pieces settle down by gravity, transfer supernatant to a new tube (keep original tube with undigested pieces), and centrifuge at 600 × g for 5 min to collect the dissociated cells. Recover the supernatant (collagenase solution) and reuse it to continue digestion of large pieces in the original tube until needed. Wash the single cell suspension two times with medium and distribute into plates. 6. Obtained dissociated sample should be small enough to be aspirated using a 10 mL pipette and as close as possible to a single cell suspension. To obtain, in some cases, deep tumor infiltrating lymphocytes it is advised to set separate cultures with the remaining small tumor pieces in CTL medium supplemented with rIL-2 (150 U/mL) and rIL-7 (10 ng/mL). Sometimes, activated lymphocytes come out of tumor fragments after a few days of culture. 7. If the size of the tumor sample is too small, do not pass the teased tissue fragments through the strainer. After teasing, wash the sample directly and culture the cells in tumor cell culture medium. After 1–2 days, collect only the cells in suspension (most melanoma tumor cells adhere firmly to tissue culture plates), wash them, and put them back in culture in CTL culture medium supplemented with IL-2 (150 UI/mL) and IL-7 (10 ng/mL). Add 2 mL of tumor cell culture medium onto the remaining adherent tumor cells. 8. In order to obtain short-term cultured TILNs, avoid culturing them for more than 15–20 days. 9. DMSO is a toxic compound, therefore all the manipulation of the freezing process should be performed on ice to slow down the DMSO entrance into the cells. 10. A total of 2–30 × 106 cells can be frozen in a final volume of 1 mL/cryotubes. Total volume should be increased up to 1.5–1.8 mL, if more than 30 × 106 cells are frozen per vials. 11. Additional options for successful cell cryopreservation are either the use of the Nalgene freezing containers (Nalgene) which allow repeatable –1◦ C/min cooling rate required for successful cell recovery or the use of a controlled rate freezing device (Thermo Scientific). 12. When using more than 10 × 106 cells, scale up the staining volumes accordingly.
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13. Multimers as well as antibodies should be titrated prior to routine use in flow cytometry to determine the optimal dilution to be used for staining. In the case of multimers, it is useful to have large cryopreserved stocks of the appropriate T-cell clones or lines. The usual working concentrations for HLA-A2/peptide tetramers are, in our experience, in the range of 5–20 μg/mL. 14. Optimal incubation times and temperatures for efficient multimer staining have been determined by comparison of signals obtained using various protocols, e.g., incubation for variable times at 4◦ C, at room temperature, or at 37◦ C. In our current experience, the best compromise for a limited background signal concomitant with highly efficient specific multimer staining can be achieved either by incubation of the cells for 1 h at room temperature or for 45 min at 4◦ C (10, 14). 15. Antibodies should be kept on ice during the preparation of the mix and rapidly stored at 4◦ C after use, in order to avoid fluorochromes dissociation. Staining should be performed avoiding exposure to direct light. 16. Aqua dye employs an amine-reactive fluorescent dye to evaluate mammalian cell viability by flow cytometry. The dye reacts with free amines both in the cell interior and on the cell surface in case of dead cells, yielding intense fluorescent staining. In viable cells, the dye’s reactivity is restricted to the cell-surface proteins, resulting in less intense fluorescence. In order to avoid quenching of staining by the BSA contained in the buffer, work at this step with plain PBS. 17. Compensation settings are performed using beads: CompBeads anti-mouse or anti-rat Ig (Becton Dickinson Biosciences) and CompBeads negative control are used according to the manufacturer’s instructions. For compensation of Aqua, use some Aqua-labeled cells. 18. An important difference between the direct ex vivo multimer staining and the LDA analysis resides in the fact that the direct staining allows the quantification of a real frequency of positive cells, while the LDA can only give an estimation of the precursor frequency of specific cells. This estimation depends on the capacity of the specific cells to proliferate during the culture period; therefore an underestimation of the effective frequency in the sample is possible. 19. No IL-2 or other cytokines are added to the CTL culture medium in order to avoid unspecific growing of T cells during the overnight incubation. 20. After every washing step, residual buffer in the wells should be removed by tapping on absorbent paper.
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21. Substrate solution should be at room temperature before use, since cold substrate slows down the enzymatic reaction. 22. Ideally, spot formation should be monitored by eye. 23. In order to avoid loss of multimer signal and therefore detection of false-negative too low frequencies of specific cells due to TCR downregulation after peptide stimulation, two multimer stainings should be performed: one before and one after incubation with the peptide (15, 16). 24. Degranulation can be visualized by flow cytometry during a short period of time following lytic effector activation by detection of the lysosomal CD107a protein on the effector cell surface (17). Therefore, the CD107a mobilization assay can be used as a surrogate assay of lytic activity. Because of the transient surface expression and rapid internalization of CD107a by the endocytic pathway (18), staining for CD107a is maximized by addition of anti-CD107a antibody during cell stimulation. By virtue of their parallel kinetics, CD107a and intracellular cytokines can be assessed at the same time using 4–6 h of stimulation. 25. After fixation and washing, cells can be kept in staining buffer at 4◦ C in the dark for 1–3 days before intracellular staining. 26. Antibodies should be previously titrated in order to define the optimal dilution to use. Intracellular staining can be performed with the mix of antibodies specific for the different cytokines at the same time. Isotype-matched control antibodies should be used to enable precise electronic gating of positive and negative events during analysis of flow cytometry data. 27. Final labeling conditions are thus 1–2 μM CFSE in PBS 1% FCS and cells at 10 × 106 /mL. However, it must be kept in mind that optimal concentrations of CFSE for efficient labeling of cells may vary for different commercial batches. Thus, it is recommended to titrate new batches of CFSE as to empirically identify the optimal concentrations for labeling. 28. This protocol enables a “3.5 log shift” in fluorescent signal intensity optimally detected at 525 nm wavelength. For compensation, use some CFSE-labeled cells. In this way, only slight adjustments in the settings are needed and still allow to discriminate up to seven cell generations (19, 20).
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References 1. Woodland, D. L. (2004) Jump-starting the immune system: prime-boosting comes of age. Trends Immunol 25, 98–104. 2. Mahnke, Y. D., Speiser, D., Luescher, I. F., et al. (2005) Recent advances in tumour antigen-specific therapy: in vivo veritas. Int J Cancer 113, 173–178. 3. Andersen, M. H., Sorensen, R. B., Schrama, D., et al. (2008) Cancer treatment: the combination of vaccination with other therapies. Cancer Immunol Immunother 57, 1735–1743. 4. Romero, P., Cerottini, J. C., Speiser, D. E. (2006) The human T cell response to melanoma antigens. Adv Immunol 92, 187–224. 5. Vujanovic, L., Butterfield, L. H. (2007) Melanoma cancer vaccines and anti-tumor T cell responses. J Cell Biochem 102, 301–310. 6. Altman, J. D., Moss, P. A., Goulder, P. J., et al. (1996) Phenotypic analysis of antigenspecific T lymphocytes. Science 274, 94–96. 7. Czerkinsky, C. C., Nilsson, L. A., Nygren, H., et al. (1983) A solid-phase enzymelinked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65, 109–121. 8. Jung, T., Schauer, U., Heusser, C., et al. (1993) Detection of intracellular cytokines by flow cytometry. J Immunol Methods 159, 197–207. 9. Baumgaertner, P., Rufer, N., Devevre, E., et al. (2006) Ex vivo detectable human CD8 T-cell responses to cancer-testis antigens. Cancer Res 66, 1912–1916. 10. Romero, P., Dunbar, P. R., Valmori, D., et al. (1998) Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes. J Exp Med 188, 1641–1650. 11. Speiser, D. E., Pittet, M. J., Guillaume, P., et al. (2004) Ex vivo analysis of human antigen-specific CD8+ T-cell responses:
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quality assessment of fluorescent HLA-A2 multimer and interferon-gamma ELISPOT assays for patient immune monitoring. J Immunother 27, 298–308. Pittet, M. J., Valmori, D., Dunbar, P. R., et al. (1999) High frequencies of naive MelanA/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals. J Exp Med 190, 705–715. Taswell, C. (1981) Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. J Immunol 126, 1614–1619. Whelan, J. A., Dunbar, P. R., Price, D. A., et al. (1999) Specificity of CTL interactions with peptide-MHC class I tetrameric complexes is temperature dependent. J Immunol 163, 4342–4348. Appay, V., Nixon, D. F., Donahoe, S. M., et al. (2000) HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. J Exp Med 192, 63–75. Appay, V., Rowland-Jones, S. L. (2002) The assessment of antigen-specific CD8+ T cells through the combination of MHC class I tetramer and intracellular staining. J Immunol Methods 268, 9–19. Betts, M. R., Brenchley, J. M., Price, D. A., et al. (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281, 65–78. Fukuda, M. (1991) Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking. J Biol Chem 266, 21327–21330. Lyons, A. B., Parish, C. R. (1994) Determination of lymphocyte division by flow cytometry. J Immunol Methods 171, 131–137. Parish, C. R. (1999) Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell Biol 77, 499–508.
Chapter 17 Large-Animal Model for Establishing E/T Ratio of Adjuvants Luuk A. Th. Hilgers Abstract To develop novel adjuvants for use in humans, the efficacy/toxicity (E/T) ratio of experimental products in large animal species can be investigated. The test model included two intramuscular immunizations in pigs at 3 weeks interval and analysis of immune responses and local reactions 1 week after the second injection. The antigen used to determine adjuvant activity was a well-defined, purified, viral glycoprotein that without adjuvant induces low immune responses and no detectable local reactions. Efficacy was determined by measuring ELISA and virus-neutralizing antibody titres. Toxicity was determined by necropsy and estimating size and severity of local reactions to each treatment. The persistence of the side effects was deduced from the difference in the local reaction 4 weeks after the first and 1 week after the second injection. For graphic representation of E/T ratios, toxicity was expressed in arbitrary units and plotted against antibody titre. The graphs provided insight into dose– and structure–response relationships and enabled the stepwise optimization of adjuvant candidates. Key words: Animal model, pig, efficacy, local toxicity, reactogenicity, E/T ratio, necropsy, risk/benefit ratio.
1. Introduction Ideally, vaccines are fully effective and completely safe but in practice, products are always a compromise between these two conflicting features. The acceptance of certain risks of widespread vaccination is dictated by risks and benefits of alternative options, including no interference. As a consequence, the profile of a vaccine against a severe disease for which there is no alternative is theoretically distinct from that against a mild disease for which various alternative options exist. It has become clear that efficacy of many vaccines depends on the use of an adjuvant. G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_17, © Springer Science+Business Media, LLC 2010
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Many compounds have shown adjuvant activity but only a few are applied in marketed vaccines. Insufficient efficacy and/or safety in target species, insufficient stability and high costs of production are a few of the possible causes for not reaching the market. Although toxicity is a key factor, early research often concentrates on the beneficial effects of novel adjuvants, with little attention to possible side effects. With the trade-off between efficacy and safety, parallel analysis of both aspects of novel adjuvants at the earliest possible stage will increase the efficiency of a screening program. It provides insight into the benefit and risk balance of the test substances and facilitates the optimization of a candidate. Analysis of the performance of adjuvanted vaccine in different animal species has led to the general conclusion that small rodents overestimate the efficacy in larger animals and humans (1). Many products that demonstrated adjuvanticity in mice or other rodents failed in larger target species. This drawback is recognized widely but costs, availability, facilities, expertise and test systems are often important decision factors. More than a decade ago, we decided to use a larger species to identify and optimize novel adjuvants for human and veterinary purposes. The pig was chosen for several reasons. There were indications that the pig immune system is quite similar to that of the human but detailed proof was lacking. Although the repertoire of immunological tools for pigs was (and still is) limited, sufficient test systems were available to analyse humoral and cell-mediated immunity. Importantly, body weight is in the same order of magnitude and ∼1,000-fold higher than that of mice. As a result there is no need to adjust the vaccine dose or the injection volume. Relative to toxicity, the large body size offers the possibility to target a single muscle, which enables macroscopic detection of minimal, abnormal local reactions. In additions, there is minimal risk of interference between separate injections. The pig model presented here was applied to establish E/T ratios of different known and newly developed adjuvants. Ongoing clinical trials may confirm the usefulness of this model.
2. Materials 2.1. Immunization
1. Animals: Outbred, male or female pigs 10–20 weeks of age with body weight between 20 and 50 kg. Inclusion and exclusion criteria were established, e.g. only healthy animals not vaccinated or administered with oil-containing products were used. Animals were marked individually and assigned at random to groups of 4–6 animals. Pigs were housed
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in appropriate lab facilities at conventional conditions and monitored daily for general behaviour and health. 2. Test antigen: For example, 32 μg per immunization of immunoaffinity-purified, recombinant E2 glycoprotein of classical swine fever virus produced in insect cells by using baculovirus expression vector as described elsewhere (2) (see Note 1). 3. Adjuvants: Different types of adjuvants have been tested in this model. References were Alhydrogel (Brenntag Biosector, Frederikssund, Denmark) as a mild adjuvant and waterin-mineral oil known as Specol (3) (ID-Lelystad, Lelystad, the Netherlands) as a strong one. The test adjuvants were aqueous formulations such as oil-in-water emulsions, aqueous suspensions of insoluble salts and solutions of soluble polymers. Among them were squalane-in-water emulsions supplemented with synthetic carbohydrate derivatives, as described elsewhere (1, 4, 5). They were prepared by passage of a mixture of squalane, Polysorbate 80, carbohydrate derivative (1, 5–8) and phosphate-buffered saline through a high-pressure emulsifier (Microfluidizer Y-110; Microfluidics Corp., Newton, USA) (6, 7). This standardized procedure resulted in stable emulsions with mean droplet size between 100 and 400 nm. 4. Vaccines: With aqueous adjuvants, vaccines were obtained by mixing antigen solution and adjuvant solution at appropriate volume ratios by simple agitation without special equipment. In case of oily adjuvants, the antigen solution was added dropwise to the adjuvant by continuously and vigorously mixing the oil phase with a blender, yielding a water-in-oil emulsion.
3. Methods 3.1. Immunization (see Note 2)
1. Collect blood samples of ∼10 mL on the day of the first immunization (pre-treatment 1; PT0) in tubes with and without heparin (see Note 3). 2. Inject pigs intramuscularly (IM) in the neck, 3 cm behind the left ear, or in the left upper hind leg, using a dose of 2.0 mL per injection per animal. 3. After 3 weeks (on the day of the second immunization), collect blood samples of ∼10 mL (post-treatment 1; PT1) in tubes with and without heparin (see Note 3). 4. On the same day but after PT1 blood sampling, immunize again 3 cm behind the right ear or in the right hind upper
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leg with the same dose of vaccine as used for the first immunization (see Note 4). 5. One week after the second injection, collect blood samples of ∼10 mL (post-treatment 2; PT2) in tubes with and without heparin (see Note 3). 6. On the same day but after PT2 blood sampling, euthanize animals for necropsy of the injection sites. Carry out macroscopic analysis of injections sites (see Note 5). 3.2. Determination of Efficacy
The efficacy of an adjuvant can be determined by the increase in specific antibody response against the test antigen. 1. Determine specific antibody titres in serum by ELISA or by virus neutralisation assay (see Note 6). 2. In accordance with the Beer–Lambert’s law, express ELISA antibody titres as the regression coefficient of the linear part of the plot of absorbance versus serum concentration. In our experiments, linearity was significant between absorbance values of 0.0 and 1.4 absorbance units. The titre corresponds to the factor of dilution of the serum sample giving an optical density of 1.0 absorbance unit above background. 3. Calculate virus-neutralising antibody titres as the serum dilutions giving 50% reduction in classical swine fever virus plaque formation. Express mean antibody titres of each group or treatment as geometric mean titre (GMT) ± SD and antilog value. 4. Determine cellular responses by measuring 3 H-thymidine incorporation of peripheral blood mononuclear cells after incubation with antigen (see Note 7).
