Measles: Pathogenesis and Control (Current Topics in Microbiology and Immunology)

Current Topics in Microbiology and Immunology Volume 330

Series Editors Richard W. Compans Emory University School of Medicine, Department of Microbiology and Immunology, 3001 Rollins Research Center, Atlanta, GA 30322, USA Max D. Cooper Department of Pathology and Laboratory Medicine, Georgia Research Alliance, Emory University, 1462 Clifton Road, Atlanta, GA 30322, USA Tasuku Honjo Department of Medical Chemistry, Kyoto University, Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan Hilary Koprowski Thomas Jefferson University, Department of Cancer Biology, Biotechnology Foundation Laboratories, 1020 Locust Street, Suite M85 JAH, Philadelphia, PA 19107-6799, USA Fritz Melchers Biozentrum, Department of Cell Biology, University of Basel, Klingelbergstr. 50–70, 4056 Basel Switzerland Michael B.A. Oldstone The Scripps Research Institute, Department of Immunology and Microbial Science, 10550 N. Torrey Pines, La Jolla, CA 92037, USA Sjur Olsnes Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello 0310 Oslo, Norway Peter K. Vogt The Scripps Research Institute, Dept. of Molecular & Exp. Medicine, Division of Oncovirology, 10550 N. Torrey Pines. BCC-239, La Jolla, CA 92037, USA

Diane E. Griffin • Michael B.A. Oldstone Editors

Measles Pathogenesis and Control

Editors: Diane E. Griffin Johns Hopkins University School of Hygiene and Public Health Department of Molecular Microbiology 615 N. Wolfe Street Baltimore, MD 21205 USA [email protected] [email protected]

ISBN 978-3-540-70616-8

Michael B.A. Oldstone Scripps Research Institute Department of Immunology and Microbial Science 10550 N. Torrey Pines Road La Jolla, CA 92037 USA [email protected]

e-ISBN 978-3-540-70617-5

DOI 10.1007/978-3-540-70617-5 Current Topics in Microbiology and Immunology ISSN 0070-217X Library of Congress Catalog Number: 2008931704 © 2009 Springer-Verlag Berlin Heidelberg This work is subject to copyright. All rights reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September, 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publisher cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Cover design: WMXDesign GmbH, Heidelberg, Germany Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

Contents

Introduction ......................................................................................................

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Making It to the Synapse: Measles Virus Spread in and Among Neurons ............................................................................ V.A. Young and G.F. Rall Modeling Subacute Sclerosing Panencephalitis in a Transgenic Mouse System: Uncoding Pathogenesis of Disease and Illuminating Components of Immune Control ............ M.B.A. Oldstone

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Measles Studies in the Macaque Model ................................................. R.L. de Swart

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Ferrets as a Model for Morbillivirus Pathogenesis, Complications, and Vaccines ................................................................... S. Pillet, N. Svitek, and V. von Messling

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Current Animal Models: Cotton Rat Animal Model ........................................................................................... S. Niewiesk

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Current Animal Models: Transgenic Animal Models for the Study of Measles Pathogenesis ................................................... 111 C.I. Sellin and B. Horvat

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Molecular Epidemiology of Measles Virus ............................................ 129 P.A. Rota, D.A. Featherstone, and W. J. Bellini

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Human Immunology of Measles Virus Infection .................................. 151 D. Naniche

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Measles Control and the Prospect of Eradication................................. 173 W.J. Moss

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Measles: Old Vaccines, New Vaccines .................................................... 191 D.E. Griffin and C.-H. Pan

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Measles Virus for Cancer Therapy ........................................................ 213 S.J. Russell and K.W. Peng

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Measles Virus-Induced Immunosuppression ........................................ 243 S. Schneider-Schaulies and J. Schneider-Schaulies

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Hostile Communication of Measles Virus with Host Innate Immunity and Dendritic Cells ................................................................ 271 B. Hahm

Index .................................................................................................................. 289

Contributors

W.J. Bellini Measles, Mumps, Rubella and Herpesvirus Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA R.L. de Swart Department of Virology, Erasmus MC, University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands, [email protected] D.A. Featherstone Immunization, Vaccines and Biologicals, World Health Organization, Geneva, Switzerland D.E. Griffin Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe St. Rm E5132, Baltimore, MD 21205, USA, [email protected] B. Hahm Departments of Surgery and Molecular Microbiology and Immunology, Center for Cellular and Molecular Immunology, University of Missouri-Columbia School of Medicine, One Hospital Dr., Columbia, MO 65212, USA, [email protected]. edu B. Horvat U758-ENS Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France, branka. [email protected] W.J. Moss Department of Epidemiology and the W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore MD, USA, [email protected]

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Contributors

D. Naniche Barcelona Center for International Health Research (CRESIB), Hospital Clinic, Institut d’Investigacions Biomedicas August Pi i Sunyer (IDIBAPS), C/Rossello 132, 4 08036, Barcelona, Spain, [email protected] S. Niewiesk College of Veterinary Medicine, Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210, USA, niewiesk.1@osu. edu M.B.A. Oldstone The Scripps Research Institute, Department of Immunology and Microbial Science, La Jolla CA, USA, [email protected] C.-H. Pan Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe St. Rm E5132, Baltimore, MD 21205, USA K.W. Peng Mayo Clinic, Department of Molecular Medicine, 200 1st Street SW, Rochester, MN 55905, USA S. Pillet INRS-Institut Armand-Frappier, University of Quebec, 531, boul. des Prairies, Laval, QC, H7V 1B7, Canada P.A. Rota Measles, Mumps, Rubella and Herpesvirus Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA, [email protected] S.J. Russell Mayo Clinic, Department of Molecular Medicine, 200 1st Street SW, Rochester, MN 55905, USA, [email protected] J. Schneider-Schaulies Institute for Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany S. Schneider-Schaulies Institute for Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany, [email protected]

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S. Svitek INRS-Institut Armand-Frappier, University of Quebec, 531, boul. des Prairies, Laval, QC, H7V 1B7, Canada C.I. Sellin U758-ENS Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France V. von Messling INRS-Institut Armand-Frappier, University of Quebec, 531, boul. des Prairies, Laval, QC, H7V 1B7, Canada, [email protected] V.A. Young Division of Basic Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA

Introduction

Measles virus, one of the most contagious of all human viruses, has been largely contained by the development and use of a vaccine that was introduced 50 years ago. These two volumes were timed to honor the introduction of the vaccine and to record the enormous advancements made in understanding the molecular and cell biology, pathogenesis, and control of this infectious disease. Where vaccine has been effectively delivered, endemic measles virus transmission has been eliminated. However, difficulties in vaccine delivery, lack of health care support and objection to vaccination in some communities continue to result in nearly 40 million cases and over 300,000 deaths per year from measles. By itself measles virus infection has and still provides some of the most interesting phenomena in biology. Following infection of dendritic cells, measles virus causes a profound suppression of the host’s immune response that lasts a number of months after apparent recovery from infection. Indeed, measles virus was the first virus to be associated with immunosuppression with many of the manifestations to be observed one hundred years later with HIV infection. Measles is also associated with development of both post-infectious encephalomyelitis, an autoimmune demyelinating disease, and subacute sclerosing panencephalitis, a slowly progressive neurodegenerative disorder. How measles virus infects cells, spreads to various tissues and causes disease, as well as the role of the immune response, generation of new vaccines, and use as a vector for gene delivery are topics covered in these two volumes. A unique highlight for readers of this series and those interested in the history of a major and profound biomedical research accomplishment is the chapter written by one of the participants who worked on the initial discovery and use of the vaccine who records the events that occurred at that time. Baltimore, MD La Jolla, CA

Diane E. Griffin Michael B.A. Oldstone

D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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Making It to the Synapse: Measles Virus Spread in and Among Neurons V.A. Young and G.F. Rall(*)

Contents Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Virus: Genome and Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Virus Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Understanding MV CNS Complications: Results from Brains of Infected Individuals and Persistently Infected Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Big Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transgenic Mouse Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vitro Culture Systems and Techniques in Cellular Transport Studies. . . . . . . . . . . . . . . Reverse Genetics for MV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Access to the CNS: Lessons from CDV, PV, and WNV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Spread and Transport in Non-neuronal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Spread in Non-neuronal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of Microtubules and Their Associated Motor Proteins to Achieve Viral Transport Within an Infected Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Spread in Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Movement Within and Among Neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Immune Responses on MV Spread in Neurons. . . . . . . . . . . . . . . . . . . . . . . . . . Remaining Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Measles virus (MV) is one of the most transmissible microorganisms known, continuing to result in extensive morbidity and mortality worldwide. While rare, MV can infect the human central nervous system, triggering fatal CNS diseases weeks to years after exposure. The advent of crucial laboratory tools to dissect MV neuropathogenesis, including permissive transgenic mouse models, the capacity to manipulate the viral genome using reverse genetics, and cell biology advances in understanding the processes that govern intracellular

G.F. Rall Division of Basic Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA, e-mail: [email protected]

D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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trafficking of viral components, have substantially clarified how MV infects, spreads, and persists in this unique cell population. This review highlights some of these technical advances, followed by a discussion of our present understanding of MV neuronal infection and transport. Because some of these processes may be shared among diverse viruses, comparisons are made to parallel studies with other neurotropic viruses. While a crystallized view of how the unique environment of the neuron affects MV replication, spread, and, ultimately, neuropathogenesis is not fully realized, the tools and ideas are in place for exciting advances in the coming years.

Abbreviations BBB CNS CSF EGFP F FIP H HSV IC IFNAR IL IN IV KO L M MAP-2 MIBE MV N NK P PIE PV PrV RAG RNP SLAM SSPE WNV YAC

Blood–brain barrier Central nervous system Cerebrospinal fluid Enhanced green fluorescence protein MV fusion Fusion inhibitory peptide MV hemagglutinin Herpes simplex virus Intracerebral Interferon alpha receptor Interleukin Intranasal Intravenous Knockout MV large (viral polymerase) MV matrix Microtubule associated protein 2 Myelin inclusion body encephalitis Measles virus MV nucleoprotein Neurokinin MV phosphoprotein Postinfectious encephalomyelitis Poliovirus Pseudorabies virus Recombinase activating gene Ribonucleoprotein Signaling lymphocyte activation molecule Subacute sclerosing panencephalitis West Nile virus Yeast artificial chromosome

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Introduction The basic steps of the infectious cycle of any pathogen’s entry into, spread within, and exit from the host offer crucial insights into how pathogens cause disease. As a result, a full understanding of the stages of a microorganism’s life cycle may inform the rational design of therapeutics to prevent or ameliorate consequent disease. Measles virus (MV) is one of the most transmissible microorganisms known; it continues to result in hundreds of thousands of infections annually throughout the world, many of which have serious pathogenic consequences that can result in death. Despite the tremendous progress that has been made in deciphering the basis of MV pathogenesis and in the creation of effective attenuated vaccines, how MV causes disease, including rare, but serious, central nervous system (CNS) complications, remains poorly understood. It is our view that defining the pathogenesis of MV in the CNS necessitates an understanding of the interaction of the virus with the host in trafficking to and spreading within the CNS. This is the focus of this review.

Measles Virus: Genome and Proteins MV is a member of the Paramyxoviridae, within the Morbillivirus genus. Its genome consists of approximately 16,000 bases of nonsegmented, single-stranded negativesense RNA, meaning that the viral genome is transcribed immediately upon entry into the cell. Virions are spherical and enveloped, and the envelope is derived from the host cell as the viral particle buds from the plasma membrane. The viral genome encodes eight proteins, the function of which are briefly noted here. Inserted into the envelope of the virion are the two MV glycoproteins, hemagglutinin (H) and fusion (F). These proteins mediate attachment to cellular receptors and fusion of the virion with the target cell or fusion of an infected cell with an adjacent uninfected cell. The matrix protein (M) lies immediately underneath the virion envelope and serves in virus assembly and budding. The two polymerase proteins, large (L) and phosphoprotein (P), are closely associated with the genome, which is encapsidated by the nucleocapsid (N) protein. The nonstructural proteins V and C, encoded within the P cistron, are also packaged within the virion; these proteins have recently been shown to play a role in counteracting host antiviral immune responses (reviewed in Griffin 2001).

Measles Virus Pathogenesis MV is a human-restricted pathogen that spreads among individuals by release of aerosol droplets. An infected individual will undergo a latent period of 10–14 days followed by a few days of fever, cough, coryza, and rash. Primary infection occurs in the upper respiratory tract, but MV will secondarily infect lymphoid cells (reviewed in Griffin 2001; Rall 2003; Schneider-Schaulies et al. 2003; Sips et al.

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2007), though a contrasting hypothesis has recently been proposed that lymphoid cells are a primary traget for MV replication, followed then by engagement of an as yet unidentified receptor on the basolateral membrance of lung epithelia (de Swart et al., 2007; Leonard et al., 2008). Although the immunological response made by most infected, immunocompetent individuals is sufficient to clear the virus and provide life-long protection, a period of transient immunosuppression is a notorious characteristic of MV infection and is likely the basis of most of the complications and the subsequent fatalities following acute infection (reviewed in Dhib-Jalbut and Johnson 1994; Griffin et al. 2008; Rall 2003). Additional consequences of acute infection include diarrhea pneumonia, and encephalitis.