3.3. Determination of Toxicity
In this model, toxicity is determined by analysing the sites of injection at necropsy (see Note 8). To facilitate comparison of different products and graphic representation of E/T ratio, toxicity was expressed as a mathematic number. The value takes into account (1) the size, (2) the severity and (3) the persistence of the local reaction. In the pig, size can be determined precisely after surgical removal of the tissue affected. Assessment and quantification of severity of the local reaction are more complicated. For this purpose, we classify the local reactions and attribute arbitrary values as described below. The local reaction of each injection site is expressed as the product of size and severity score. Finally, to include the persistence of toxicity, the score 4 weeks after the first injection (4 weeks PT1) is multiplied by a factor representing the difference in time interval between analysis and first and second treatment, and added to that of 1 week after the second treatment (1 week PT2). Per group, the geometric mean toxicity (GMX)
Large-Animal Model for Establishing E/T Ratio of Adjuvants
255
(± SD) is calculated. The method for quantification is described below in further detail. 3.3.1. Quantification of Local Reaction
1. After euthanasia of the animals, surgically remove the muscular tissues with the injection site in the centre. 2. Label and store muscle samples at 4◦ C. 3. Within 24 h, cut each tissue sample into slices of 0.5–1 cm and inspect each slice macroscopically for abnormal tissue reactions. 4. Measure length, width and depth (cm × cm × cm) of abnormal tissue with a ruler guide or a marking gauge (see Note 9). 5. Classify the observed local reactions by attributing arbitrary scores as follows: 0: no abnormal reactions visible 1: discoloration, oedema, fibrosis and/or loss of muscular structure 3: connective tissue and/or granuloma 9: necrosis, pus and/or abscess 12: vaccine residue 6. If different types of local reactions are present, express the relative size of each type as 1/6th, 2/6th, 3/6th, 4/6th or 5/6th of the total area of abnormal tissue. For example, a local reaction of 2 × 4 × 3 cm consisting of 4/6th discoloration and oedema and 2/6th connective tissue gave the following sore: (2 × 4 × 3) × ((4/6 × 1 (discoloration/oedema) + 2/6 × 3 (connective tissue)) = 40. 7. Combine the results of the first and second injections. To give extra weight to the persistence of the local reaction, multiply (arbitrarily) the score at 4 weeks PT1 by a factor of 4 and add to the score 1 week PT2, yielding a single score for the two treatments with the test substance (see Note 10). For example, a score of 40 one week PT2 and 12 four weeks PT1 results in an individual score of 88. Finally, calculate logs of individual values.
3.4. References and Controls
1. For each experiment, include a group of animals immunized with antigen alone (see Note 11). 2. Before the first immunization, collect blood samples from at least 20 animals for analysis of the immune responses prior to treatment (see Note 12).
3.5. Data Processing
1. Determine the E/T ratio of individual animals and groups (see Note 13).
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2. Calculate for each group, geometric means of antibody titres (GMT) and of log values of toxicity scores (GMX) ± standard deviations (SD) and antilog values thereof (10GMT or 10GMX ). 3. Make a graphic representation of efficacy (10GMT ) versus toxicity (10GMX ) ± SD.
4. Notes 1. This glycoprotein elicits virus-neutralising antibodies against classical swine fever virus (8). The dose used per immunization was 32 μg. Without adjuvant, immune responses were low and local reactions were absent. 2. A pig model was developed for estimating E/T ratios of newly developed adjuvants for human vaccines. To illustrate this model, E/T ratios of an arbitrary selection of distinct types of adjuvants are depicted in Fig. 17.1. Each square represents a group of pigs immunized with a test substance. Efficacy ranged from 10 to > 10,000 and toxicity score from 5 to 5,000. The graph demonstrates that products are clustered around an imaginary diagonal, that the test system is capable of detecting a wide range of E/T ratios and that there are different products with clearly distinct E/T ratios. Validation of an animal method is complex and in this case is limited to the analysis of
Toxicity Fig. 17.1. E/T ratio of distinct aqueous adjuvants including insoluble salts, water-soluble polymers and oil-in-water emulsions.
Large-Animal Model for Establishing E/T Ratio of Adjuvants
257
dose–response and structure–response relationships. With some of the aqueous adjuvants tested, significant effect of dose on efficacy and toxicity could be noted and E/T plots demonstrated linear and nonlinear relationships. Analysis of structure–response relationship of adjuvant formulations varying in only one respect revealed distinct E/T ratios, indicating that modification of a single component of the formulation had a stronger effect on efficacy or on toxicity. Further validation might include analysis of products with known performance in humans. Finally, the pig was selected in the assumption that they better predict adjuvant performance in humans than do mice or other small laboratory animal species. Although there is still no clear-cut evidence for this hypothesis, the larger body size offered several important advantages including the possibility for detailed analysis of local reactions. This was crucial to quantify toxicity, to determine E/T ratio and to identify and optimize adjuvant candidates. 3. Blood samples were collected in tubes without and with heparin for the measurement of antibody titres and lymphocyte proliferation, respectively. 4. Injection in the hind upper leg offered the advantage that a single muscle with homogenous structure was targeted, which facilitated macroscopic detection of the least detectable abnormal tissue reaction. 5. Observations can be confirmed if required by microscopic histochemistry of dissected tissue. 6. ELISA is described by Blom and Hilgers (1) and virus neutralization assay is described by Terpstra et al. (9) see also Chapter 13 of this volume. 7. See Ref. (1) for details. The test system, however, was not capable of discriminating between adjuvants that caused moderate and strong antibody responses. 8. In humans, vaccine safety is assessed by monitoring local and systemic adverse events such as erythema, induration, swelling, ecchymosis, local pain, fatigue or myalgia, malaise, shivering, sweating, arthralgia, nausea or vomiting, diarrhoea and temperature (10). Some of them are graded as, e.g. mild, moderate, severe and life threatening. With a few exceptions, these symptoms are rare or difficult to monitor in pigs. 9. The precision in our experiments was about 10% for values less than 5 and 0.5 cm for values greater than 5 cm. 10. The factor 4 represents the difference in time intervals between necropsy and first and second treatment.
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11. A group of untreated controls can also be included. 12. The various independent studies revealed the following general data. The detection limits of the test systems for efficacy and toxicity were low. Antibody responses before immunization and in control animals that received no antigen were below detection limit. Two injections with antigen and without adjuvant revealed GMTs between 1.0 and 1.3. Two injections with antigen plus an oily adjuvant (water-in-mineral oil emulsion) gave high GMTs > 4.0 (antilog > 10,000). Variance (% SD/GMT) decreased with increased GMT and was between 10 and 30% in groups with low antibody titres and less than 10% in groups with high titres. Relative to toxicity, no significant local reactions to phosphate-buffered saline or antigen without adjuvant were noted except in a few individuals, which displayed discoloration and loss of structure of < 1 cm3 1 week PT2, resulting in toxicity score of <1. Alhydrogel and oily emulsion applied as references gave toxicity values of between 0 and 40 and > 20,000, respectively. Oily emulsions manifested necrosis, abscess, fibrosis, granulomatous and purulent inflammation, and vaccine residues with little difference between 1 week PT2 and 4 weeks PT1. The negative and positive controls indicated a large window for the analysis of both efficacy and toxicity of adjuvants. 13. A typical study included between 10 and 25 groups of five animals per group. As animals were marked individually, E/T ratio of individual animals and groups could be determined. References 1. Mestas, J., Hughes, C. W. (2004) Of mice and not men: differences between mouse and human immunology. J Immunol 172, 2731–2738. 2. Hulst, M. M., Westra, D. F., Wensvoort, G., Moormann, R. J. M. (1993) Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera. J Virol 67, 6479–6486. 3. Bokhout, B. A., Bianchi, A. T., van der Heijden, P. J., Scholten, J. W., Stok, W. (1986) The influence of a water-in-oil emulsion on humoral immunity. Comp Immunol Microbiol Infect Dis 9, 161–168. 4. Hilgers, L. A., Lejeune, G., Nicolas, I., Fochesato, M., Boon, B. (1999) Sulfolipocyclodextrin in squalane-in-water as a novel and safe vaccine adjuvant. Vaccine 17, 219–228. 5. Blom, A. G., Hilgers, L. A. T. (2004) Sucrose fatty acid sulphate esters as novel vaccine
adjuvants: effect of the chemical composition. Vaccine 23, 743–754. 6. Hilgers, L. A. T., Platenburg, P. L. I., Luitjens, A., Groenveld, B., Dazelle, T., FerrariLaloux, M., Weststrate, M. W. (1994) A novel non-mineral oil-based adjuvant. I. Efficacy of a synthetic sulfolipopolysaccharide in squalane-in-water emulsion in laboratory animals. Vaccine 12, 653–660. 7. Hilgers, L. A. T., Platenburg, P. L. I., Luitjens, A., Groenveld, B., Dazelle, T., Weststrate, M. W. (1994) A novel non-mineral oil-based adjuvant. II. Efficacy of a synthetic sulfolipopolysaccharide in squalane-in-water emulsion in pigs. Vaccine 12, 661–665. 8. Bouma, A., de Smit, A. J., de Kluiver, E. P., Terpstra, C., Moormann, R. J. M. (1999) Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus. Vet Microbiol 66, 101–114.
Large-Animal Model for Establishing E/T Ratio of Adjuvants 9. Terpstra, C., Bloemraad, M., Gielkens, A. L. J. (1984) The neutralizing peroxidase-linked assay for detection of antibody against swine fever. Vet Microbial 16, 123–128. 10. US Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research.
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(2009). Guidance for Industry: toxicity grading scale for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials (Draft guidance 2005). Available at http://www.fda.gov/cber/gdlns/toxvac.htm [accessed January 2009].
Chapter 18 Determining the Activity of Mucosal Adjuvants Barbara C. Baudner and Giuseppe Del Giudice Abstract Mucosal vaccination offers the advantage of blocking pathogens at the portal of entry, improving patient’s compliance, facilitating vaccine delivery, and decreasing the risk of unwanted spread of infectious agents via contaminated syringes. Recent advances in vaccinology have created an array of vaccine constructs that can be delivered to mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts using intranasal, oral, and vaginal routes. Due to the different characteristics of mucosal immune response, as compared with systemic response, mucosal immunization requires particular methods of antigen presentation. Welltolerated adjuvants that enhance the efficacy of such vaccines will play an important role in mucosal immunization. Among promising mucosal adjuvants, mutants of cholera toxin and the closely related heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli present powerful tools, augmenting the local and systemic serum antibody response to co-administered antigens. In this chapter, we describe the formulation and application of vaccines using the genetically modified LTK63 mutant as a prototype of the family of these mucosal adjuvants and the tools to determine its activity in the mouse model. Key words: Mucosal vaccination, mucosal adjuvants, LT mutants, ELISA, antibody response, T-cell response, ELISPOT.
1. Introduction One of the most notable benefits of mucosal immunization is the fact that both systemic and mucosal immunity are triggered, which is particularly advantageous in the case of vaccination against diseases caused by mucosal pathogens. The vast majority of infections occur, or start from, mucosal surfaces, which leads to the hypothesis that protective immunization against these pathogens needs a successful mucosal immune response. G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_18, © Springer Science+Business Media, LLC 2010
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Therefore, the mucosal route appears to be the most appropriate means for immunization. However, most clinically relevant vaccine candidates show weak immunogenicity when delivered mucosally and poor transport characteristics across biological barriers. This implies the need for adjuvants to potentiate their protective immune response or to improve their presentation and targeting. Mucosal adjuvants are components which are co-administered with a vaccine to enhance the immunogenicity of vaccine antigens (1). A better understanding of mucosal immunity, cellular immunology, and molecular biology during the past decades and novel technologies led to the discovery of new adjuvants (2, 3). Some of the more promising adjuvants include microspheres, immune-stimulating complexes (ISCOMS), liposomes, CpG DNA, cytokines, monophosphoryl lipid A, virus-like particles, and modified bacterial toxins (1). Among bacterial toxins, heat-labile enterotoxin (LT) from Escherichia coli and cholera toxin (CT) from Vibrio cholerae are known to be potent mucosal immunogens and have shown to serve as excellent adjuvants for co-administered antigens (4–6). LT and CT belong to the family of adenosine 5 -diphosphate (ADP)-ribosylating bacterial toxins and have an A–B structure (7, 8). The A subunit is enzymatically active and is responsible for increased intracellular accumulation of cyclic adenosine monophosphate (cAMP), thought to be responsible for the toxicity of both LT and CT. The pentameric B subunit of LT and CT binds to cell membrane surface receptors by its receptorbinding site, through interaction mainly with GM1 (9–11). This allows the transfer of the A subunit to the cytoplasm of the cell. Consequently, both LT and CT are toxic in their native state, and several mutants have been generated with reduced toxicity while maintaining the adjuvanticity of these molecules (12, 13) and are able to enhance local IgA, systemic IgG, and cellular immune responses to co-administered vaccine antigens after both oral and nasal immunization (13–19). The LTK63 and LTR72 mutants have been successfully used to immunize a variety of animal species with various vaccine antigens. These mucosal vaccine formulations have proven efficacious each time challenge with infectious pathogen or lethal toxin was feasible. Table 18.1 gives a summary of the available studies with relevant references (17, 19–36). More recently, LTK63 has been proven to exert its mucosal adjuvanticity in human volunteers after intranasal immunization with influenza vaccine (37). This chapter will focus on the use of LTK63 mutant as a prototype of this family of mucosal adjuvants. Its activity/adjuvanticity for the induction of systemic and mucosal antibodies, and increased T-cell and B-cell activity against co-administered vaccine antigens will be evaluated.
PO
PO
IN
IN
H. pylori urease
H. pylori NAP
Myc. Tub. Ags
Hib conjugates
IN
IN
SC
Measles antigens
HIV-1 gag
HIV-1 gag
IN
PO
H. pylori VacA
Pertussis antigens
PO
H. pylori CagA
IN
IN
H. pylori CagA
IN/SC
TC
Diphtheria toxoid
Pneumo conjugates
IN
Tetanus fragment C
MenC conjugates
Route
Antigen
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Guinea pig/mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Species
+
ND
ND
+
+
+
+
+
+
+
+
+
+
+
+
Serum
ND
ND
ND
+
+
+
+
+
ND
ND
+
+
+
ND
+
Mucosal
Antibody response
ND
ND
ND
+
+
ND
ND
ND
ND
ND
ND
ND
+
ND
ND
CD4+
T-cell response
+
+
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
CD8+
ND
ND
ND
Yes
Yes
ND
ND
Yes
Yes
Yes
Yes
Yes
Yes
ND
Yes
Protection
Table 18.1 LTK63 induces strong, protective immune responses in various animal models following various mucosal routes
(continued)
(27)
(27)
(26)
(17)
(19, 25)
(24)
(21)
(23)
(22)
(22)
(22)
(22)
(22)
(21)
(20)
References
Determining the Activity of Mucosal Adjuvants 263
IN
PO
IM
IN
IN
IN
Flu HA
Flu HA
Flu HA
Toxoplasma gondii antigens
Malaria antigens
Ricin A
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Goat
Moue
Mouse
Species
+
+
+
+
+
+
+
+
ND
Serum
+
+
+
-
+
+
+
+
ND
Mucosal
Antibody response
+
+
+
ND
ND
ND
ND
ND
ND
CD4+
T-cell response
Notes: IN, intranasal; PO, per os; IM, intramuscular; SC, subcutaneous; TC, transcutaneous; ND, not determined.
IN
Vaginal
Goat HSV-1
IN
RSV M2 peptide
HSV-2 gD2
Route
Antigen
Table 18.1 (continued)
ND
+
ND
ND
ND
ND
ND
ND
+
CD8+
Yes
Yes
Yes
ND
ND
Yes
Yes
ND
Yes
(36)
(35)
(34)
unpublished data
(33)
(32)
(31)
(30)
(28, 29)
Protection References
264 Baudner and Giudice
Determining the Activity of Mucosal Adjuvants
265
The mutant LTK63 is generated by site-directed mutagenesis of single-stranded DNA by classic techniques using defined oligonucleotides (38) and procedures have been extensively reported in laboratory textbooks (12, 39, 40); furthermore other mutants such as the LTR72 which contains some residual enzymatic activity have been generated (13). LTK63 is the result of a substitution of serine 63 in the A subunit with a lysine which dramatically reduces its enzymatic activity (14–17). LTK63 has been shown to be a potent mucosal adjuvant for inducing CTL with a co-administered peptide immunogen (14). The residual toxicity of LT mutants can be very easily assessed in vitro and in vivo. LT exhibits toxicity on Y1 adrenal cells, which undergo rounding when cultured as a monolayer in the presence of these toxins (41). Furthermore the residual ADPribosyltransferase activity can be addressed biochemically in vitro using appropriate substrates (42). The in vivo toxicity of these
Fig. 18.1. Schematic representation of determining the activity of mucosal adjuvants.