Immunosuppression Immunosuppression following MV infection can last for weeks, extending beyond the classical MV symptoms. The chief risk of immunosuppression is that it renders infected individuals more susceptible to secondary infections, though precisely how this occurs is not fully understood. In humans, MV-induced immunosuppression is characterized by a loss of delayed type hypersensitivity responses to recall antigens (e.g., tuberculin; Tamashiro et al. 1987), a limited response of lymphocytes to mitogens when cultured ex vivo (Hirsch et al. 1984), and impaired responses to new antigens (Coovadia et al. 1978). To date, a number of mechanisms have been proposed based on animal and cell culture studies, all of which could be pertinent in the natural infection. For example, Griffin and colleagues showed that MV infection of antigen-presenting cells suppresses interleukin-12 (IL-12) production, which then in turn skews the CD4+ T cell response toward a Th2 profile (Karp et al. 1996). This altered CD4+ T cell response leads to inappropriate priming of T cells and a failure of T cells to proliferate following interaction with MV-infected dendritic cells (Fugier-Vivier et al. 1997; Servet-Delprat et al. 2000). These studies correlate well with serum profiles in MV-infected macaques and humans that also show a skewing toward Th2-like cytokines (Atabani et al. 2001; Moss et al. 2002; Polack et al. 2000). In addition to influencing the Th1/Th2 balance, acute MV infection may precipitate immunosuppression by causing an overall lymphopenia, due to effects on T cell proliferation and progression through the cell cycle (Naniche et al. 1999; Niewiesk et al. 1999, 2000; Schnorr et al. 1997), as well as specifically inhibiting immune function via the production of unidentified, immunosuppressive molecules from infected T cells (Sun et al. 1998). A detailed discussion of MV-induced immunosuppression can be found in other chapters within this volume.

CNS Complications Following Acute MV Infection Approximately 1 in 100,000 acutely infected individuals later go on to develop CNS complications. These diseases differ in terms of immune status of the affected

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host, onset of symptoms, presence of MV within the CNS, host survival rate, and neuropathological findings. These are briefly discussed below.

Subacute Sclerosing Panencephalitis Subacute sclerosing panencephalitis (SSPE) is a slow, progressive disease that is invariably fatal, and can occur from 1 to 15 years following acute MV infection (Dubois-Dalcq et al. 1974). Children are far more likely to develop this complication than adults (reviewed in Johnson 1998). The disease initially manifests as subtle cognitive losses, progressing to more overt cognitive dysfunction, followed by motor loss, seizures, and eventual organ failure in virtually all affected individuals. The rate of SSPE occurrence ranges from 1 in 10,000–300,000 acute MV infections (reviewed in Rima and Duprex 2005; Takasu et al. 2003). Neurons are predominantly infected, though at late times of infection, oligodendrocytes, astrocytes, and endothelial cells may also be involved (reviewed in Rima and Duprex 2005). SSPE affects both gray and white matter and is histologically characterized by the presence of cellular inclusion bodies, inflammation, glial activation, loss of blood–brain barrier (BBB) integrity, and neuronal loss (reviewed in Dhib-Jalbut and Johnson 1994; Rall 2003). A serologic hallmark of SSPE, as compared to the other CNS complications, is the elevation of measlesspecific antibodies in the blood and cerebrospinal fluid (CSF) (Dubois-Dalcq et al. 1974). Importantly, evidence from brain biopsies of SSPE patients indicates that infected neurons do not release budding virus (Paula-Barbosa and Cruz 1981). Based on extensive sequencing studies of MV from these specimens and from cells persistently infected with MV isolates from SSPE patients, it has been proposed that the failure of infected neurons to produce complete extracellular virus may be due to defects in protein expression caused by extensive point mutations in the envelope-associated genes, H, F, and M (Cattaneo et al. 1988; reviewed in DhibJalbut and Johnson 1994; Rima and Duprex 2005), though what role these viral proteins play in neuronal spread of MV and how mutations may affect MV biology in infected neurons are not known.

Postinfectious Encephalomyelitis Postinfectious encephalomyelitis (PIE) occurs more frequently than SSPE, affecting approximately 1 in 1,000 infected individuals. Symptoms of PIE normally appear 5–14 days after the characteristic MV rash but can predate the rash (reviewed in Johnson 1998). This complication is thought to be an autoimmune reaction, perhaps to myelin basic protein (Johnson et al. 1984). MV antigen and nucleic acids have not been detected in PIE brain biopsies by immunohistochemistry or in situ hybridization (Johnson et al. 1984; reviewed in Dhib-Jalbut and Johnson 1994; Norrby and Kristensson 1997), supporting the notion that this

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is an autoimmune disease. Additional hallmarks of PIE include perivascular inflammation and demyelination (reviewed in Norrby and Kristensson 1997). Unlike SSPE, intrathecal production of MV antibodies has only been found in a few cases of PIE. Affected individuals present with seizures, deafness, ataxia, and movement disorders. There is an approximate 25% mortality rate associated with PIE, and survivors are likely to suffer from frequent neurologic sequelae.

Measles Inclusion Body Encephalitis Measles inclusion body encephalitis (MIBE), a rare CNS complication following acute MV infection, has been described in children and adults receiving immunosuppressive drugs and therefore is thought to chiefly affect immunocompromised hosts. The neurologic disease appears 3–6 months after the acute MV rash (reviewed in Dhib-Jalbut and Johnson 1994; Johnson 1998). As the name suggests, MIBE is characterized by inclusion bodies in both neurons and glia, with accompanying neuronal loss but an overall lack of inflammation (reviewed in Dhib-Jalbut and Johnson 1994; Johnson 1998; Norrby and Kristensson 1997; Rall 2003). Measles antigen is present in the brain, and virus has been isolated directly from the brains of affected individuals (Johnson 1998). MIBE differs from SSPE in the absence of elevated serum and cerebrospinal fluid neutralizing antibodies (reviewed in Dhib-Jalbut and Johnson 1994; Rima and Duprex 2005). The disease course is relatively short, lasting from days to weeks, causing seizures, motor deficits, and stupor, often leading to coma and death (reviewed in Johnson 1998). Importantly, even though only a small percentage of acute MV infections will go on to develop CNS complications, a few studies have detected MV RNA in various organs, including brain, upon autopsy of elderly individuals who died of nonviral and non-CNS causes (Katayama et al. 1995, 1998). These findings suggest that MV may persist in the brains (and other organs) of healthy individuals, and that the frequency with which MV invades the CNS cannot be determined by summing the occurrence of the above-described CNS complications.

Understanding MV CNS Complications: Results from Brains of Infected Individuals and Persistently Infected Cell Lines The development of a robust live attenuated vaccine has profoundly decreased the infection rate in vaccinated populations. Vaccination has not been associated with SSPE, and only wild-type virus sequences have been isolated from SSPE tissues. However, vaccine strains have been isolated from the CNS of immunocompromised patients with MIBE (Bitnun et al. 1999; reviewed in Rima and Duprex 2005), implying that the attenuated vaccine strain can traffic to the brain under conditions of poor immune surveillance.

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Both SSPE and MIBE are characterized by mutations that apparently render the virus defective, though spread in neurons may still occur. As noted above, it has long been hypothesized that these mutations are the reason why infectious virus is not released from infected brain cells, especially since the mutations map to the envelope-associated proteins M, F, and H (Baczko et al. 1988; Billeter et al. 1994; reviewed in Johnson 1998). In SSPE, M protein expression is reduced, likely through one of two mechanisms: either a steeper gradient of transcription (Cattaneo et al. 1987a, 1987b) or increased read-through across the P–M junction of the MV genome (reviewed in Rima and Duprex 2005). In support of these data, hyperimmune antibody responses in SSPE are directed to all MV proteins with the key exception of M (reviewed in Rima and Duprex 2005). Mutations in F have been described for both SSPE and MIBE cases (Baczko et al. 1988; Billeter et al. 1994; reviewed in Johnson 1998). Interestingly, in all SSPE cases, mutations in F characteristically consist of the loss of the C-terminal pentadecapeptide, a sequence that is strictly conserved among morbilliviruses and is therefore thought to play an essential role in F protein function. This region contains the basolateral sorting signal of F (Maisner et al. 1998; reviewed in Rima and Duprex 2005), suggesting that missorting of F may contribute to altered MV spread and lack of complete MV assembly in neurons of SSPE patients. Interestingly, a study of ten SSPE cases by immunohistochemistry and in situ hybridization suggests that MV likely spreads transneuronally, an observation subsequently validated by in vitro model studies (Lawrence et al. 2000; Oldstone et al. 1999). The analysis of these infected brains revealed that MV infection in neuronal processes was predominantly dendritic, though there were signs of occasional axonal involvement as well (Allen et al. 1996). Whether mutations in viral proteins influence how MV is transported within neurons is currently under investigation.

The Big Questions Years of research on MV CNS complications have provided key insights into MV neuronal spread and have identified future areas of study concerning the relationship between viral spread and pathogenesis. For example: how does MV move from the site of entry to the perinuclear space and then again to the site of viral egress? Once present at the synaptic membrane, what cellular and viral proteins mediate MV transsynaptic spread? Is the mechanism of interneuronal spread related to the pathogenesis of MV within the brain? The establishment of permissive animal and cell culture models, coupled with advances in manipulating the viral genome, and the advent of cell biology resources to explore intracellular trafficking have been essential for key developments over the past few years. A discussion of these recent observations serves as the basis for the remainder of this review. We first describe some of these important technical advances and then discuss how they have been applied to MV neuronal spread.

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Tools Transgenic Mouse Models To date, two human receptors for MV have been identified: CD46 (Dorig et al. 1993; Naniche et al. 1993) and signaling lymphocyte activation molecule (SLAM; Erlenhoefer et al. 2001; Hsu et al. 2001; Tatsuo et al. 2000). While this is still an evolving field, and many believe that other receptors will be identified in the future, the general consensus is that CD46 is principally a receptor for vaccine strains, such as Edmonston, whereas SLAM, though restricted to hematogenous cells, permits entry of wild-type MV. As the mouse and rat homologs of CD46 and SLAM do not confer susceptibility to MV infection (Dorig et al. 1993; Manchester et al. 1994; Naniche et al. 1993; Ono et al. 2001b), transgenic mice expressing human CD46 or SLAM were established, with the hypothesis that expression of these human proteins would overcome the initial barrier to viral entry (Mrkic et al. 1998; Oldstone et al. 1999; Rall et al. 1997; Sellin et al. 2006; reviewed in Manchester and Rall 2001). Importantly for neuron-focused studies, these transgenic mice also provide a source of primary neurons for ex vivo experiments to complement observations made in vivo (Lawrence et al. 2000; Makhortova et al. 2007). A brief introduction to some of the transgenic model systems follows. NSE-CD46 mice, developed in the laboratory of Michael Oldstone, were engineered to express the BC1 isoform of human CD46 under the control of the neuronspecific enolase (NSE) promoter, restricting CD46 expression to CNS neurons. These mice can be infected intranasally (IN) or intracranially (IC) with a vaccine strain of MV (e.g., Edmonston); as predicted, only CD46-expressing neurons are initially permissive for infection. Adult immunocompetent mice mount an aggressive T cell response and survive infection, whereas NSE-CD46 neonatal mice, or adults on an immunodeficient background succumb to CNS disease. CD46+ primary hippocampal neurons can be cultured from transgenic embryos, providing a parallel in vitro culture system to corroborate and extend the in vivo observations (Lawrence et al. 1999, 2000; Makhortova et al. 2007; Patterson et al. 2002, 2003; Rall et al. 1997). Another CD46 transgenic model that was developed in Oldstone’s lab is the YAC-CD46 mouse, in which CD46 is expressed from its own promoter, more closely mirroring expression in humans. Indeed, CD46 distribution is found throughout the mouse, and this model has been used for both CNS and immunosuppression studies. Importantly, in this model, all four major isoforms of CD46 are expressed to levels and in locations similar to those seen in humans. Furthermore, overall expression of CD46 was shown to be greater than that previously reported for other transgenic mouse models, including the NSE-CD46 mice (Oldstone et al. 1999; Patterson et al. 2001). Mrkic et al. engineered mice to express CD46 on a mouse background lacking the interferon-α receptor [IFNAR–/– (Duprex et al. 2000; Ludlow et al. 2007; Mrkic et al. 1998)]. IFNAR–/– mice can be infected intranasally with MV, though