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molecules can be tested in ligated ileal loops in rabbits, where, if toxic, they induce accumulation of fluid that increases with the amount of toxin inoculated (43). For the in vivo evaluation of the mucosal adjuvanticity of LTK63 vaccine, antigens will be formulated with and without LTK63 and given by various mucosal routes (intranasal, intragastric, vaginal, or rectal) using a mouse model. Mice are immunized according to standard schedules and mucosal washes and tissues as well as serum samples and splenocytes are harvested and prepared for stimulation (Fig. 18.1). Methods for assaying adjuvant- and antigen-specific antibody titers by ELISA, cell-mediated immunity by T-cell proliferation, and the frequency of antibody-secreting cells by ELISPOT will be described (Fig. 18.1).
2. Materials 2.1. Preparation of Immunogen/ Adjuvant Vaccine Formulations
1. Immunogen, for example, an existing or an experimental vaccine 2. Adjuvant: LTK63 or other LT mutants (Novartis vaccines) 3. Phosphate-buffered saline (PBS) 4. Sodium bicarbonate
2.2. Immunization of Animals
1. Female, 6- to 8-week-old inbred BALB/c mice (Charles River) or other strain of mice as appropriate based on the vaccine model 2. Vaccine formulations (see Sections 2.1 and 3.1) 3. Gavage needles for intragastric immunizations (Luer-Lock stainless-steel, 50-mm × 1-mm gavage with a round tip) 4. Sodium bicarbonate 5. Sterile saline 6. Syringe (<23-gauge needle) for intramuscular immunizations (controls)
2.3. Serum and Mucosal Sample Collection and Storage
1. Phosphate-buffered saline (PBS). 2. Bovine serum albumin (BSA). 3. Phenylmethylsulfonyl fluoride (PMSF) (Fluka, Bucks, Switzerland). 4. Sterile surgical instruments (scissors and forceps). 5. c-RPMI medium: (Roswell Park Memorial Institute) RPMI-1640 medium, supplemented with 10% (v/v)
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heat-inactivated fetal calf serum (FCS), 100 mM Lglutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μM 2-mercaptoethanol. Sterile filtered (0.2 μm). 2.4. Assessment of Adjuvant Activity on Antibody Responses by Enzyme-Linked Immunosorbent Assay (ELISA)
1. Plate washer (for example, ELx405TM Microplate Washer; BioTek)
2.4.1. Antibody Response to Immunogen and LTK63 or Other LT Mutants
1. Ninety-six-well, polysorb flat-bottomed ELISA plates (Greiner, Kremsmunster, Austria)
2. Plate reader (for Microplate Reader)
example,
ELx800TM
Absorbance
3. Incubator 37◦ C
2. GM1 ganglioside (Sigma Chemical Co., St Louis, MO) 3. Phosphate-buffered saline (PBS) 4. Tween 20 5. Bovine serum albumin (BSA) 6. Adhesives to cover plates 7. PBS–T washing buffer: PBS containing 0.05% (v/v) Tween 20 8. LTK63 or other LT mutants (Novartis vaccines) 9. Vaccine antigens 10. PBS–BSA blocking buffer: PBS containing 1% (w/v) BSA 11. Serum samples/mucosal washes 12. PBS–T–BSA dilution buffer: PBS containing 0.05% (v/v) T and 0.1% (w/v) BSA IgG: 13. Alkaline phosphatase-conjugated goat anti-mouse γ chain antibodies (Sigma) 14. Buffer for substrate: 10% (w/v) diethanolamine buffer containing 0.54 mM magnesium chloride, pH 9.8 (97 mL diethanolamine, 800 mL distilled water, 100 mg MgCl2 × 6 H2 O; adjust to pH 9.8 with 1 M HCl; bring volume up to 1.0 L with distilled water). Store at 4◦ C in the dark. 15. Alkaline phosphatase substrate solution for immunoassays: p-nitrophenyl phosphate, disodium, hexahydrate (available as pre-weighed tablets from Sigma), 1.0 mg/mL dissolved in diethanolamine buffer (see Step 14). The substrate solution must be prepared freshly (shortly before use) and be protected from light, bring to 25◦ C (RT) before use. IgA: 16. Biotin-conjugated goat anti-mouse α chain (Sigma) 17. Horseradish peroxidase (HRP)-conjugated streptavidin (Dako, Glostrup, Denmark)
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18. O-Phenylenediamine (OPD) available as pre-weighed tablets (Sigma) 19. Citrate buffer (0.1 M), pH 5.0, containing 0.01% hydrogen peroxide IgG subclasses: Appropriate enzyme or biotin-conjugated antisera for IgG subclasses (IgG1 and IgG2a specific), e.g., from Pharmingen, Cambridge, MA, or BioScience, Cambridge, UK. 2.5. Assessment of Adjuvant Activity on T-Cell Responses
1. Sterile surgical instruments for removal of spleens/lymph nodes and tissues. 2. Sterile-filtered (0.2 μm) c-RPMI medium: RPMI-1640 medium, supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 100 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μM 2-mercaptoethanol. 3. Cell strainer (BD Falcon) or nylon mesh or a sterile wire. 4. Petri dishes. 5. Plunger of a 5-mL syringe. 6. Falcon tubes (BD Falcon). 7. ACK buffer (red blood cell lysis buffer): Solution containing 0.15 M NH4 Cl, 10 mM KHC03 , 0.1 mM Na2 EDTA in distilled H2 O. Sterile filtered (0.2 μm). 8. Immunogens and adjuvant (LTK63 or other LT mutants). 9. Phosphate-buffered saline (PBS). 10. Ninety-six-well, flat-bottomed, plates (Costar, Corning, NY).
microtiter
cell-culture
11. Humidified 37◦ C, 5% CO2 incubator. 12. Cell-counting chamber. 13. 3 H-Thymidine (Amersham Biosciences, GE Healthcare). 14. Cell harvester (FilterMate Harvester; PerkinElmer). 15. Filter plates for scintillation counting (Unifilter-96 GF/C; PerkinElmer). 16. Scintillation cocktail for (MicrosintTM O; PerkinElmer).
scintillation
counting
17. Scintillation counter (TopCount NXT; PerkinElmer). 18. Cytokine ELISA kits (Pharmingen). 2.6. Assessment of Adjuvant Activity on B Cells (ELISPOT)
1. MultiScreen 96-well filtration plates (Millipore) 2. GM1 ganglioside (Sigma) 3. Immunogens and adjuvants (LTK63) (Novartis vaccines) 4. Phosphate-buffered saline (PBS)
Determining the Activity of Mucosal Adjuvants
269
5. Tween 20 6. PBS–T: PBS with 0.05% (v/v) Tween 20 7. Bovine serum albumin (BSA) 8. Humidified 37◦ C, 5% CO2 incubator 9. Sterile-filtered (0.2 μm) c-RPMI medium: RPMI-1640, supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 100 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μM 2-mercaptoethanol 10. Cell suspensions 11. Biotinylated goat anti-mouse IgG IgA and/or IgM immunoglobulins (Southern Biotechnology Associates, Birmingham, AL) 12. Avidin–peroxidase (BD Pharmingen, San Diego, CA) 13. DAB buffer (pH 7.5): 490 mL DI water + 10 mL 1× Tris buffer + 14.62 g NaCl 14. HRP color development reagent DAB (Bio-Rad) solution: add 100 mL DBA buffer to 250-mL bottle, warm buffer in 37◦ C water bath, add 50 mg DAB to bottle, and swirl until dissolved. Then filter it into another 250-mL bottle and label it appropriately. 15. DI water: de-ionized H2 O 16. ELISPOT reader (AELVIS).
3. Methods 3.1. Preparation of Immunogen/ Adjuvant Mixture
Importantly, the volume of the vaccine formulations and the final concentrations of the antigen and the adjuvant vary with the route of immunization (see Note 1). All formulations are performed by simply mixing the compounds and should be prepared on the day of immunization. Essential for the correct evaluation of the activity of mucosal adjuvants is the presence of appropriate control groups within the immunization protocol. As a negative control a group of mice should be immunized mucosally with only the immunogen (without the LTK63 adjuvant). As a positive control group, mice can be immunized systemically (e.g., intramuscularly or subcutaneously) with the same vaccine.
3.1.1. Immunogen/ Adjuvant Mixture for Intranasal Immunization
If possible the final immunization volume for intranasal immunization should not exceed 10 μL per mouse (5 μL per nostril) (see Note 2).
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1. The vaccine antigen/s and the LTK63 adjuvant are suspended in phosphate-buffered saline (PBS), in order to obtain a final concentration of 1 μg of each of the antigen/s per mouse dose and 1 μg of LTK63 per mouse dose (in 10 μL) (see Note 1). 2. For negative controls, prepare vaccine formulations containing only the influenza antigens without the LTK63 adjuvant. 3.1.2. Immunogen/ Adjuvant Mixture for Intragastric Immunization
For intragastric immunizations, a volume of 200 μL per mouse is usually administered (see Note 1). 1. For oral immunizations, the vaccine antigens are suspended in phosphate-buffered saline (PBS), with 3% bicarbonate solution at a final concentration of 100 μg of each of the antigens per mouse dose (in 200 μL). 2. The LTK63 mutant is given at a dose of 10 μg per mouse dose (within 200 μL of final immunization volume). 3. For negative controls, prepare vaccine formulations containing only the vaccine antigens without the LTK63 adjuvant.
3.1.3. Immunogen/ Adjuvant Mixture for Vaginal and Rectal Immunizations
For vaginal and rectal immunizations, a volume of 20 μL per mouse is administered. 1. The vaccine antigens are suspended in phosphate-buffered saline in order to obtain a final concentration of 1 μg of each of the three antigens per mouse dose (in 20 μL). 2. The LTK63 mutant is then added to this suspension at a dose 1 μg per mouse and the formulations are adjusted to a final immunization volume of 20 μL per mouse with phosphate-buffered saline. 3. For negative controls, prepare the formulations containing only the vaccine antigens without the LTK63 adjuvant.
3.1.4. Immunogen/ Adjuvant Mixture for Intramuscular Immunizations
For intramuscular immunizations, a volume of 100 μL (50 μL per left side and 50 μL per right side) per mouse is administered. 1. The vaccine antigens are suspended in phosphate-buffered saline in order to obtain a final concentration of 0.1 μg of each of the vaccine antigens per mouse dose (in 100 μL) (see Note 3).
3.2. Immunization of Animals
All in vivo procedures must comply with national regulations and locally approved ethical guidelines. The choice of vaccination procedures is complex, and the results may be dependent on many factors such as dose, type of antigen and adjuvant, route of immunization, time between inoculation, and species used to evaluate efficacy (44, 45). In addition, immune responses to an antigen vary with age, gender, and health status, factors that the vaccination regimen needs to take into account (46–48).
Determining the Activity of Mucosal Adjuvants
271
1. Female, 6- to 8-week-old inbred BALB/c mice (Charles River) are quarantined for 1 week prior to immunization (see Note 4). 2. Animals are vaccinated at days 1, 21, and 35 (see Note 5). 3.2.1. Intranasal Immunization of Mice
1. Manually restrain the non-anesthetized animal in an upright position so that the nose is pointed upward (see Note 6). 2. Drop the vaccine into the nose slowly and dropwise using a micro-pipette, dispensing a maximum dose of 5 μL into each nostril. Allow each drop to disperse before proceeding to the next drop. If due to insufficient antigen concentration, volumes higher than 10 μL need to be administered (see Note 2), add 5 μL of vaccine per application to each nostril, repeating alternately until the full dose has been applied (allow each drop to disperse before proceeding to the next drop/allow the mouse to recover for at least 15 min).
3.2.2. Intragastric Immunization of Mice
For intragastric immunizations, we use a Luer-Lock stainless-steel (50 mm × 1 mm) gavage with a round tip. 1. Two hours before immunization and 1 h after immunization, mice are deprived of food. Five minutes prior to immunization, mice are fed with 300 μL of 10% sodium bicarbonate to neutralize stomach acidity and then given 200 μL of inoculum. 2. Pick up the mouse by the nape of the neck; keep it in a vertical position, gently introduce the gavage needle through the mouth into the stomach, hyperextending the neck during transit to facilitate smooth passage. The procedure is restarted if resistance is encountered while introducing the gavage. 3. For intragastric immunization the procedure is repeated weekly two to three times more (see Note 5).
3.2.3. Vaginal Immunization of Mice
1. For vaginal immunization, wash the vagina several times with 25 μL sterile saline by inserting a fine pipette tip 1–2 mm into the vagina until the mucous clump is removed (see Note 7). 2. Administer 20 μL vaccine formulation with a micro-pipette. 3. Then immobilize mice for 2–3 h in a cylinder to avoid licking.
3.2.4. Rectal Immunization of Mice
1. For rectal immunization, mice are starved for 12 h before immunization. 2. Wash the rectum twice with 50 μL sterile saline administered from a pipette with a fine tip inserted 1–2 mm into the rectum.
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3. With a micro-pipette, deliver 20 μL of vaccine formulation into the rectum (see Note 8). 4. Then, mice are positioned with the rectum facing upward for about 45 min to reduce leakage of the inoculum. 3.2.5. Intramuscular Immunization of Mice
1. Fill syringe with vaccine formulation and remove bubbles. 2. Manually restrain the animal and insert the needle into heavy musculature of the quadriceps or posterior thigh (the same muscle should be used for all immunizations). 3. Aspirate briefly and ensure correct placement of needle (i.e., to prevent intravenous or intra-arterial injection). 4. Inject 2 × 50 μL (50 μL left side, then 50 μL right side) with moderate pressure and speed to prevent tissue damage.
3.3. Mucosal Sample Collection and Storage
The analysis of the mucosal washes is preferentially performed immediately after sample collection, otherwise samples should be snap frozen on dry ice and stored at −80◦ C until use (see Note 9). See also Ref. (49).
3.3.1. Nasal Washes
Nasal washes are collected by holding the head in an upright position in order that one nostril faces upward and applying 500 μL of ice-cold PBS containing 0.1% BSA and 1 mM phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor through the nostril pointing upward and collecting the wash from the other nostril.
3.3.2. Intestinal Washes
Intestinal washes are collected by flushing the small intestine with 4 ml of ice-cold PBS containing 0.1% BSA and 1 mM PMSF.
3.3.3. Vaginal Washes
Vaginal washes are carried out by flushing in and out the vagina with a total volume of 100 μl of ice-cold PBS containing 0.1% BSA and 1 mM PMSF.
3.3.4. Blood Collection
Serum samples are taken from the tail vein or by retro-orbital plexus puncture (see Note 10), according to conventional techniques (49). It is advised that serum samples are collected before each immunization and 10–15 days after each immunization. The serum samples are stored at 4◦ C until use (see Note 11).
3.3.5. Organ/Tissue Collection
After mucosal immunizations, isolation of lymphocytes from the systemic immune compartmentthe spleen, the intestinal tract Peyer’s patches and lamina propria cells, the respiratory tract, involving lung-derived intraparenchymal cells and draining lymph nodes of the upper and lower respiratory tractcan be performed. Suspensions of organs and tissues are prepared in the classic manner (see Note 12). For example, for splenocyte isolation
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1. Sacrifice mice by cervical dislocation. 2. Disinfect the fur by thoroughly saturating it with 70% (v/v) ethanol. 3. With scissors, make an incision in the abdominal skin and, grasping either side of this incision with gloved forefingers and thumbs, pull back the skin until the spleen can be observed through the abdominal musculature. Make an incision over the spleen and remove spleen with forceps, carefully cutting away the connecting tissue. 4. Transfer the organ to sterile medium c-RPMI (see Note 13). 3.4. Assessment of Adjuvant Activity on Antibody Responses by Enzyme-Linked Immunosorbent Assay (ELISA)
The enzyme-linked immunosorbent assay is a highly sensitive and quantitative serological assay used to evaluate specific antibody activity of sera.