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replication of MV is limited. Engineering mice to express the MV receptor CD46 on the IFNAR–/– background resulted in higher levels of MV infection in the respiratory tract with subsequent inflammation, providing a more relevant model for the acute MV infection seen in humans. A perplexing observation from the establishment of a number of other CD46 transgenic mice was that, despite CD46 expression and ability to infect such cells when cultured ex vivo, little if any infection was observed in vivo. For example, Blixenkrone-Moller et al. generated a CD46 transgenic mouse that expressed a genomic copy of CD46 on a Bl/6 × SJL mouse background (Blixenkrone-Moller et al. 1998). Despite apparently robust CD46 expression, MV replication could not be detected following intraperitoneal (IP) or IN inoculation, though kidney and lung cell cultures from these mice were permissive. An important observation made by these authors was that, in these cultures, MV did not reach full replication levels as compared to MV-infected Vero cells. Furthermore, while IC challenge of CD46 transgenic mice did result in limited CNS infection, infection was also observed in nontransgenic mice, suggesting that factors other than CD46 play a role in mediating MV uptake into murine cells, and that key intracellular factors are needed to achieve optimal levels of MV replication. This work was supported by the findings of Horvat et al. and Evlashev et al. who found that cells from CD46 transgenic mice showed cell-type specific susceptibility to MV infection, indicating a role for host factors other than CD46 in mediating productive MV infection (Evlashev et al. 2001; Horvat et al. 1996). Thus far, four different SLAM-expressing transgenic mouse models have been created (Ohno et al. 2007; Sellin et al. 2006; Shingai et al. 2005; Welstead et al. 2005). However, to date, only one group has used these mice to look at MV CNS infections. Sellin et al. engineered mice to ubiquitously express SLAM. Wild-type and vaccine strains of MV could infect transgenic mice, either by IC or IN routes, but vaccine strains were less virulent (Sellin et al. 2006).

Other Animal Models Other investigators have focused on rodent-adapted MVs to infect nontransgenic (i.e., wild-type) mice, rats, or hamsters (Castro et al. 1972; Duprex et al. 1999a; Griffin et al. 1974; Moeller-Ehrlich et al. 2007; Schubert et al. 2006; Johnson and Swoveland 1977; Johnson and Norrby 1974; Roos et al. 1978). Such studies have been ongoing since the 1970s and have allowed researchers to study MV in a small animal model prior to the identification of the human MV receptors. Importantly, despite the mutations that occurred in adapting MV to rodents, the pathogenesis of such strains is remarkably similar to that seen following MV infection of humans or transgenic mouse models. Efforts to create animal models to study MV infection and pathogenesis have not been limited only to mice. For example, MV-infected cotton rats recapitulate the immunosuppression seen in MV-infected humans (Niewiesk 1999; Niewiesk et al.

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1999; Wyde et al. 1992), even with wild-type (clinical) isolates (Wyde et al. 1999). Furthermore, studies on rat brain slices grown in culture and infected with MV have revealed important clues about how MV spreads through an infected brain (Ehrengruber et al. 2002). Interestingly, the generation of a transgenic CD46expressing rat demonstrated that although MV was taken up by CD46-expressing cells, subsequent intracellular blocks in MV replication prevented robust infection of the animal (Niewiesk et al. 1997). Ferret models have also been used by some researchers as an SSPE model of MV infection in the CNS (Brown et al. 1985, 1987; Mehta and Thormar 1979; Thormar et al. 1983). Finally, while rhesus macaques have been chiefly used to study immune responses to MV, immune suppression induced by MV and efficacy of possible vaccines against MV (de Swart et al. 2007; Pan et al. 2005; Pasetti et al. 2007; Polack et al. 2000), some investigators have used tissues collected from infected monkeys to model human CNS diseases (Albrecht et al. 1977; Steele et al. 1982). How results from all of these model systems have been used to advance our understanding of MV interneuronal spread and neuropathogenesis will be discussed in the final section of the chapter.

In Vitro Culture Systems and Techniques in Cellular Transport Studies Primary Neuron Cultures and Neuron-Like Cell Lines The ability to culture primary neurons from various CNS substructures (e.g., hippocampus, cortex, dorsal root ganglia, cerebellum) of transgenic mice has been a powerful tool in dissecting aspects of intra- and interneuronal MV transport. These cultures are validated as neurons in their expression of characteristic neuronal markers such as MAP-2 and NeuN, their ability to form synapses in culture, and the fact that, once plated, these cells are mitotically inactive. While in general quite pure neuron cultures can be obtained (90%–95%), contaminating glial cells, culture-to-culture variability, difficulty in establishing these cultures and a fairly short lifespan in culture (10–14 days, typically), are some of the disadvantages. Cells lines such as NT2 (human teratocarcinoma cells that can be terminally differentiated into neuronal cells by retinoic acid treatment), human astrocytoma cells, and mouse neuroblastoma cells offer an alternative that is free of the complexities and challenges of primary neuron cultures (Duprex et al. 1999b, 2000; Lawrence et al. 2000; Ludlow et al. 2005; McQuaid et al. 1998). The ease of using these established lines of CNS cells is balanced by concerns about whether these cells accurately reflect primary CNS cell biology.

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Slice Cultures The introduction of organotypic monolayer cultures of nervous tissue has added an intermediate technique to the neurologist’s toolbox, falling between the complexity of a whole animal model and the culturing of cell lines or pure primary cultures that do not recapitulate the cellular heterogeneity of the CNS. Advantages to organotypic brain slice cultures are the ability to take cells from a defined region of the brain, the ability to use phase microscopy on the monolayer of resulting brain cells, the relevance of culturing primary cells without performing single cell dissociation, and the power of studying heterogeneous cell populations and how such diverse cells impact viral spread (Gahwiler 1981). This approach has been used to assess the spread of rMV-EGFP through neurons of cultured rat brain slices (Ehrengruber et al. 2002) as well as the spread of SSPE isolates through hamster cerebellum slices (Sheppard et al. 1975).

Reagents to Study the Role of Motor Proteins in Viral Spread Progress in our understanding of the cellular cytoskeleton and how organelles, signaling complexes, and proteins are transported within cells has greatly advanced our understanding of how viruses are transported within cells (Jouvenet et al. 2004; reviewed in Leopold and Pfister 2006; Mackenzie et al. 2006; reviewed in Ploubidou and Way 2001; Rietdorf et al. 2001; Ward and Moss 2004). For example, the development of dominant negatives to genetically manipulate the cellular transport system, as well as the increased availability of genetically altered mice with defined motor protein deficiencies, have enabled a dissection of which cellular proteins are utilized by viruses to enable transport. Although pharmaceutical approaches to ablate specific microtubule motors or microtubules themselves are broadly used (e.g., vanadate, colchicine, cytochalasin, nocodazole, etc.), the harsh and somewhat nonspecific consequences of using these cytotoxic reagents was always a limitation that can now be obviated with more precise and less caustic genetic strategies. For example, the generation and characterization of mice via ENU-mutagenesis containing a single point mutation in dynein heavy chain has helped to define the role of dynein in the spread of mouse-adapted scrapie prions from the periphery to the CNS of infected mice (Hafezparast et al. 2004). These loa (legs at odd angles) mice are viable as heterozygotes, and are currently being used by a number of neurovirology labs to assess the role of dynein in virus transport (Ahmad-Annuar et al. 2003; Hafezparast et al. 2003, 2004). Another tool to define viral–cytoskeletal interactions are dominant negative constructs. For example, overexpression of p50 dynamitin, a member of the multiprotein complex dynactin, which acts as an accessory factor to dynein, specifically disrupts dynein function (Ahmad et al. 1998; Burkhardt et al. 1997; Echeverri et al. 1996). This approach was used by Dohner et al. to show that herpes simplex virus type 1 (HSV-1) utilizes dynein following its entry into a cell to travel to the nucleus (Dohner et al. 2002). Similar reagents have been established for kinesins,

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motors that govern intracellular retrograde transport (Verhey et al. 1998; Verhey et al. 2001).

Reverse Genetics for MV The establishment of a reverse genetics system for vaccine strains of MV has also furthered our understanding of MV replication in neurons, and in MV intra- and interneuronal spread (Devaux et al. 2007; Radecke et al. 1995; Schneider et al. 1997). Future studies using this approach will be key to identifying what roles each of the viral proteins play in neuronal infection, and will define whether some viral proteins may be dispensable for neuronal infection. Moreover, given the speculation that point mutations found in SSPE isolates might make these viruses more neuropathogenic, reverse genetics offers an opportunity to directly test these hypotheses in a controlled setting. Already, MV reverse genetics has been used to address the role of the H protein of rodent brain-adapted MV in conferring neurovirulence to MV Edmonston in nontransgenic mice (Duprex et al. 1999a). Moreover, Maisner’s group engineered viruses with altered basolateral sorting signals in F and H and showed that these domains are important for MV propagation through lymphoid cells, in addition to their previously described role in MV spread through epithelial cells (Runkler et al. 2008). Furthermore, the ability of SSPE-associated viral genes to confer certain phenotypes on wild-type or vaccine strains of MV has been tested through reverse genetics approaches. For example, a recombinant MV expressing the M gene of an SSPE isolate was found to replicate less efficiently and led to a CNS infection in YAC-CD46 transgenic mice with a longer course than that seen following infection with MV Edmonston. This MVexpressing SSPE M also showed defects in viral assembly and subsequent budding of progeny virus from infected cells (Patterson et al. 2001). Finally, the power of reverse genetics has provided some experimental convenience in that we can now molecularly tag MV with marker proteins such as GFP to more easily follow MV infections in vivo, in brain slices, and in cell culture (de Swart et al. 2007; Duprex et al. 1999b, 2000; Ehrengruber et al. 2002; Ludlow et al. 2007; Plumb et al. 2002; Schubert et al. 2006).

Access to the CNS: Lessons from CDV, PV, and WNV Despite this broad palette of tools that are available to study MV–neuron interactions, many of the reagents and strategies have been developed only recently, and as a result we have more questions than answers about how MV gets to the CNS, spreads within CNS cells, and mediates neurological disease. How MV gains access to the CNS from the periphery is not known, though it has been previously proposed that MV spreads into the brain by infecting endothelial cells during secondary viremia (Esolen et al. 1995). Alternatively, there is speculation that MV may infect

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the CNS by infiltration of infected lymphoid cells, e.g., Alternatively there is speculation that MV may infect the CNS by infiltration of infected lymphoid cells, e.g., infiltrating macrophages. Despite a lack of certainty regarding MV neuroinvasion, clues about how MV spreads to the CNS can be gained by looking at the spread of other neurotropic viruses from the periphery to the CNS. Two routes of CNS infection have been proposed for a related morbillivirus (CDV): the hematogenous route and anterograde trafficking via the olfactory nerve. Like MV, CDV can infect lymphocytes during the systemic phase of infection, thus providing an opportunity for infected lymphocytes to traffic across the BBB and release infectious virus to initiate infection within the brain parenchyma. Importantly, the work by Rudd et al. showed that entry of CDV into the CNS can also occur via the olfactory bulb and suggested that this may be a common mechanism of entry for neurotropic paramyxoviruses (Rudd et al. 2006). Similar portals of entry have been proposed and tested for both polio virus (PV) and West Nile virus (WNV). One hypothesis is that these viruses cross the BBB; the second is that PV or WNV is transmitted via peripheral nerves (Ohka et al. 1998; Samuel et al. 2007). Yang et al. demonstrated that circulating PV crosses the BBB at a high rate that is independent of polio virus receptor (PVR) expression (Yang et al. 1997). More recently, studies in transgenic PVR-expressing mice have shown that PV is transported through the sciatic nerve by fast retrograde axonal transport. Furthermore, PV accesses the sciatic nerve from its intramuscular inoculation site by a process dependent on PVR (Ohka et al. 1998), and the mechanism by which PV is transported retrogradely along the sciatic nerve is thought to be an interaction of the PVR cytoplasmic domain with dynein light chain Tctex1 (Ohka et al. 2004). As with polio, axonal transport of WNV can also occur, mediating entry into the CNS. However, WNV can spread to the CNS even when the sciatic nerve has been transected, suggesting that WNV likely uses multiple routes to access the brain (Samuel et al. 2007). One of these routes of WNV entry has been shown to be dependent on Toll-like receptor 3 (TLR3)-mediated inflammation and subsequent opening of the BBB (Wang et al. 2004). Thus, as for CDV (Rudd et al. 2006), multiple non-mutually exclusive routes of spread are available for PV and WNV entry into the CNS. How MV penetrates into the CNS, and perhaps more importantly, how often this occurs in a human population, remain to be determined.