3.4.1. Antibody Response to LTK63 and to Other LT Mutants
1. Coat 96-well, flat-bottomed plates by adding 100 μL of GM1 ganglioside at a concentration of 1.5 μg/mL in PBS, pH 7.4 (0.15 μg/well), cover the plates, and incubate overnight at 4◦ C. 2. Empty the wells and wash with PBS–T (300–450 μL/well); this washing procedure is repeated three times (if possible, use a plate washer, otherwise empty the wells by flicking the plate over a sink). 3. Add 100 μL/well of purified LTK63 mutant at a concentration of 1.0 μg/mL in PBS, pH 7.4 (0.1 μg/well), cover the plates, and incubate for 2 h at 37◦ C. 4. Empty the wells and wash with PBS–T (300–450 μL/well); this washing procedure is repeated three times (see Step 2). 5. Saturate unreacted sites for 1 h at 37◦ C with 200 μL of PBS–BSA blocking buffer. 6. Empty the wells and wash with PBS–T, (300–450 μL/well); this washing procedure is repeated three times (see Step 2). 7. Add (100 μL/well) serial dilutions (dilution Step 2) of individual serum or mucosal samples in dilution buffer (PBS–T–BSA). Serum samples are usually tested starting from a dilution of 1:100 to 1:1000 and mucosal samples from a starting dilution of 1:5 to 1:10. In detail: add 100 μL/well of PBS–T–BSA to each well (except row A) using the multi-channel pipette. Then add 200 μL/well of serum/mucosal sample to be tested into column 3–12 of row A, 200 μL/well of the reference serum into
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column 2 of row A (see Note 14), and 200 μL/well of the blank (buffer) into column 1 of row A (see Note 15), and mix carefully by taking the solutions up and down with the multi-channel pipette for a minimum of three times (avoid splashing or the introduction of air bubbles). After mixing, transfer 100 μL/well of the solution from each of the first wells (from row A) into the second wells (into row B), using the multi-channel pipette, and repeat the mixing procedure. Transfer 100 μL/well from each of the second wells to the third wells, and so on, down to the last row H of wells from each of which 100 μL/well of solution is removed and discarded. Thus, a twofold dilution series is created. Cover the plates and incubate for 2 h at 37◦ C. 8. Empty the wells and wash with PBS–T (300–450 μL/well); this washing procedure is repeated three times (see Step 2). Continue with IgG responses or IgA responses. IgG responses 9. Incubate plates (from Step 8) for 2 h at 37◦ C with alkaline phosphatase-conjugated antibody to mouse IgG to be diluted between 1:1000 and 1:5000 times in PBS–T–BSA (see Note 16). 10. Empty the wells and wash with PBS–T (300–450 μL/well), this washing procedure is repeated three times. 11. Add 100 μL/well of substrate solution (see Section 2.4.1, Step 15) to every well. Keep the plate at room temperature and observe for color development. 12. When color development is judged to be optimal or after a maximum of 30 min to 1 h, read the plate, using a multiscan photometer, at a wavelength of 405 nm. (For multiple plates, plates should be read in the same sequence as the substrate is added to minimize plate-to-plate variation. Alternatively, a suitable stop reagent can be used.) IgA responses 13. Incubated plates (from Step 8) for 2 h at 37◦ C with 100 μL of a biotin-conjugated goat anti-mouse α chain (Sigma), diluted 1:3000 in PBS–T–BSA (see Note 16). 14. Empty the wells and wash with PBS–T (300–450 μL/well); this washing procedure is repeated three times (see Step 2). 15. Add HRP-conjugated streptavidin (100 μL/well) diluted 1:2000 in PBS–T–BSA to the plates and incubate for 2 h at 37◦ C.
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16. Empty the wells and wash with PBS–T (300–450 μL/well); this washing procedure is repeated three times (see Step 2). 17. Antigen-bound antibodies are revealed by addition of 100 μL/well of o-phenylenediamine at 0.75 mg/mL in 0.1 M citrate buffer, pH 5.0, containing 0.01% hydrogen peroxide. After 20–30 min, plates are read for absorbance at 450 nm. The data can be analyzed in several ways, as end point or cutoff titers relative to the reference serum or by parallel-line assay methods (51). The readings from the antigen-negative controls provide the background values (see Note 15). Furthermore, antibody titers can be determined arbitrarily as the reciprocal of the last sample dilution giving an optical density of 0.3. Samples with optical densities below this value at the first dilution are considered negative. 3.4.2. Antibody Response to Vaccine Antigens
The ELISA method described above can be easily applied to the evaluation of IgG and IgA antibody titers, in serum and in mucosal samples, against the vaccine antigens used to immunize mice, by following the following steps: 1. Coat the 96-microwell plates directly with the influenza antigens used for immunization. Coat 100 μL/well of vaccine antigens at a concentration of 2 μg/mL in PBS (0.2 μg/well), cover the plates, and incubate overnight at 4◦ C. Be aware that the optimal amount of coating and incubation temperatures can vary from one antigen to another. Preparatory experiments should be carried out to optimize this amount. 2. Empty the wells and wash with PBS–T (300–450 μL/well); this washing procedure is repeated three times (if possible, use a plate washer, otherwise empty the wells by flicking the plate over a sink). 3. Follow protocol(s) above (Section 3.4.1) from Step 5 onward. It should be remembered that the results obtained from the immunosorbent assay of serum or mucosal samples may not correlate with its functional activities (see Section 3.7). Furthermore, the optimal conditions for the ELISA may vary from one antigen to another, although the method reported above has proven to be useful in our laboratories for several antigens tested.
3.5. Assessment of Adjuvant Activity on T Cells
Lymphocytes and other immune cells can be used to assess cellmediated immunity in general or, via antigen-specific stimulation, to detect previous exposure to an antigens and to monitor the response to vaccination. Cells previously exposed to an antigen proliferate in vitro when re-stimulated with the specific
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antigen. Proliferation can be measured by the incorporation of 3 H-thymidine into newly synthesized DNA. 3.5.1. Preparation of Single-Cell Suspensions
Suspensions of, for example, spleens (a systemic lymphoid tissue) and cervical lymph nodes (which drain the nasal mucosa) are prepared in the classic manner (see Section 3.3.5 and Note 12). All procedures should be carried out in sterile environment such as biological safety cabinet class II or laminar flow workstation. 1. Collect mouse spleens/lymph nodes in 15-mL Falcon tubes in 5 mL c-RPMI. 2. Mesh spleens/lymph nodes, etc. through cell strainer in a Petri dish to make single-cell solution (we use syringe pistons to squeeze them through), wash strainer and dish with another 7 mL of c-RPMI, and re-collect all in new 15-mL Falcon tube. 3. Spin down for 10 min at 1,200 rpm at 4◦ C and wash twice with 10 mL c-RPMI, then re-suspend the pellet in 3 mL ACK buffer for 3 min at room temperature (for red blood cell lysis, see Note 17). After 3 min, add 11 mL of c-RPMI, spin down (pellet should be white now), discard the supernatant, re-suspend cells in 2 mL c-RPMI, and count cells with a cell-counting chamber.
3.5.2. T-Cell Proliferation Assay
1. After counting the cells, adjust to 4×105 cells/100 μL and add 100 μL into each well of 96-well flat-bottom plates. 2. Add 100 μL/well LTK63 adjuvant in triplicates at two (or more) different concentration (e.g., 1 and 0.3 μg/mL) or add 100 μL/well vaccine antigens in triplicates at two (or more) different concentration (e.g. 1 and 0.3 μg/mL). Use medium without any antigen as a negative control in at least six wells (100 μL/well) to measure the background proliferation level. As a positive control, stimulate cells with the mitogen ConA (see Note 18) at a final concentration of 5 μg/mL (100 μL/well in triplicates). The final volume per well is now 200 μL. 3. Incubate cells for 4–6 days in a humidified atmosphere containing 5% CO2 at 37◦ C. 4. Pulse cells with 20 μL/well 3 H-thymidine (1 μCi/well) for 16 h (see Note 19). 5. Harvest cells onto filter plates by using a cell harvester with a 96-well microtiter format. Dry filter plates at room temperature. 6. Add scintillation cocktail (Microscint) to wells (35 μL/well).
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7. Determine the 3 H-thymidine incorporation by liquidscintillation counting using a scintillation counter. 8. Express proliferative T-cell responses as counts per minute (cpm) or as stimulation index (SI). Cpm values (cpm) are calculated by subtracting the mean of cpm values obtained without antigen (medium only) from the mean of triplicate cpm values obtained in the presence of antigen. SI is defined as the ratio of the mean of cpm triplicate values obtained in the presence of antigen to the mean cpm value obtained in the absence of antigen. SI values exceeding 3 or 5 are generally considered as positive responses. 3.5.3. Cytokine Production Assay
1. Carry out Steps 1–6 as described for the preparation of single-cell suspensions and T-cell proliferation assay (Sections 3.5.1 and 3.5.2) (see Note 20). Include wells for antigens of interest, control antigens and mitogens (ConA/positive control), medium alone, and nonstimulated cells (medium and cells). 2. At a specific time point (or multiple time points) during cell culture, aspirate supernatants from the wells and centrifuge at 4◦ C to pellet any cell debris. 3. Freeze the supernatants in aliquots at –80◦ C until use. 4. Thaw supernatants and measure cytokine concentrations using commercially available cytokine-specific ELISA kits (freeze and thaw only once).
3.6. Assessment of Adjuvant Activity on B Cells
B cells play an important role in protection against pathogens, and they secrete specific antibodies in serum and mucosal secretions upon antigenic stimulation, contributing to immune exclusion and clearance of pathogens. The enzyme-linked immunosorbent spot assay (ELISPOT) allows to enumerate B cells secreting antigen-specific antibodies. The frequency of antibody-secreting cells in specific organs is often a reflection of the route of antigen exposure, i.e., systemic, oral, or intranasal as well as of antigenic load.
3.6.1. Assessment of LTK63 Mutant-Specific and Vaccine Antigen-Specific Antibody-Secreting Cells (ASCs) by ELISPOT
Day 1 1. Coat MultiScreen 96-well filtration plates (Millipore) with specific adjuvant (LTK63) or vaccine antigen: (a) For LTK63, pre-coat plates with 100 μL/well of GM1 ganglioside at a concentration of 1.5 μg/mL. Incubate for 2 h at room temperature. Empty the wells by flicking the plates over a sink, then wash plates four times with PBS. Then add 100 μL/well LTK63 at a concentration of 1 μg/mL.
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(b) For vaccine antigens, add 100 μL/well of antigen at a concentration of 1 μg/mL in PBS. (This amount should be optimized for each vaccine antigen.) 2. Incubate plates overnight at 4◦ C with lids on in a humid chamber. Day 2 3. Prepare single-cell suspensions (see Section 3.5.1). 4. Empty the wells by flicking the plates over a sink, then wash plates three times with PBS–T. Dry carefully. Then wash three times again with PBS. Remove all liquid from plates and dry carefully. 5. Add 100 μL/well c-RPMI medium and block/incubate plates at 37◦ C in a CO2 incubator for 30 min to 1 h. 6. Add 100 μL/well of 2×105 cells (2×106 cells/mL) in duplicates to the first row A of blocked plates (containing 100 μL media/well—final concentration 1×105 cells/well) and perform 1:1 dilution by mixing row A cells and media four times and transferring 100 μL from row A to row B. Repeat for other row pairs. Incubate at 37◦ C in CO2 incubator overnight (see Note 21). 7. Following overnight incubation of cells, empty the wells by flicking the plates over a sink and wash plates three times with PBS–T. Dry carefully. Then wash three times again with PBS. Remove all liquid from plates and dry carefully (see Note 22). 8. Add 65 μL/well biotinylated antibody (biotinylated goat anti-mouse IgG or IgA immunoglobulin) diluted 1:2000–8000 in PBS/0.1% BSA and incubate plates in humid chamber with lids on at room temperature for 2 h. 9. Empty the wells by flicking the plates over a sink and wash plates three times with PBS–T. Dry carefully. Then wash three times again with PBS. Remove all liquid from plates and dry carefully. 10. Add 65 μL/well avidin–peroxidase at 1:1000 dilution in PBS/0.1% BSA. Incubate plates for 1 h at 37◦ C. 11. Empty the wells by flicking the plates over a sink and wash plates three times with PBS–T. Dry carefully. Then wash again three times with PBS. Remove all liquid from plates and dry carefully. 12. Prepare DAB solution (during Step 10). Just before adding to plates, add 5 μL of 30% H2 O2 and mix well. Spots are visualized by adding 100 μL/well DAB solution for 30 min.
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13. The plates are washed with de-ionized H2 O and airdried. The spots were counted by an automatic ELISPOT reader. Two to four wells per group and per tissue are counted. The data are expressed as the number of antigenspecific antibody-secreting cells per 106 mononuclear cells (see Note 23). 3.7. Measurement of Functionally Active Antibodies In Vitro and In Vivo Evaluation of Efficacy
Assays to evaluate the efficacy of mucosal immunization depend on the antigen used and, as a consequence, on the availability of an appropriate animal model to investigate efficacy in vivo. It is clear that these models vary enormously among themselves, and it would be more appropriate to describe them in sections devoted to these models or to the pathogens they refer to. In addition to measuring antibodies by ELISA or other antigen–antibody binding assays, one should measure antibody function by neutralization, opsonophagocytic, or bactericidal assays, if available. However, the most decisive test is protection against experimental in vivo challenge. To give a few examples, it has been clearly shown in mice that intranasal immunization with fragment C of tetanus toxin plus nontoxic mutants of LT induces an immune response able to protect the model against a lethal challenge with tetanus toxin (20). Furthermore, oral immunization of mice with selected recombinant Helicobacter pylori antigens in conjunction with the LTK63 mutant significantly improved the level of protection against an infectious challenge, both prophylactically and therapeutically (18, 56). See also Table 18.1. Importantly, the induction of protective immunity might depend upon the quality rather than the quantity of antibodies, which means the induction of antibodies of the appropriate isotype and fine-epitope specificity. In conclusion, unique interactions between the adjuvant, the antigen, and the host determine the efficacy of a vaccine. Thus the search for an effective vaccine must involve both antigens and adjuvants from the start of preclinical development, and one needs to keep in mind that no adjuvant can be considered a gold standard.
4. Notes 1. Since the intragastric route of immunization is much weaker in inducing specific immune responses as compared with, for example, the intranasal route, usually the amount of antigen/adjuvant is at least 10 times higher for the intragastric immunization than for the intranasal immunization.
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In our laboratory, we usually give 1–10 μg of antigen plus 1 μg of LTK63 mutant for each intranasal, vaginal, or rectal dose. For intragastric immunizations, 100–1,000 μg of antigen plus 10–100 μg of LTK63 mutant are usually given per dose. This implies that, in order to immunize mice intranasally using 10 μL volumes, the antigens must be sufficiently concentrated (2.5–3 mg/mL or more). The antigen concentration is less critical for intragastric immunizations because of the larger volumes employed. 2. If the volume for intranasal immunizations exceeds 10 μL due to insufficient antigen concentration, higher volumes up to 40–50 μL might be used, if carefully administered in 5 μL aliquots to alternated nostrils of the mouse. Importantly, take into consideration that immunization with volumes greater than or equal to 50 μL is associated with the vaccine deposited in the lung, with spillage into the gastrointestinal tract, presumably through swallowing. 3. The best amount of antigen can vary depending on the nature of the vaccine. This amount can range between 0.1 and 10–20 μg/dose. 4. Most of the studies on mucosal immunization with soluble antigens given together with LTK63 have been performed in female, 6- to 8-week-old inbred BALB/c, C57BL/6, or CBA inbred mice. Male, specific pathogen-free, CD-1 outbred mice have also been successfully used in our laboratory. The mouse is the most commonly used animal model and is usually a suitable choice. However, if it proves to be unsatisfactory, other species should be tried. LTK63 has been successfully given to guinea pigs and monkeys, and vaginally to goats (see Table 18.1 and relative references). 5. No single immunization schedule is suitable for all applications. The most useful schedule is not necessarily equivalent to that used in humans or the one that induces the strongest immune responses. It may well be the schedule that shows the greatest differentiation between the responses of vaccines of differing efficacies. 6. Intranasal immunization can be performed with or without light anesthesia. Importantly, the volume applied to the nasal mucosa and the use of anesthesia can lead to the induction of immune responses either to the upper respiratory tract and distal mucosal sites (through stimulation of the common mucosal immune system) or to the lower respiratory tract. Intranasal administration of the vaccine to anesthetized mice results in the delivery into the lung and toward stimulation primarily of pulmonary immune responses.