MV Spread and Transport in Non-neuronal Cells MV Spread in Non-neuronal Cells MV infection is initiated when H binds to one of its cellular receptors, CD46 or SLAM (Dorig et al. 1993; Naniche et al. 1993; Tatsuo et al. 2000). The receptor usage correlates with the source of the MV H protein: wild-type isolates typically use SLAM (Erlenhoefer et al. 2001; Ono et al. 2001a, 2001b; Tatsuo et al. 2000), whereas attenuated vaccine strains, such as Edmonston and the strains derived from

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it, preferentially use CD46. Receptor selection is not exclusive, however: SLAM has been shown to be an effective receptor for MV entry for vaccine strains and conversely a few wild-type strains have the ability to bind to CD46 (Erlenhofer et al. 2002). It has been suggested that the receptor usage of a given MV isolate may depend on the cell line used to isolate and amplify the virus rather than the wildtype or vaccine strain status of the MV isolate (Manchester et al. 2000). Furthermore, genetic and structural studies of MV H proteins have illustrated that the H binding domains to CD46 and SLAM are spatially distinct (Colf et al. 2007; Erlenhofer et al. 2002), providing support for the idea that H can interact with both CD46 and SLAM, albeit with different affinities for each. In any case, given that SLAM expression is limited to lymphoid cells (McQuaid and Cosby 2002), it is likely that other MV receptors await discovery. In non-neuronal cells, MV buds from the apical surface, resulting in the release of free virus or in cell fusion (reviewed in Griffin 2001; Fig. 1.1A). Transport of the viral nucleocapsid to the plasma membrane is dependent on levels of M protein and its accumulation at the cell surface (Runkler et al. 2007). Apical budding occurs despite the preferential sorting of MV glycoproteins F and H to the basolateral membrane through a tyrosine-based sorting signal (Blau and Compans 1995; Maisner et al. 1998; Moll et al. 2001). Appropriate budding is achieved by restricted expression of M at the apical surface, which retargets some of F and H in polarized cells (Naim et al. 2000; Riedl et al. 2002). The interaction of the cytoplasmic tails of F and H with M mediates virus assembly (Cathomen et al. 1998a, 1998b). However, the predominant presence of the glycoproteins F and H at the basolateral membrane has been hypothesized to play a key role in MV spread through an infected individual. The tyrosine-based sorting signals in F and H are not only required for basolateral sorting, but are also required for the fusogenicity of the F/H complex in polarized epithelial cells (Maisner et al. 1998; Moll et al. 2001). Consequently, it has been proposed that the basolaterally expressed F/H complex promotes cell–cell fusion, thus allowing the virus to spread to underlying tissues

Fig. 1.1 A, B Immunohistochemistry for MV antigen with hematoxylin counterstain. A MVinfected Vero cells form syncytia, or multinucleated giant cells. B MV-infected primary hippocampal neurons from NSE-CD46 mice do not form syncytia

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and facilitating systemic MV dissemination (Moll et al. 2004). This hypothesis emphasizes that apically released budding virus and basolaterally mediated cell– cell fusion are both important components of MV systemic dissemination. It should be noted that the classical MV spread in non-neuronal cells (extracellular progeny virus budding and cell–cell fusion) is dependent on MV glycoproteins. Thus, MV H interaction with its receptor likely triggers F protein fusogenic activity, initiating viral infection (Griffin 2001; Lamb and Kolakofsky 2001). As we point out later, these events may be substantially altered upon MV infection of neurons.

Use of Microtubules and Their Associated Motor Proteins to Achieve Viral Transport Within an Infected Cell Introduction to Microtubules How are the glycoproteins and RNP complexes shuttled through a cell to achieve appropriate assembly and egress? Microtubules are part of the cellular cytoskeleton and play a critical role in mediating movement of cellular proteins and organelles to their proper destinations. In neurons, their role is even more critical, as neurons rely on microtubules for neurite extension, synaptic vesicle trafficking, and synapse formation (Zhai and Bellen 2004). Microtubules are long, hollow cylindrical polymers of tubulin that assemble with a head-to-tail orientation (reviewed in Henry et al. 2006). The plus-end of microtubules typically extends to the plasma membrane and away from the nucleus, or toward the axon or dendrite and away from the cell body in neurons. In non-neuronal cells, microtubule minus-ends are bound and stabilized at the microtubule organizing center (MTOC) near the nucleus, whereas in neurons the minus-ends are oriented to the cell body, but lack an MTOC. The protein motors that move cargo along these microtubules are grouped into two families: the predominately plus-end-directed kinesin family of motors, which move cargo away from the cell body (anterograde transport), and the minus-end directed dynein family of motors, which move cargo to the cell body (retrograde transport), often as part of the lysosomal pathway. Cytoplasmic kinesin (or conventional kinesin) consists of two heavy chains and two light chains. The heavy chains contain the motor domains, which allow for the generation of force and subsequent movement along the microtubule. Furthermore, the heavy chains facilitate dimerization and cargo binding. The kinesin light chains may also facilitate binding to cargo (reviewed in Mandelkow and Mandelkow 2002). Cytoplasmic dynein binds to microtubules via its two heavy chains, but other components of this complex (including six light chains, two light intermediate chains, and two intermediate chains) mediate cargo binding and contribute to the specificity of the cargo that is transported. Furthermore, cytoplasmic dynein associates with another protein complex dynactin, which acts as a co-factor to facilitate cargo binding and processivity along the microtubule (reviewed in Leopold and Pfister 2006; Radtke et al. 2006).

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Viruses of many families utilize the microtubule pathway during infection. Some viruses associate with microtubules and retrograde motors to move to the nucleus, including murine polyomavirus, adeno-associated virus, adenovirus, herpes simplex virus 1, and human immunodeficiency virus (reviewed in Greber and Way 2006; Radtke et al. 2006). Other viruses are associated with anterograde transport, as has been shown for both Vaccinia virus and African swine fever virus transport to the plasma membrane through an interaction of viral cargo with kinesin-1 (Jouvenet et al. 2004; Rietdorf et al. 2001). Presumably, for these viruses, interaction with anterograde motors facilitates viral egress. Moreover, recent work with the alphaherpesvirus pseudorabies, a large DNA virus, illustrated fast axonal transport through neurons, a process mediated by microtubule motors (Smith et al. 2001). However, little is known about how neurotropic RNA viruses, including MV, interact with molecular motors in neurons. MV and the Cytoskeleton It has been known for some time that actin plays a key role in MV trafficking within cells. Actin is packaged within MV virions (Tyrrell and Norrby 1978), and treatment of infected cells with the actin-disrupting agent cytochalasin B resulted in disruption of MV virion formation (Stallcup et al. 1983). Electron microscopy studies of cytoskeletal preparations of MV-infected cells showed that the growing end of actin filaments protruded into budding virions. The authors suggested that the vectorial action of actin promotes MV budding and may also be involved in the transport of nucleocapsids to the cell surface (Bohn et al. 1986). Furthermore, actin was associated with transcriptionally silent MV nucleocapsids in vitro, supporting a role of actin in the budding of mature (i.e., not transcriptionally active) MV nucleocapsids. The same study showed that tubulin promoted MV RNA synthesis in in vitro RNA synthesis assays and could be co-immunoprecipitated with MV L, implying a further requirement for tubulin in MV replication (Moyer et al. 1990). These points, in addition to the large body of work illustrating the essential role of microtubules in facilitating longdistance transport within cells (reviewed in Greber and Way 2006), strongly support the notion that MV engages the microtubule network for transport within infected neurons. Ongoing work in a number of MV-focused laboratories is addressing this hypothesis, recognizing that it is hard to imagine a neurotropic virus achieving transport from one end of a neuron to the other without hijacking the cell’s railroad, the microtubule system. Transport of Other Viruses in Neurons The best-studied viruses, with regard to neuronal transport and spread, are the alphaherpesviruses, including herpes simplex virus 1 (HSV1) and pseudorabies virus (PRV). Cell imaging studies with both viruses have revealed by time-lapse