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7. Some groups synchronize the estrous cycle of mice by subcutaneous administration of progesterone. Progesterone is administered 5–7 days prior to vaginal immunization to synchronize the estrous cycle within groups of experimental mice in order to reduce variability in secretory IgA detection during different phases of the estrous cycle. 8. For rectal immunization, alternatively insert a fine, blunt end urinary cat catheter or disposable feeding needle attached to a 1-mL tuberculin syringe, approximately 0.5 cm beyond the rectum, and slowly inject vaccine formulation. 9. For mucosal washes, Eppendorf caps for sample collection should be saturated overnight with PBS containing 1% BSA. 10. It is best to assay sera as individual samples rather than as pools, since this allows statistical analysis of the results. 11. Although sera may be stored at 4◦ C for several days, or with the addition of antibacterial agent for many weeks, they should be kept at –20◦ C for prolonged storage. Repeated thawing and freezing should be avoided. Sera that are to be compared should have been stored under similar conditions. 12. Suspensions from several mice can be pooled to reach a sufficient number of cells per culture. 13. For details of collection and processing of mouse cells, see Ref. (50). 14. Using a reference serum and expressing the results in terms of the reference serum allow the direct comparison of data from assays performed at different times and in different laboratories. 15. When using an ELISA to quantify antibody responses, it is important to incorporate a serum-negative well on each plate in order to determine the background values. 16. Using appropriate enzyme-conjugated antisera, one can also determine the IgG subclasses specific for the LTK63 mutant induced for the immunization. 17. Overexposure of spleen cells to ACK buffer will significantly reduce the viability of the spleen cell preparation. It is important not to exceed 3 min of incubation on ice in order to maintain optimal viability of the lymphocytes. 18. Concanavalin A (ConA from Sigma) is used at a concentration of 1–5 μg/mL as a positive control to induce maximal T-cell stimulation. This mitogen should always be added last to minimize cross-contamination between wells.
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19. A shorter time period could also be used but minimum 4 h. The plates may be frozen after the thymidine incubation step and then thawed immediately before harvesting. Follow the general guidelines for the use of 3 H-thymidine. 20. Because the cell-culture procedures of the proliferation and cytokine measurements are identical, the performance of these assays can be combined by using the same cellculture plates. The optimum time point for measuring each cytokine of interest must be determined for the cytokine and antigen(s) being studied. This is done by measuring the levels of cytokine released over time using daily time points over the 6-day assay period. The supernatants should be maintained at 4◦ C until frozen. 21. Make sure that the incubator is not shaken or moved during the incubation period, because if cells move during incubation, spot duplication may occur. 22. Cytokine production assay: Carry out Steps 1–6 as described for the ELISPOT assay. After the overnight incubation, remove 65 μL/well of supernatants and transfer into appropriately labeled tissue culture plates. Freeze plates and store at –80◦ C until use. Thaw supernatants and measure cytokine concentrations using commercially available cytokine-specific ELISA kits. 23. Please note that ELISPOT assays have been further adapted for various tasks, as, for example, the identification and enumeration of cytokine-producing cells at the singlecell level. At appropriate conditions the ELISPOT assay allows visualization of the secretory product of individual activated or responding cells. Each spot that develops in the assay represents a single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information (52–55).
Acknowledgment We are grateful to Markus Hilleringmann for critical reading of the manuscript and Derek O’Hagan for general support. References 1. Baudner, B. C., Verhoef, J. C., Junginger, H. E., Del Giudice, G. (2004) Mucosal adjuvants and delivery systems for oral and nasal vaccination. Drugs Future 29, 721–732. 2. McNeela, E. A., Mills, K. H. (2001) Manipulating the immune system: humoral versus
cell-mediated immunity. Adv Drug Deliv Rev 51, 43–54. 3. Czerkinsky, C., Anjuere, F., McGhee, J. R., George-Chandy, A., Holmgren, J., Kieny, M. P., Fujiyashi, K., Mestecky, J. F., PierrefiteCarle, V., Rask, C., Sun, J. B. (1999)
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Chapter 19 Adjuvant Activity of Cytokines Michael G. Tovey and Christophe Lallemand Abstract The activity of several potent adjuvants, including incomplete Freund’s adjuvant, CpG oligodeoxynucleotides, and alum, has been shown to be due at least in part to the induction of cytokines, including type I interferons (IFNs), IFN-γ, interleukin-2 (IL-2), and IL-12, that play key roles in the regulation of innate and adaptive immunity. The relatively short half-life of recombinant homologues of cytokines has limited their use as vaccine adjuvants. These difficulties have been overcome by encapsulation into liposomes and the use of cytokine expression vectors co-administered with DNA vaccines. Although a number of cytokines including IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, and Flt-3 ligand have been shown to potentiate the immune response to vaccination in various experimental models, the full potential of cytokines as vaccine adjuvants remains to be established. Key words: Adjuvant, cytokines, interferons, interleukins, adaptive immunity, innate immunity, Toll-like receptors.
1. Introduction The search for effective non-toxic adjuvants capable of enhancing the immune response to an antigen has heightened with the advent of vaccines based on recombinant proteins rather than whole cells or virus-based vaccines since the primary immune response to a soluble protein is usually weak in the absence of an adjuvant. Furthermore, many recombinant vaccines require induction of a strong T-helper (Th) or cytotoxic T-lymphocyte (CTL) cellular immune response in addition to a strong humoral response. A number of potent adjuvants including incomplete Freund’s adjuvant (IFA), CpG oligodeoxynucleotides, polyphosphazenes (1), and aluminum salts commonly known as alum (2) have been G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_19, © Springer Science+Business Media, LLC 2010
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shown to induce the production of a varying array of cytokines including the type I interferons (IFNs), IFN-γ, interleukin-2, IL-12, and IL-18 (1, 2) that play an important role in the innate immune response and the establishment of adaptive immunity. Thus, although the action of adjuvants such as IFA or alum is complex involving carrier, depot, targeting, and immunomodulatory functions, studies using mice lacking a functional type I IFN receptor have shown that the adjuvant activity of IFA and certain CpG oligodeoxynucleotides is due at least in part to their ability to induce the production of type I IFNs (3). It is not surprising therefore that cytokines such as type I IFNs should in their own right exhibit potent adjuvant activity. The subject of this chapter is to review the data suggesting that certain cytokines produced by recombinant DNA technology may constitute a novel group of candidate adjuvants for use in vaccination against infectious agents. Although a number of cytokines including IL-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, and IFN-γ have been reported to exhibit adjuvant activity in tumor models and in experimental antitumor vaccines in man, antitumor vaccination strategies often differ markedly from vaccination against infectious agents and are beyond the scope of this review. 1.1. Pattern-Recognition Receptors, Cytokines, Innate Immunity, and Establishment of the Adaptive Immune Response
Specific populations of cells including dendritic cells (DCs) distributed throughout the peripheral tissues act as sentinels capable of recognizing conserved microbial-associated molecular patterns (MAMPs) characteristic of infectious agents through patternrecognition receptors (PRRs). Exposure of DCs, specialized antigen-presenting cells (APCs), to PRR ligands induces rapid cellular activation and maturation followed by migration of APCs to peripheral lymph nodes and priming of naïve T cells (500). Pattern-recognition receptors include the cell-surface and endosomal family of Toll-like receptors (TLRs), each member of which detects a unique or a specific set of MAMPs, which together confer on the host the ability to recognize a wide range of infectious agents. Recently, a second plasma membrane/endosomal PRR has been identified, Dectin-1, a C-type lectin that binds β-1,3-glucans present in the cell walls of fungi and some bacteria (4). The largest group of intracellular PRRs are the nucleotidebinding oligomerization domain or Nod-like receptors (NLRs) which include members (Nod1, Nod2, NAIP, and IPAF) that can recognize peptidoglycans from bacterial cell walls (5) and receptors such as Nalp3 which can recognize endogenous markers of cellular damage. Nalp3 is also responsible for the detection of alum (2), through a process involving activation of the Nalp3 inflammasome and production of IL-1β and IL-18 (2). The retinoic acid-inducible gene I (RIG-I)-like cytosolic receptor proteins RIG-I, melanoma differentiation antigen-5 (MDA5), and Laboratory of Genetics and Physiology-2 (LGP2) cytosolic
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receptors play a critical role as cytoplasmic sensors of viral RNA, in the induction of the type I IFNs, and the establishment of the innate immune response (6). Evidence also exists for the presence of cytosolic DNA sensor(s), although the exact identity of the sensor remains controversial (6, 7). A number of TLR agonists exhibit potent adjuvant activity characterized by an efficient T-helper 1 (Th1) CD4+ immune response to antigen; production of IL-2, IL-12, and IFN-γ; activation of CD8+ cytotoxic T cells; and generation of complementfixing antibody IgG2a and IgG3 (8). In contrast, adjuvants such as alum induce a Th2-type response characterized by production of IL-4, IL-5, and IL-6 and production of IgG1 (8). Some of these cytokines have been tested individually for their ability to stimulate the immune response to vaccines against infectious agents. These data will be reviewed below for those cytokines that have been studied sufficiently for their potential as vaccine adjuvants to be evaluated and in particular for type I IFNs which have been studied extensively.
2. The Human Interferon Superfamily 2.1. Type I Interferons
In man, the type I IFN gene family is located on the short arm of chromosome 9 and encodes 12 functional IFN-α and single IFN-β, IFN-ω, IFN-ε, and IFN-κ subtypes. All type I IFNs bind to a common high-affinity cell-surface receptor composed of two trans-membrane polypeptides IFNAR1 and IFNAR2. IFN binding results in the phosphorylation and activation of the Janus kinases Jak1 and Tyk2, which in turn phosphorylate and activate the latent cytoplasmic signal transducers and activators of transcription 1 (STAT1) and STAT2, leading to the formation of the transcription complex ISGF3 in association with IFN regulatory factor 9 (IRF9). Translocation of this complex to the nucleus results in the transcriptional activation of a specific set of genes that encode the effector molecules responsible for mediating the biological activities of the type I IFNs. All type I IFNs induce comparable qualitative biological activities, although quantitative differences in the activity of different subtypes have been reported (9).
2.2. Type II Interferons
In man, type II interferon or interferon gamma (IFN-γ) is encoded by a single copy gene located on chromosome 12. IFN-γ binds to a specific cell-surface receptor composed of two trans-membrane polypeptides, IFNGR1 and IFNGR2, and signals through a STAT1 homodimer, resulting in the
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transcriptional activation of a specific set of genes that encode the effector molecules responsible for mediating its biological activity. IFN-γ is produced primarily by T cells and NK cells in response to specific antigen or mitogens and plays a key role in T-cell activation and the establishment of the adaptive immune response (8). 2.3. Type III Interferons
Type III IFNs or IFN-λ1, IFN-λ2, and IFN-λ3, also known as IL-29, IL-28A, and IL-28B, respectively, are encoded by genes located on chromosome 19 in man. IFN-λ binds to a specific cell-surface receptor comprising a unique IFN-λR1 chain and the IL-10R2 chain common to IL-10, IL-22, and IL-26 (10). In common with the type I IFNs, type III IFNs share a common signaling pathway and activate the transcription of a similar set of genes encoding effector proteins (10). In contrast to type I IFNs, expression of the type III IFN receptor is, however, restricted to certain cell types (10).
2.4. Activation of the Type I Interferon Pathway
Recognition of specific conserved molecular constituents of microbial pathogens by pattern-recognition receptors such as TLRs gives rise to the production of type I IFNs. Thirteen members of the TLR family have been identified in mammals (11). Each TLR mediates a distinctive response in association with different combinations of four Toll/IL-1 receptor (TIR) domain-containing adaptor proteins, myeloid differentiation factor-88 (MyD88), the MyD88 adaptor-like protein (Mal), the TIR domain-containing adaptor protein inducing IFN-β (TRIF), TRIF-related adaptor protein (TRAM), and the sterile α- and armadillo-motif-containing protein (SARM). All the TLRs except TLR3 interact with MyD88. TLR3, which recognizes double-stranded viral RNA (dsRNA), the single-stranded RNA (ssRNA) receptor TLR7, and the CpG DNA receptor TLR9, is localized in the endosomes of myeloid DCs and requires acidification of vesicles for activation. TLR3 signals via TRIF and activates TBK1/IKKε which phosphorylates the interferon regulatory factor 3 (IRF3) and NFκB, resulting in the production of IFN-β (12, 13). The cytosolic dsRNA receptor MDA5 and the 5 -triphosphate RNA receptor RIG-I-like are DExD/H box RNA helicases which carry caspase activation and recruitment domain (CARD)-like motifs at the N terminus (14). The CARD domain interacts with IFN-β promoter stimulator 1 (IPS-1), resulting in the activation of the transcription factors IRF3, NFκB, AP1 and the production of IFN-β. Thus, activation of PRRs leads to the production of pro-inflammatory cytokines including type I IFNs and the activation of the innate immune response. Recent studies have shown that both TLR3 and RIG-I/MDA5 are required for a robust response to dsRNA and that the adjuvant activity of polyI:C is dependent upon TRIF and IPS1. Thus, enhanced
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antibody production and antigen-specific CD8+ T-cell activation in response to polyI:C were markedly reduced in mice deficient in both TRIF and IPS-1. This was accompanied by reduced production of IFN-α and IFN-β in response to polyI:C. IFN-γ production was also inhibited, reflecting reduced T-cell activation and a less vigorous adaptive immune response (15). Dendritic cells signal principally through TLRs, while RIGI-like receptors predominate in other cell types. Two major DC subsets can be distinguished in man, CD11c(+) monocytederived myeloid DCs, present in most tissues, and CD11c(–) plasmacytoid DCs (pDCs), present principally in lymph nodes. Plasmacytoid DCs are the principal producers of type I IFNs in response to viruses (16). Plasmacytoid DCs express high levels of TLR7/8 and TLR9 that recognize single-stranded RNA (ssRNA) and unmethylated viral or bacterial CpG DNA, respectively (17–19). Activation of both TLR7/8 and TLR9 leads to the formation of a complex with MyD88 and phosphorylation of IRF7 and production of high levels of type I IFNs (11). 2.5. Effect of Type I Interferons on the Innate Immune Response and Induction of Adaptive Immunity
Type I IFNs stimulate DC maturation by increasing the expression of co-stimulatory molecules including CD40, CD80, and CD86 and major histocompatibility complex (MHC) antigens (16). Upon activation, DCs migrate from the peripheral tissues to the secondary lymphoid organs, where they present pathogenderived antigens to naïve T cells, leading to the adaptive antigenspecific immune response. Trafficking of DCs is also enhanced by type I IFNs which increase the expression of several chemokines and chemokine receptors (20). Type I IFNs also play an important role in cross-priming (21–23). Thus, following activation, DCs present foreign virus-derived peptides on MHC class I antigens to CD8+ T cells, leading to CD8+ T-cell activation (21). IFN-stimulated cross-priming appears to be independent of the CD40 ligand–CD40 interaction between DCs and CD+ T cells (21–23). Thus, type I IFNs produced primarily by pDCs play a key role in the innate immune response to virus infection and in the induction of the primary adaptive immune response. Type I IFNs are also powerful polyclonal B-cell activators which induce a strong primary humoral immune response characterized by isotype switching and protection against virus challenge (3). Thus, type I IFNs have been shown to induce B lymphocytes to differentiate into antibody-producing plasma cells and to be necessary for the production of both specific and polyclonal IgGs in response to influenza infection (24). Furthermore, type I IFNs have been shown to enhance the primary antibody response to a soluble antigen in vivo and to enhance the production of all IgG sub-classes (3, 22). Type I IFNs also enhance long-term antibody production and immunological memory (3, 22). Type I IFNs are also usually powerful adjuvants when admixed with influenza
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vaccine and injected intramuscularly (25). The adjuvant activity of type I IFNs has been shown, using mice deficient in the type I IFN receptor on specific cell populations, to involve both direct effects of IFN on B cells and effects on T cells, most probably CD4+ T cells (21, 22). Production of B-cell stimulatory factors including BAFF and APRIL by IFN-stimulated DC may also play a role in the adjuvant activity of the type I IFNs (26). Although the precise mechanism(s) of the IFN stimulation of the humoral response remains to be elucidated, possible mechanisms include enhanced antigen-triggered proliferation and differentiation of B cells and protection of both B cells and helper T cells from apoptosis, leading to enhanced and prolonged antibody production (22). Type I IFNs exert both direct and indirect effects on T cells and play a key role in the fine-tuning of their activity, by delivering both stimulatory and inhibitory signals. In this manner, type I IFNs contribute to the homeostasis of the immune response. Thus, treatment of naïve CD4+ T cells with type I IFNs delays their entry into the cell cycle after TCR triggering. Indirect effects of the type I IFNs on T-cell activation include induction of co-stimulatory molecules, CD80, CD86, on antigen-presenting cells (APCs) which in the presence of antigen stimulate T-cell activation and prevent induction of anergy (27). IFN also acts directly on memory T cells to increase their survival and indirectly through stimulation of IL-15 production by APCs that contributes to the survival of memory T cells (27, 28). Virusstimulated pDCs produce large amounts of IFN-α and prime naïve CD4+ T cells to differentiate into IFN-γ/IL-10-producing cells possessing anergic and regulatory properties (29, 30). Induction of this functional phenotype is dependent on the presence of IFN-α. Type I IFNs also attenuate the generation of antigenspecific CD8+ T cells through the induction of CD4+ Tr1 cells (31). Thus, type I IFNs may play an important role in suppressing excessive inflammatory responses while at the same time stimulating the humoral response. 2.6. Effect of Type I Interferons on Apoptosis and Antigen Presentation
Human DCs phagocytose virus-infected apoptotic cells and present peptide epitopes on MHC class I and class II molecules, resulting in the activation of viral antigen-specific CD8+ T cells or CD4+ T cells, respectively, a process known as cross-priming (32). Many cytokines can modulate this process in two ways. First, TNF-α and type I and II IFNs can directly stimulate the transcription of genes involved in antigen processing, such as TPA1 (transporter associated with antigen processing) (33, 34), LMP2 (low molecular mass polypeptide 2), or LMP7 (35, 36, 34), resulting in enhanced antigen presentation. Second, many cytokines can directly promote the apoptosis, increasing numbers of apoptotic vesicles and enhancing presentation of viral
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(antigenic) peptides. Thus, the TNF family member IL-21 induces the TRADD/FADD apoptotic pathway and upregulation of the BH3 family member BIM (37). Type I and II IFNs can activate a number of components involved in the apoptotic pathway. In contrast, type I and type II IFNs do not trigger apoptosis per se. Thus, pro-caspase 2 and 8, FAS, FAS ligand, and the transcription factors IRF1 and IRF3 are all transcriptionally activated following interferon treatment (20). IRF1 and IRF3 play a central role in the regulation of the interferon system. They are the principal transcriptional activators of the type I IFNs and are themselves in turn induced by type I IFNs constituting a positive feedback loop. Furthermore, IRF1 and IRF3 are also involved in the pro-apoptotic activity of the type I IFNs. Thus, the presence of infectious virus, inactivated viral particles, or viral components such as dsRNA induces post-translational modification (e.g., phosphorylation) of IRF1 and IRF3, resulting in marked transcriptional activation of the apoptotic gene NOXA, resulting in apoptosis of virus-infected cells (38), increased numbers of apoptotic vesicles, and enhanced presentation of viral peptides by IFN-activated DCs. Induction of CD69 by both type I IFN and IFN-γ (39) has also been reported to stimulate apoptosis by a mechanism that remains unclear (136). Together, these observations suggest that type I IFNs can stimulate antigen presentation both by inducing the maturation of myeloid DCs and by enhancing the presentation of viral peptides as well as by enhancing apoptosis, resulting in increased numbers of apoptotic vesicles that can be processed directly by DCs. 2.7. Type I Interferons and Mucosal Immunity
Nasal immunization with inactivated whole-virus vaccines or virus-like particles induces secretory IgA and systemic IgG antibody responses that afford protection from respiratory infections and prevent dissemination of the infection to extrapulmonary sites (40, 41). Mucosal administration of soluble proteins in general does not, however, elicit a strong antibody response (42, 43). Type I IFNs admixed with the influenza subunit vaccine and administered intranasally have been shown to afford complete protection of animals against virus challenge, while vaccine alone was only partially effective (44). Type I IFNs are produced at mucosal surfaces as part of the innate immune response to infectious agents. Intranasal administration of recombinant IFN-α mimics mucosal production of IFN and has been shown to confer protection against virus infection and tumor cell multiplication (45). Protection occurs in the absence of detectable circulating levels of IFN-α (137) by a mechanism thought to involve cellular immunity (45, 46). Treatment of mice with recombinant IFN-α was found to markedly enhance the humoral response to influenza vaccine when admixed with the vaccine and injected intramuscularly (46).
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Intranasal administration of IFN-α concomitantly with intramuscular injection of vaccine alone was also found to enhance the antibody response to influenza vaccination (46). The results of experiments using transgenic mice expressing an EGFP reporter gene regulated by an IFN-responsive promoter suggest that IFN treatment increased trafficking of IFN-activated antigenpresenting cells to the site of influenza vaccination (46). These results may explain at least in part the mechanisms underlying the adjuvant activity of IFN-α. 2.8. Type I Interferons: Clinical Studies
The results of pilot clinical studies using parenterally administered IFN-α either have failed to demonstrate an adjuvant effect of IFN-α in healthy unvaccinated individuals (47) or at best have given some indication of efficacy in immunocompromised renal transplant patients vaccinated with hepatitis B virus (HBV) vaccine (48). A trend toward an increase in seroconversion was also observed in healthy non-responders to a previous HBV vaccination treated with IFN-α (49). Parenteral administration of recombinant IFNs is also associated with significant toxicity including a flu-like syndrome (50, 51). More serious myelosuppression and neuro-psychiatric effects also appear after long-term treatment (50, 51). To determine whether sublingual IFN can enhance the immune response to vaccination at a dose with an acceptable toxicity profile, a randomized, double-blind, placebo-controlled clinical trial was initiated to evaluate the effect of sublingual administration of IFN-α on the immune response to influenza vaccination in the elderly. Sublingual administration of 10 million IU of IFN-α, immediately prior to vaccination, was found to reduce the virus-specific antibody response to a single component, the New York strain of influenza A, of the standard subunit influenza vaccine in elderly institutionalized individuals without detectable drug-related local or systemic toxicity (52). At the dose tested, IFN treatment did not inhibit the immune response to the other components of the vaccine: the New Caledonia strain of influenza A virus or the Jiangsu strain of influenza B virus (52). It is of interest that the population studied had not previously been exposed to the New York strain of influenza A virus in previous vaccination campaigns. Thus, at the dose tested, IFN-α would appear to reduce the “primary” humoral response to the New York strain of influenza A virus but not the secondary immune response to either the New Caledonia strain of influenza A virus or the Jiangsu strain of influenza B. The results obtained in these clinical studies contrast with previous reports of the adjuvant activity of IFN-α in mice injected with influenza vaccine (25, 46). Although this may reflect the poor predictive value of rodent models, other factors may also be important, including the dose of IFN employed and the time and mode of IFN administration. Thus, high doses of parenterally or sublingually
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administered IFN-α have been reported to inhibit, while lower doses stimulated, the immune response to influenza vaccination in mice (46). It is also difficult to compare effects observed in small studies carried out in patient populations (elderly, immunocompromised individuals, healthy young adults) differing in their response to vaccination (49, 52). Further dose-ranging, higherpowered studies will be necessary in order to determine whether sublingual administration of IFN-α will find application as an adjuvant for influenza vaccination in either elderly individuals or healthy young subjects. 2.9. Type II Interferons: Interferon γ (IFN-γ)
IFN-γ plays a pivotal role in the regulation of the immune response including the activation and differentiation of T cells, B cells, NK cells, and macrophages. IFN-γ is produced primarily by activated T cells and NK cells in response to specific antigen, mitogens, IL-2, and IL-12. IL-18, a pleiotropic cytokine secreted by activated macrophages, has been shown to prime NK cells to produce IFN-γ upon subsequent stimulation with IL-12 (53). IFN-γ production is critical for the establishment of Th1 immunity and protection against virus infection. IFN-γ also inhibits IL-4 production by Th2 cells, thereby enhancing the production of IgG1 relative to that of IgE. IFN-γ enhances the expression of MHC class II antigens on APC and promotes antigen presentation to helper T cells (53). IFN-γ production is also the hallmark of CD8+ T-cell activation. Despite the key role that IFN-γ plays in the establishment of the adaptive immune response and its importance in conferring protective immunity against a variety of virus infections, IFN-γ has not found wide application for use as a vaccine adjuvant. This is despite early reports indicating that IFN-γ can increase the response rate of hemodialysis patients to HBV vaccination (54) and increase the rate of protection of rhesus macaques against pathogenic SHIV challenge following vaccination with a nef-deleted SHIV vaccine (55, 56). IFN-γ has also been shown to act as a potent adjuvant in mice vaccinated with Plasmodium yoelii malaria vaccine (57). The relatively few studies on the use of exogenous IFNγ as a vaccine adjuvant is due in part to the rapid biodegradation and clearance of IFN-γ that limits its efficacy. Liposomal encapsulation of human IFN-γ has been shown, however, to reduce degradation and improve its pharmacokinetics (58). Thus, administration of IFN-γ has been shown to increase the humoral response to subunit preparations of the hemagglutinin and neuraminidase surface proteins of influenza virus without affording protection against virus infection in experimental mice (59). In contrast, in the same study, incorporation of IFN-γ into liposomes was shown to markedly enhance the antibody response to the viral surface proteins and to protect a majority of animals against virus challenge (59). Association of IFN-γ with albumin
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nanoparticles has also shown promise as a carrier for delivering biologically active cytokines (60, 61). Expression of IFNγ in conjunction with DNA vaccines has also been shown to enhance the humoral and cell-mediated immune response to a variety of virus antigens. Thus, an IFN-γ–HIV-1 gp120 fusion protein produced stronger primary antibody and T-cell responses to the gp120 protein than did vaccination with gp120 alone (62). Similarly, co-immunization of rhesus macaques with a plasmid expressing IFN-γ was shown to enhance the antigen-specific T-cell response to immunization with HIV-1 env/rev or SIV gag/pol (63). In other studies, although co-immunization of rhesus macaques with a plasmid expressing IFN-γ together with plasmids expressing SIV Env and Gag was shown to enhance the immune response to the virus antigens, the animals were not protected to against a lethal challenge with SIV (64). Expression of IFN-γ has also been shown to enhance an IgG2-biased humoral response to influenza hemagglutinin (65) and HBV DNA-based vaccines (66). Immunization of ducks with an IFN-γ expressed vector together with a duck hepatitis B virus (DHBV) vaccine has also been shown to increase protection of animals against infection with DHBV (67). Co-administration of plasmid DNA encoding IL-18 has also been shown to enhance a Th1-biased immune response to a HSV glycoprotein B DNA vaccine, resulting in enhanced production of specific IgG2 antiviral antibodies, enhanced IFN-γ-producing T-cell responses, and enhanced production against vaginal challenge (68). Protection was shown to be dependent upon production of IFN-γ, as mice with an inactivated IFN-γ gene remained susceptible to challenge (68). Similarly, the adjuvant activity of certain CpG oligonucleotides has been shown to be dependent upon induction of IFN-γ (69, 70). Recent results also suggest that IL-18 may be an effective adjuvant when co-expressed together with a schistosomiasis DNA vaccine (71). Excessive production of IFN-γ has been shown, however, to reduce the anti-HIV-1 Env cellular immune response in mice vaccinated with a vector based on the Western Reserve (WR) strain of vaccinia virus (VV) expressing both proteins (72). Furthermore, co-expression of IFN-γ and IL-12 abrogated the increase in the immune response to HIV-1 Env observed when either cytokine was expressed separately in combination with a modified MVA vector expressing HIV-1 Env (72). Furthermore, co-administration of IL-18 expression vector together with a PrV gB-encoded DNA vaccine has been reported to strongly inhibit the antiviral antibody response and to increase susceptibility to a virulent virus challenge (73). Thus, together these results emphasize the potent effects that IFN-γ, or IFN-γ-inducing cytokines, exerts on the immune response and emphasize the difficulty in modulating these effects in order to produce an effective and safe vaccine adjuvant.
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3. Interleukin 2 (IL-2) IL-2 is a key T-cell growth factor responsible for the clonal expansion of T cells following activation by specific antigen and for the differentiation and survival of effector T cells and NK cells (74). IL-2 can also limit T-cell expansion by downregulation of both the γc chain of the trimeric IL-2 receptor (IL-2R) and the anti-apoptotic protein Bcl2, leading to apoptosis of proliferating T cells (75). IL-2 is also required for the survival of FoxP3+ regulatory T cells (76). Thus, IL-2 can have very different effects on antigen-specific T cells depending upon the time of administration, the state of T-cell differentiation, and the presence or the absence of antigen (77). Although IL-2 has been used to enhance the T-cell response to both viral antigens and tumor antigens in patients infected with HIV or metastatic solid tumors (78, 79), considerable toxicity including vascular leakage has been observed particularly at high doses (78, 79). Immunization of mice with a recombinant herpes simplex virus type I (HSV-1) expressing IL-2 did not increase the humoral response to the vaccine but did enhance the cytotoxic T-lymphocyte (CTL) response (80). Immunization of chickens with whole-virus influenza vaccine bearing membrane-bound IL-2 was found to elicit higher levels of antiviral antibody than did immunization of animals with whole-virus alone (81). Co-administration of IL-2 cDNA with DNA vaccines has also been shown to induce a Th1-type cellular response with increased production of IFN-γ and a higher ratio of IgG2/IgG1 (82). Co-expression of IL-2 and influenza peptides has also been shown to protect mice against lethal influenza virus infection (83). The use of recombinant IL-2 to enhance the immune response to such DNA vaccines remains to be tested, however, in man.
4. Interleukin 12 (IL-12) IL-12 plays a key role in the innate immune response and the regulation of adaptive immunity. Thus, IL-12 has been shown to stimulate NK-cell activity and the production of IFN-γ by T cells, to enhance CD8+ cytotoxic T-cell activity, and to stimulate the development of CD4+ Th1 responses (84). In keeping with these findings, IL-12 has been shown to act as a potent adjuvant capable of generating both efficient humoral and T-cell responses in combination with peptide antigens, virally delivered antigens, or cDNA vaccines (85). Since IL-12 is cleared rapidly from the
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circulation, attempts have been made to encapsulate IL-12 into microspheres in order to maintain the adjuvant activity of IL-12 in peptide or protein vaccines designed to enhance protective immunity against infectious agents (86). An alternative approach has been to use poly (gamma-glutamic acid) nanoparticles to target DCs and macrophages to induce the production of IL-12p40 and IL-6 (87) or to co-express IL-12 with antigen in cDNA vaccines (88). These approaches remain to be validated by appropriate clinical studies.
5. Interleukin 15 (IL-15) IL-15 is a member of the four alpha helix bundle family of cytokines that plays a key role in T-cell and NK-cell activation and function (89). IL-15 binds to a heterotrimeric receptor consisting of the IL-15-specific IL-15Rα chain, the IL-2/IL-15Rβ chain, and the common γ chain (γC) also shared by IL-4, IL-7, IL-9, and IL-21 (89). IL-15 is unusual in that it exhibits both “cis” and “trans” receptor presentation (89). For example, although IL15Rα alone can mediate signal transduction in some cell types, the IL-2/15Rβγc heterodimer is essential for signal transmission in T cells and NK cells, whereas the IL-15Ra chain is not required for signaling in these cells. Thus IL-15 bound to the high-affinity IL15α subunit on one cell type can be presented in trans to T cells or NK cells expressing the IL-2/15Rβγc heterodimer, thereby inducing IL-15-dependent responses in these cells. Signal transduction via the IL-2/15Rβγc heterodimer involves activation of Jak1 and Jak3 and STAT3/STAT5 pathway as well as activation of Ras/Raf/MAPK and PI-3 K/Akt pathways (90–92). IL-15 together with IL-7 plays a key role in the generation and maintenance of memory CD8+ T cells (93). Thus, IL-7 is required for the survival of both naïve and memory CD8+ T cells, while IL-15 is essential for their proliferation (93). The effect of IL15 on enhancing and sustaining memory CD8+ T-cell responses, in particular its ability to inhibit TRAIL-induced apoptosis by downregulating Bax and increasing expression of anti-apoptotic proteins such as Bcl-xL in CD8+ T cells (94, 95), suggests that IL-15 may be effective as a vaccine adjuvant. In view of the relatively low serum half-life of IL-15, most studies have concentrated upon the use of IL-15 expression vectors as adjuvants for DNA vaccines. IL-15 expression vectors have been shown to enhance both the humoral and cellular immune response to HIV-1 Gag and gp120 DNA vaccination in mice (96, 97) and to potentiate CD8+ T-cell-mediated long-term immunity (96–99). An IL15 expression vector has also been reported to exert a synergistic
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increase in DNA vaccine-induced recall responses to HIV-1 Env glycoprotein when combined with an IL-21 vector (100) and to enhance T-cell memory subsets in macaques primed with SIV DNA (101). An IL-15 expression vector has also been reported to increase the cellular immune response to simian/human immunodeficiency virus (SHIV) vaccination in macaques (102). In other studies, although an IL-15 expression vector was found to induce some minor increases in humoral and cell-mediated immunity in macaques in response to SIV gag DNA vaccination, IL-15 did not improve outcome following SHIV challenge (103). Treatment of macaques with exogenous IL-15 during acute SIV infection has also been reported to enhance virus replication and to accelerate onset of disease in spite of an increase in NK cells and SIV-specific CD8+ T cells (104). Other studies have shown that IL-15 is effective at increasing the anti-tetanus toxoid IgG1 and IgG2 response when coexpressed together with the antigen on the surface of liposomes (105). Exogenous IL-15 has also been shown to enhance tetanus toxoid- and influenza-specific CD8+ T-cell responses in rhesus macaques (106) and the antigen-specific antibody response in mice vaccinated with staphylococcal enterotoxin B vaccine (107). IL-15 expression vectors have also been shown to enhance protection against smallpox in animals vaccinated with the Wyeth strain of variola (108), to enhance CTL priming in animals vaccinated with a DNA vaccine encoding HBsAg (109), and to increase the longevity of the CD8+ T-cell response to HBV core antigen DNA vaccine (110). IL-15 expression vectors have also been shown to increase long-term CD8+ T-cell cellular immunity and protection against influenza virus in an influenza DNA vaccination model (99) and to enhance the mucosal and systemic immune response to intranasal vaccination with a foot and mouth disease virus (FMDV) DNA vaccine (111). Intranasal administration of an IL-15 expression vector together with a DNA vaccine encoding HSV glycoprotein B has also been reported to enhance both humoral immunity and primary and memory CD8+ T-cell responses and protection against virus challenge in mice (112). Expression of IL-15 has also been shown to increase the memory CD8+ T-cell response to a HSV glycoprotein B peptide vaccine (113). A recombinant BCG vaccine secreting a BCG 85B antigen–murine IL-15 fusion protein has been shown to exhibit enhanced CD4+ and CD8+ T-cell responses relative to a vector secreting antigen alone (114). An IL-15 expression vector has been shown to reduce parasite replication in chickens vaccinated with an Eimeria acervulina DNA vaccine relative to vaccine alone (115). Recombinant IL-15 has also been shown to reverse the decline in the long-term CD8+ T-cell immune response and enhance protection in mice immunized with a vaccine strain of Toxoplasma gondii (116).
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Taken together, the results of the studies carried out to date suggest that IL-15 expression vectors hold promise as adjuvants for DNA vaccination.
6. Interleukin 18 (IL-18) IL-18 is a pleiotropic cytokine secreted by activated macrophages that was originally described as IFN-γ-inducing factor (117). The adjuvant activity of IL-18 is reviewed together with that of IFN-γ in Section 2.9.
7. Interleukin 21 (IL-21) IL-21 is a type I cytokine produced by activated CD4+ T cells that shares significant homology with IL-2, IL-4, and IL-15 (118) and binds to a heterodimeric receptor composed of the IL-21specific receptor chain IL-21R and the common cytokine receptor gamma chain (gammac). Binding of IL-21 to its receptor induces activation of the Jak1 and Jak3 that in turn phosphorylate and activate STAT1 and STAT3 (119). IL-21 is a key regulator of the cellular immune response and affects the growth, survival, and activation of B cells, T cells, and NK cells (119). In particular IL-21 has been shown to promote differentiation of naïve CD8 T cells into cells with a highly effective cytolytic activity, in response to antigen and co-stimulation (120). IL-21 together with IL-4 also plays an important role in IgG1 and IgG3 isotype switching. Thus, IL-21 has been shown to induce predominately IgG3 production in CD40L-stimulated naïve human B cells and to act synergistically with IL-4 to induce IgG1 production (121). The role played by IL-21 in both innate and adaptive immunity suggests that IL-21 may find application as a vaccine adjuvant. Thus, treatment of mice with an expression vector for either IL21 alone or in combination with an IL-15 expression vector was shown to enhance the cellular immune response to a HIV-1 Env DNA vaccine (100). Expression of IL-21 has also been shown to potentiate both the humoral and cellular immune responses to intravascular vaccination with a DNA vaccine encoding glycoprotein B of HSV-1 (122) and to enhance the immunogenicity of a DNA vaccine against TB (123). As in the case of some of the other more recently characterized cytokines, further studies are needed to determine whether exogenous IL-21 or IL-21
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expression vectors will prove to be effective adjuvants for conventional or DNA-based vaccines, respectively.
8. Granulocyte Macrophage ColonyStimulating Factor (GM-CSF)
GM-CSF has been shown to recruit APCs to the site of antigen processing, to stimulate the maturation of myeloid DCs, and to enhance the antigen-specific CD8+ T-cell response, suggesting that GM-CSF may hold promise as a vaccine adjuvant. Thus, the results of a meta-analysis have shown that administration of GMCSF together with a HBV vaccine increased the seroprotection rate in patients with end-stage renal disease (124). Co-injection of GM-CSF cDNA together with a DNA vaccine based on glycoprotein B of pseudorabies virus (PrV) was found to induce a Th2-type humoral response characterized by production of IgG1 while at the same time inducing a Th1-type cellular response characterized by increased production of IL-2 and IFN-γ by T cells and protection against lethal PrV challenge (125). Vaccination of animals with modified vaccinia virus Ankara (MVA)-expressing GM-CSF has also been shown to enhance the cellular immune response to vector-encoded antigens and to increase the MVAspecific antibody response relative to vaccination in the absence of GM-CSF expression (126). GM-CSF was effective when administered during primary immunization but not in booster immunizations, suggesting that the cytokine is more effective at stimulating naïve T cells than promoting recall of memory T-cell responses (126). Co-expression of GM-CSF and antigen in DNA prime and adenovirus boost immunizations has also been shown to be effective in stimulating an antigen-specific CTL response (127). Further studies are warranted to determine the full potential of GM-CSF as an adjuvant for specific antigens.
9. Flt-3 Ligand The Fms-like tyrosine kinase-3 ligand (Flt-3L) is a hematopoietic growth factor that has been shown to efficiently induce DC expansion in vivo (128) and to increase the cell-mediated immune response to HIV-1 DNA vaccines (129). In spite of the ability of Flt-3L to act as a DC growth factor and hence increase antigen presentation, and the large number of studies using Flt-3L as an adjuvant in conjunction with tumor antigens in antitumor vaccination studies, Flt-3L has received limited attention as an
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adjuvant for antiviral vaccines (70, 129–132). Further studies are needed to determine whether addition of exogenous Flt-3L or co-expression of Flt-3L will prove to be an effective adjuvant for conventional or DNA-based antiviral or anti-bacterial vaccines.
10. Conclusions Cytokines that play key roles in the regulation of innate and adaptive immunity would be expected to enhance the immune response to vaccination. Thus, recombinant analogues of a number of cytokines including IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, and Flt-3 ligand have been reported to exhibit adjuvant activity in vaccination models. The results obtained for a given cytokine have, however, for the most part, been variable. The relatively short serum half-lives of recombinant analogues of certain cytokines may contribute to a less than optimal adjuvant activity and a number of approaches have been adopted to overcome these difficulties. These include incorporation of cytokines into liposomes and co-administration of cytokine expression vectors together with DNA vaccines. Perhaps more importantly, cytokines act locally as part of a network of intercellular messengers and the results obtained by administration of a single recombinant cytokine may not be the same as those exerted by the endogenous counterpart acting in conjunction with other cytokines and chemokines. Furthermore, cytokines such as the type I IFNs can exert opposing effects on the humoral immune response to vaccination depending upon the dose administered. Thus, low doses of IFN-α have been reported to enhance the humoral response to vaccination, while high doses of IFN-α have been reported to inhibit immunoglobulin production (25, 46, 52). Furthermore, systemic administration of high doses of certain cytokines including IL-2, IL-12, IFN-α, and IFN-γ is associated with significant toxicity (50–52, 77, 133–135), which would preclude their use as vaccine adjuvants at such doses. Thus, the challenge will be to find the means of overcoming these difficulties in order to reveal the full potential of cytokines as vaccine adjuvants. References 1. Mutwiri, G., Benjamin, P., Soita, H., Babiuk, L. A. (2008) Co-administration of polyphosphazenes with CpG oligodeoxynucleotides strongly enhances immune responses in mice immunized with Hepatitis B virus surface antigen. Vaccine 26, 2680–2688.
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306
65.
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69.
70.
71.
72.
73.
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SUBJECT INDEX
A
Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Caspase–1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7, 114 CD11c . . . . . . . 107–108, 110–112, 153, 163–164, 174, 179, 181, 183–184, 204, 291 CD14 . . . . . 22, 106–112, 115, 125, 134–135, 141, 143, 145 CD1d . . . . . . . . . . . . . 150–152, 154–156, 159–161, 163–165 CD4+ . . . . . . . . . 5, 104, 112, 150–151, 226–227, 263–264, 289, 292, 297, 299–300 CD40 . . . . . . . . . 11, 106–107, 109–110, 123, 128–129, 132, 136–139, 153, 163–165, 291, 300 CD80 . . . . . . . . . . . . . 11, 106, 107, 109–110, 123, 135–137, 139–140, 142–143, 145, 153, 163, 164, 291–292 CD86 . . . . . . . . . 11, 106, 107, 109–110, 123, 129, 135–139, 143, 153, 163–164, 291–292 Cell-mediated immunity . . . . . . . . . . . . . . 114, 252, 266, 299 Cellular response . . . . . . . . . . . . . . . . . . . . . . . . . . 254, 297, 301 CervarixTM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16–17, 26 CFSE dilution assay . . . . . . . . . . . . . . . . . . . . . . . . . . 217, 223, 224 proliferation assay . . . . . . . . 232, 235, 244–245, 276–277 Chemokine . . . . . . . . . 6, 106, 109, 119, 123–124, 136–137, 145, 165, 170, 291, 302 Cholera toxin (CT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126, 262 Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74–75 Clinical trial . . . . . . . . . . 19, 22, 25–26, 29, 36–37, 252, 294 Co-inhibitory signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Complement . . . . . . . 6, 8, 10, 201, 202, 205, 207–210, 289 Complete Freund’s adjuvant (CFA) . . . . . . . . . . . 5, 7, 59–70 Confocal microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 Correlates of protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Co-stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–4, 300 Co-stimulatory signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 CpG . . . . . . . . . . . 8, 24, 30, 94, 96, 106, 108, 110, 112, 114, 118, 132, 134, 136, 138–140, 144–146, 169, 262, 288–291, 296 Critical micelle concentration (CMC) . . . . . . . . . . . . . . . . . 80 Cross-priming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291–292 CTLA-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3, 11 C-type lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9, 288 Cytokines . . . . . . . . . . 9–11, 22, 33, 105–107, 109–114, 119, 122–126, 128, 131–132, 134–138, 140, 142–143, 145, 150–151, 153, 161–162, 165, 169–170, 214–216, 218–221, 225–227, 232–235, 243–244, 247–248, 262, 268, 277, 282, 287–302 Cytometric bead analysis (CBA) . . . . . . . . . . . 153, 162, 214, 216, 219, 221, 227, 280 Cytotoxic T lymphocytes (CTL) . . . . . . . . . . . . . . 7, 92, 151, 233–235, 237, 239–242, 244, 246–247, 265, 287, 297, 299, 301
Adju-PhosTM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41, 50 Adjuvant system. . . . . . . . . . . . . . . . . . . . . .16–18, 24, 25, 114 Adsorption . . . . . . 17, 24, 41, 44–46, 48–49, 51–56, 84, 92, 94–95, 97–98, 100, 204 AlhydrogelTM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Alum . . . . . . . . . 3, 6–7, 42, 54, 103–104, 114, 117, 287–289 Aluminium adjuvants . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61, 70 Aluminium hydroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61, 65 Aluminum phosphate . . . . . . . . . 6, 17, 41–42, 45–50, 54–55 Aminoalkyl glucosaminide phosphates (AGP) . . . . 105, 113 Animal model . . . . . . . . . 20, 33, 38–39, 171, 200, 251–258, 263–264, 279–280 Antibody anti-meningococcal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 avidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9, 104, 200, 232 isotype . . . . . . . . . . . . . . . . . . 7, 10, 42, 248, 279, 291, 300 neutralising . . . . . . . . . . . . . . . . . . . . . . . . . . . 200, 254, 256 secreting cells (ASC) . . . . . . . . . . . . . . . . . . . 266, 277, 279 secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 titre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187–197, 201 Antigen recall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215, 219 Antigen-presenting cells (APC) . . . . . . . 3, 8–9, 16–17, 103, 107, 110–111, 150, 152–153, 159, 161–163, 170–171, 175, 180–181, 218, 225–226, 234–235, 288, 292, 295 Apoptosis . . . . . . . . . 136–137, 139–140, 163, 204, 292–293, 297–298 AS04 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15–26 Avidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9, 104, 200, 232
B B7–1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 B7–2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Bacillus Calmette Gu´erin (BCG) . . . . . . 61, 63, 67–68, 119, 127–129, 299 Balb/c mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19, 69, 266, 271 B cell proliferation . . . . . . . . . . . . . . . . . . . . . . 135, 136, 143, 145 B cell antigen receptor (BCR) . . . . . . . . . . . . . . 131, 137–138 Bicinchoninic acid assay (BCA) . . . . . . . . . . . . . . . . . . . . . . . 51 Broncho-alveolar lavage (BAL) . . . . . . . . 118, 120, 124, 126 Buffy coat . . . . . . . . . . . . . . . . . . . . . . . . . . . 105, 108, 124, 134, 140–141
C Carboxyfluorescein succinimidyl ester (CFSE). . . . . . . .135, 143–144, 175, 182, 215, 217, 223–224, 228, 235, 244–245, 248
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312 Subject Index D
H
Danger signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194, 223 Dectin-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4, 288 Dehydration-rehydration . . . . . . . . . . . . . . . . . . . . . . . . . 80, 87 Delivery vehicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91–93 Denaturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Dendritic cells (DC) preparation . . . . . . . . . . . . . . . . . . . . . . . 105–106, 108–109 stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109, 112 Depot effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Detergent removal technique . . . . . . . . . . . . . . . . . . . . . . . . . 80 Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 76, 80–81, 83, 86, 88 Diphtheria toxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .205–206 DNA vaccines . . . . . . . . . . . . . . . . . . . . . . 31–32, 42, 296–302 DOTAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75, 134, 140–141 Drug Master File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Heat-labile enterotoxin (LT) . . . . . . 262, 265–268, 273, 279 Heat shock proteins (Hsp) . . . . . . . . . . . . . . . . . . 3, 4, 10, 118 HEK 293 human embryonic kidney cell . . . . . . . . . . . . . . 105 Hepatitis B surface antigen (HBsAg) . . . . . . . . . . . . . . 19–21 Hepatitis B virus (HBV) . . . . . . . . . . 42, 294–296, 299, 301 High pressure homogenization . . . . . . . . . . . . . . . . 78–79, 86 His-tagged Eα-GFP preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172–173 HL60 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 HLA-DR . . . . . . . . . . . . . . . . . . . . . . . . . . . 106, 109, 111, 139 Human immunodeficiency virus HIV . . . . . 39, 92–95, 263, 296–301 Human papillomavirus (HPV) . . 16, 20–21, 24–26, 42, 104 Human peripheral blood mononuclear cells (PBMC) . . . 7, 25, 108, 112, 114, 124, 134, 136, 140–145, 155–156, 159–160, 164, 236 Humoral response . . . . . 10, 19, 65, 287, 292–297, 301–302
E Efficacy/toxicity (E/T) ratio . . . . . . . . . . . . . . . . . . . . 251–258 ELISA (Enzyme-linked immunosorbent assay) plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190, 192–194, 267 coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192–193 washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 Emulsifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60, 62–64, 253 Emulsion oil-based . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3, 5, 10 Encapsulated microparticles . . . . . . . . . . . . . . . . . . . . . . 96–97 Eosinophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–7 Ethanol injection technique . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Extrusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75, 78, 81, 86 Eα-GFP . . . . . . . . . . . . . . . 171–173, 176, 178, 181, 183–184
F FACS . . . 107, 111, 134–135, 141, 143–144, 151, 154–157, 159–160, 163, 175, 180–181, 218, 223–225 FENDrixTM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16, 17, 26 Ficoll . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134, 140 Filtration . . . . . . . . 18, 20, 46, 54, 74–76, 78–81, 85–88, 97, 268, 277 Flagellin . . . . . . . . . . . . . . . . . . . . 118–120, 125, 132, 139, 141 Flow cytometry . . . . . . . . . 22, 109–110, 120, 123, 134, 138, 140–144, 146, 152, 155–157, 171–172, 175–176, 180–181, 184, 203, 214, 232, 238, 247–248 Flt3Ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 Flu-like syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294 Formulation . . . . . . . 6, 18–20, 23–24, 30, 34, 36–37, 41–56, 73–89, 91–100, 144, 169, 188, 199–200, 253, 257, 262, 266, 269–272, 281 Functional antibody assays . . . . . . . . . . . . . . . . . . . . . . 199–210
G α-GalCer . . . . . . . . . . . . . . . . . . . 150–156, 159–162, 164–165 Gel-liquid crystalline transition temperature (Tc) . . . . . . 75, 77–80, 86, 263–264 Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34, 36–37 Geographical concept of immunity . . . . . . . . . . . . . . . . . . . . . 5 Glycolipid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149–165 GM-CSF . . . . . 106, 108–109, 121, 125, 175, 288, 301–302 Granulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6, 301 Granuloma . . . . . . . . . . . . . . . . . . . . . . . 6, 60, 62, 69, 255, 258 Guidelines . . . . . . . . . . . . 20, 30–33, 36, 43, 45, 85, 270, 282
I IFN-α . . . 109–110, 132, 139, 145–146, 289, 292–295, 302 IFN-γ . . . . 10, 104, 106, 109–112, 132, 139, 145–146, 162, 214–220, 222, 225–226, 234, 240, 242–243, 288–293, 295–297, 300–302 IgA . . . . . . . . . . . 10, 189, 262, 267, 274–275, 278, 281, 293 IgE . . . . . . . . . . . . . . . . . . . . . . . . . . . 7, 10, 190–192, 194, 295 IgG1 . . . 7, 42, 136, 138, 189, 191, 204, 268, 289, 295, 297, 299–301 IgG . . . 24–25, 133, 137–138, 174, 178–179, 189–191, 194, 262, 267–269, 274–275, 278, 281, 291, 293 IgG2a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10, 19–20, 268, 289 IL-10 . . . . . . . . . . . . . . . 11, 22, 106–107, 109, 111–112, 121, 135–138, 145, 290, 292 IL-12. . . . . .10–11, 104, 123, 127, 129, 138, 165, 169, 214, 288–289, 295–298, 302 IL-13 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10–11 IL-15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288, 292, 298–300 IL-18 . . . . . . . . . . . . . . . . . . . . 7, 114, 288, 295–296, 300, 302 IL-1R1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 IL-1β . . . . . . . . . . 7, 9–11, 22, 104, 106–107, 109, 111–112, 114, 117, 119, 121, 123, 125–127, 129, 132, 135–138, 145, 165, 169, 214–216, 219–220, 288–290, 292, 295–300, 302 IL-21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288, 293, 298–300 IL-2. . . . . . . . . . . . . . 152–153, 160–162, 216, 218, 225–226, 233–235, 237, 240, 243, 245–247, 288–289, 297–298, 300–302 IL-4 . . . 6, 7, 10–11, 17, 106, 108–109, 111, 162, 214, 216, 289, 295, 298, 300 IL-5 . . . . . . . . . . . . . . . . . . . . . 10–11, 162, 216–217, 222, 289 Immune response . . . . . 2–5, 7, 9–11, 15–20, 25, 30, 33–36, 43, 61–63, 85, 91, 103–104, 109–110, 114, 131–132, 138, 151, 169–171, 189, 201, 205, 208, 231–232, 255–256, 261–263, 270, 279–280, 287–302 Immunization intragastric. . . . . . . . . . . . . . . . . . .266, 270–271, 279–280 intramuscular . . . . . . . . . . . . . . . . . . . . . . . . . 266, 270, 272 intranasal . . . . . . . . . . . . . . . 262, 266, 269, 271, 279–280 rectal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270–271, 281 vaginal . . . . . . . . . . . . . . . . . . . . . . 270–271, 280–281, 296 Immunofluorescence . . . . . . . . . . . . . 171–172, 174, 176, 179 Immunogenicity . . . 2, 16, 19, 30, 33–34, 39, 189, 262, 300 Immunopotentiator . . . . . . . . . . . . . . . . . . . . . . . . 2, 61, 74, 97 Incomplete Freund’s adjuvant (IFA) . . . . . . . . . . . . . 287–288
VACCINE ADJUVANTS Subject Index 313 Inflammasome . . . . . . . . . . . . . . . . . . . . . 7, 103, 114, 125, 288 Influenza . . . . . . . 10–11, 37, 60, 66, 88, 203, 262, 270, 275, 291, 293–297, 299 INKT hybridoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Innate immunity . . . . . . . . . . . . 114, 118, 213–214, 287–288 Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 Interferon gamma (IFN-γ) . . . 10, 111–112, 162, 214–220, 222, 225–226, 234, 240, 242–243, 288–293, 295–297, 300–302 Interferon (IFN) . . . . . . . 3, 10, 33, 104, 106, 108–109, 146, 288–295 Intracellular cytokine staining . . . . . . . . . . . . . . 232, 235, 243 ISCOM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169, 262 ISCOMATRIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Isoelectric point (IEP) . . . . . . . . . . . . . . . . . . . . . . . . 45–46, 56
L Ligand exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46, 55 Limiting dilution analysis (LDA) . . . . . . . . . . . . . . . 232, 234, 238–239, 241, 247 Limulus amebocyte lysate (LAL) test . . . . . . 19, 54, 83, 113 Lipoplexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Lipopolysaccharide (LPS) . . . 3, 7–8, 17–18, 21–23, 25–26, 55–56, 61, 74, 88, 92, 104, 106, 109, 110, 112–113, 118–120, 125–129, 139–141, 161, 169, 175, 181–182 Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73–88, 169 Local reaction . . . . . . . . . . . . . . . . . . . . . . . . . . 6, 252, 254–258 LTK63 . . . . . . . 262–263, 265–270, 273, 276–277, 279–281 Luminex . . . . . . . . . . . . . . . . . . . . . . . . . 33, 106–107, 109–111 Lymph node (LN) cells . . . . . . . . . . . . . . . . . . . . 219, 222–223
M Macrophage BAL lavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124, 126 Bone marrow derived (BMDM) . . . 118–119, 122, 126, 161, 175, 182 Magnetic beads . . . . . . . . . . . . . 106–108, 112, 121, 125, 159 Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . 2, 17, 37 Memory B cells . . . . . . . . . . . . . . . 24–26, 131–133, 136–138 Meningitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 Meningococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 MF-59 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6, 169 MHC class I . . . . . . . . . . . . . . . . . . . . . . . . . . 43, 150, 291–292 MHC-class II . . . . . . . . . . . . . . . . . . . . . . . . . . 4, 109, 131, 295 MHC-I multimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232, 238 Microparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92–98 Mineral oil . . . . . . . . . . . . . . . . . . . . . . 60–65, 68–70, 253, 258 Mitogen-activated protein kinases (MAPK) . . . . . . . . . . 8–9 Modified vaccinia virus Ankara (MVA) . . . . . . . . . . 296, 301 Monoclonal . . . . . . . . . . . . . 42, 106–109, 120, 134, 136, 154 Monocyte-derived dendritic cells . . . . . . . . . . . . . . . . . . . . 105 Monocytes . . . . . . . . . . 6, 105–111, 113–114, 118–119, 121, 124–125, 143, 145, 161, 291 Mononuclear cells (MNC) . . . . . . . 7, 22, 33, 108, 112, 125, 152–154, 157–161, 233, 236, 254, 279 Monophosphoryl lipid A (MPL) . . . . . . . . . 17–18, 104, 262 Montanide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62–64, 68 Mouse weight gain test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 MTT assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123, 127 Mucosal adjuvants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261–282 Multilamellar Vesicles (MLVs) . . . . . . . . . . . . . . . . 78–79, 88 Multi-photon laser scanning microscopy (MPLSM) . . . 172 Multiplex analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 Muramyl dipeptide (MDP) . . . . . . . . . . . . . . . . . . . 8, 74, 125
Mycobacteria . . . . . . . 60–61, 64, 67–68, 118, 120, 127, 129 MyD88 . . . . . . . . . . . . . . . . . . . . 7, 9, 104, 120, 132, 290–291
N Nanoparticles . . . . . . . . . . . . . . . . . . . . . . 30, 91–100, 296, 298 Natural killer T cell (NKT) activation . . . . . . . . . . . . . . . . . . . . 153, 163, 165, 172, 176 culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152, 160 isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152, 157 Neutrophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6, 204 NF-κB . . . . . . . 8–9, 104, 108, 113–115, 132, 145–146, 162, 216, 218, 225 NOD-like receptors . . . . . . . . . . . . . . . . . . . . . . . 4, 9, 114, 288 Non-clinical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29–39 Non-human primate (NHP) . . . . 33, 38, 155, 159–160, 164 No-Observed-Adverse-Effects-Level (NOAEL) . . . 35, 38
O Oil-in-water emulsions (O/W) . . . . . . 6, 63–64, 92, 96, 98, 253, 256 Oligodeoxynucleotides (ODN) . . . 132, 134, 136, 138–142, 144–146, 287–288 Opsonization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8, 10 Opsonophagocytic antibodies . . . . . . . . . . . . . . . . . . . . . . . 203 Osmolarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Ovalbumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5, 59, 63, 66
P Particle size distribution (PSD) . . . . . . . . . . . . . . . . . . . 82–83 Pathogen. . . .1, 4, 8–11, 16–17, 35, 92, 104, 132, 169, 199, 201–202, 205, 208, 213, 261–262, 277, 279–280, 290–291, 295 Pathogen-associated molecular patterns (PAMPS) . . . . . . 4, 8–9, 16, 18 Pathogen recognition receptors (PRRs) . . . 4, 8–9, 213, 288, 290 Peptidoglycan . . . . . . . . . . . . . . . . . . . 8, 56, 61, 118, 139, 288 Peripheral blood mononuclear cells (PBMC) . . . . . . . . 7, 22, 24–26, 33, 108, 112, 134, 140–145, 152, 155–156, 159–160, 164, 215, 218–219, 222–223, 233–234, 236, 238, 240–245, 254 Peripheral mononuclear cells (PMN) . . . . . . . 7, 22, 33, 108, 134, 159, 233, 236, 254 Pharmacopoeia . . . . . . . . . . . . . . . . . . . 45, 49, 68–69, 82, 201 Phorbol myristate acetate (PMA) . . . . . . . 23, 235, 243–244 Phosphatidylserine (PS) . . . . . . . . . . . . . . . . . . . . . . . . . 74, 140 Phospholipid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46, 74, 118 Plasma cell . . . . . . . . . . . . . . . . . . . . . . . . . 24–25, 61, 131, 291 Point of zero charge (PZC) . . . . . . . . . . . . . . . . . . . . . . . 45–46 PolyI:C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141, 290–291 Polylactide-co-glycolides(PLG) . . . . . . . . . . . . . . . . . . . 92–99 Polymerase chain reaction (PCR) . . . . . . 110–111, 121, 126 PRR ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4, 288 Pyrogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19, 37, 66–69
Q QS-21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 QuilA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
R Rabbit pyrogenicity assay . . . . . . . . . . . . . . . . . . . . . . . . . 19, 66 RAMOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134, 142, 145 Reactogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35, 43, 62
VACCINE ADJUVANTS
314 Subject Index
Reference serum . . . . . . . . 190, 193, 203, 205, 273, 275, 281 Regulatory T cells (Treg) . . . . . . . . . . . . . . . . . . . . . . 9, 11, 297 Repeat-dose toxicity . . . . . . . . . . . . . . . . . . . . . . . 34–35, 37–38 Resident peritoneal macrophages . . . . . . . . . . . . . . . . . . . . 124 RIG-like helicases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
S Safety pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Saponin . . . . . . . . . . . . . . . . . . . . . . . . . . 2, 10, 30, 74, 235, 244 Saposin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153, 161, 165 Seroconversion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .294 Serum bactericidal antibodies (SBA) . . . . . . . . 200–205, 207 SHIV vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0, 4 Signal 1 facilitators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–11 Signal 2 facilitators . . . . . . . . . . . . . . . . . . . . . . . 2–4, 8–11, 170 Simian immunodeficiency virus (SIV) . . . . . . . . . . . 296, 299 Single-dose toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Smallpox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Small Unilamellar Vesicles (SUVs) . . . . . . . . . . . . . 78–81, 87 Solvent evaporation method . . . . . . . . . . . . . . . . . . . 92, 95–96 Sonication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78, 86, 88 Specol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Spleen cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 Standard curve . . . . 51, 53, 55, 65, 188, 190–191, 193–195, 220–221 Stranger motifs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Sublingual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294–295 Systemic toxicity . . . . . . . . . . . . . . . . . . . . . . . . . 20, 30, 68, 294
TNF-α . . . . . . . . . . . . . . . . . . . . . . . . . . . 22–23, 106–107, 109, 111–112, 121, 137–138, 162, 216, 218, 225–226, 243, 292 Toll-like receptors (TLR) . . . . 4, 7–9, 11, 16–18, 22–24, 38, 104–106, 108–114, 118–120, 122–124, 128–129, 132–134, 138–141, 144–145, 213, 288–290 Toxicity developmental and reproductive . . . . . . . . . . . 35–36 Toxin neutralisation . . . . . . . . . . . . . . . . . . 200–201, 205–206 Trehalose dimycolate (TDM) . . . . . . . . . . . . . . . . . . . . . . . . 74 Tumor infiltrated lymph nodes (TILN) . . . . . . . . . . 233, 236 Two photon imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . 175, 182 Two-signal model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3–4 Type III interferon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 Type II interferon . . . . . . . . . . . . . . . . . . . . . . . . . 289–290, 295 Type I interferon . . . . . . . . . . . . . . . . . . . . . 104, 146, 288–294
U U937 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22–23 Ultracentrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76, 81 Uric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–7, 10
V Vaccine efficacy . . . . . . . . . . . . . . . 33, 39, 105, 232, 251, 279, 294 safety. . . . . . . . . . . . . . . . 2, 22, 25, 30–31, 34, 38–39, 257 Vero cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200–201, 205–206 Virus neutralisation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
T
W
T cell . . . . . 2, 4, 7, 9, 17, 104, 109, 112, 117, 122, 125–126, 131–132, 137, 140, 149–165, 170–172, 175, 180, 182, 184, 213–228, 231–248, 253, 262–264, 266, 268, 275–277, 281, 288–292, 295–301 Tetanus toxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Tetramer staining. . . . . . . . . . . . . . . . . . . . .154–156, 160–161 TGF-β. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .214 Th1 . . . . . . . 2, 9–11, 43, 104, 114, 117, 132, 150–151, 162, 165, 214, 216, 221, 227, 289, 295–297, 301 Th2 . . . . 7, 9, 11, 17, 43, 114, 150–151, 162, 165, 214, 216, 221, 227, 289, 295, 301 Therapeutic vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Thiogycollate activated macrophages . . . . . . . . . . . . . . . . . 124 TLR ligands . . . . . . . . . . . 4, 9, 104–106, 108–111, 113–114, 119–120, 132, 134, 138–141, 144
Wash intestinal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 nasal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 vaginal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 Water-in-oil emulsion (W/O) . . . . . . 5–6, 63–64, 68, 95–98 Water in oil in water emulsion (W/O/W) . . . . . . 92, 96, 98 Whole blood cytokine production . . . . . . . . . . . . . . . . . . . . . . . . 107, 111
Y Y-Ae antibody preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172, 175
Z Zeta potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82–84