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fluorescence microscopy that these viruses travel retrogradely down the dendrite to the cell body (Bearer et al. 2000; Feierbach et al. 2007). This intracellular trafficking of the virus along microtubules can be disrupted by inhibitors of microtubule assembly, such as colchicine, vinblastine, or nocodazole (Sodeik et al. 1997; Topp et al. 1994). HSV-1 capsids entering a cell associate with the dynein complex, and overexpression of the dynactin component dynamitin (p50) blocks the transport of HSV1 to the nucleus (Dohner et al. 2002). Two somewhat contradictory models have emerged to explain virus transport away from the nucleus and subsequent viral egress. In the first model, herpesviral capsids and glycoproteins are transported separately, and assembly takes place somewhere along the axon shaft or at the axon terminus. The second model proposes that fully assembled enveloped virions are transported down the axon in vesicles (reviewed in Diefenbach et al. 2008; Lyman et al. 2007). As of last count, five different laboratories, three for HSV, two for PRV, have demonstrated contradictory findings as to which model is correct, and the controversy continues as the differences between variables, viruses, types of neurons, kinetics, and technical differences are sorted out (reviewed in Diefenbach et al. 2008). In either case, these two models provide the RNA virologist with an impetus for thought as to how smaller enveloped RNA viruses achieve egress from an infected neuron.

MV Spread in Neurons MV Movement Within and Among Neurons MV-infected CD46-expressing mice illustrated that, in vivo, neurons are the primary cell of the CNS infected by MV, even in animals where the expression of CD46 was not neuronally restricted (Oldstone et al. 1999). Similarly, neurons are the main target of MV infection in the brain of infected SLAM transgenic mice (Sellin et al. 2006), as is the case for infected human CNS tissues (Allen et al. 1996). The Edmonston strain of MV engineered to express EGFP (rMV-EGFP) spread through brains of infected CD46+/IFNAR–/– neonatal mice by neuronal processes. Furthermore, the authors detected EGFP-positive cells whose morphology was not neuronal and that the authors believed to be ependymal cells and neuroblasts (Duprex et al. 2000). This finding is of significance since it widens the population of cells susceptible to MV in the CNS of this animal model, and the result differs from the predominant neuronal infection described in other CD46-expressing animal models. Interestingly, the first report describing the generation and characterization of CD46+/IFNAR–/– mice indicated that IC infection with MV Edmonston also resulted in infection of ependymal cells, oligodendrocytes, and neurons (Mrkic et al. 1998). One key difference among these studies and those of other CD46 transgenic mouse models and of infected humans is the absence of an intact type I interferon system, which may allow the virus the opportunity to access other cell populations.

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MV does not bud from infected neurons and MV-infected primary neurons do not form syncytia (Fig. 1.1B). The presence of nucleocapsids in the axons and at the presynaptic membranes of infected neurons suggests a contact-dependent, trans-synaptic spread of MV (Lawrence et al. 2000; Oldstone et al. 1999). This finding is consistent with data from autopsy specimens of SSPE cases, where MV budding is not apparent (Paula-Barbosa and Cruz 1981). Moreover, spread of MV-Edmonston in a neuronal population is independent of the receptor CD46 (Lawrence et al. 2000); thus, in this model, while CD46 was needed for viral entry, it was dispensable for neuron-to-neuron transmission. MV spread via neuron–neuron contact has been confirmed in studies using rat organotypic hippocampal slices, as well as in tissue culture studies using differentiated human NT2 neurons (Lawrence et al. 2000; Ludlow et al. 2005; McQuaid et al. 1998). The spread of rMV-EGFP through hippocampal slices followed neuronal tracts, and no release of extracellular virus particles was observed. In this study, the interneuronal spread of rMV-EGFP was determined to be retrograde based on the spread of MV-EGFP from CA1 to CA3 pyramidal cells to granule cells within the slices and the known synaptic connectivity of these CNS substructures. Furthermore, the MV envelope proteins (F, H, and M) and P proteins were detected in dendrites of infected neurons (Ehrengruber et al. 2002). The mutations that accumulate upon MV adaptation to rodents (Rima et al. 1997; Vanchiere et al. 1995) do not seem to affect neuronal spread, as these rodent-adapted MVs have yielded data consistent with observations in humans and data from MV-infected CD46-expressing mice and primary neurons cultured from such mice (Duprex et al. 1999a; Mrkic et al. 1998; Schubert et al. 2006). For example, the hamster-neurotropic strain (HNT) of MV was used to infect Balb/c mice, and the resulting infection was limited to neurons and was not cytolytic. No virus assembly was detected (Van et al. 1979). Other work with HNT supports the lack of virus assembly following infection of weanling or adult mice (Griffin et al. 1974). The infection of weanling hamsters with HBS, another rodent-adapted MV isolated from an SSPE case, also revealed no budding virions in infected brains (Johnson and Swoveland 1977). As mentioned earlier, MV neuronal spread can occur independent of the expression of CD46, suggesting that H may not be required for MV trans-synaptic spread. Recently, Makhortova et al. indirectly investigated the role of MV F in trans-synaptic spread by using FIP, fusion inhibitory peptide (Makhortova et al. 2007). FIP, a synthetic tripeptide (z-D-Phe-L-Phe-Gly), prevents fusion by multiple viruses, though its strongest activity is against MV (Norrby 1971; Richardson et al. 1980; Richardson and Choppin 1983). FIP reduced MV infection and subsequent spread through primary CD46-expressing neuron cultures, implicating a role for F in trans-neuronal transport. Furthermore, the neurotransmitter substance P, whose active site is identical to FIP, also blocked MV neuronal spread. Genetic deletion of the substance P receptor, neurokinin-1 (NK-1), or pharmacological inhibition of NK-1 decreased MV replication and subsequent disease in MV-infected mice. Overall, these data suggest that MV F may interact with NK-1 at the synapse to mediate trans-synaptic spread of MV (Makhortova et al. 2007). The idea that MV engages a cellular receptor other than SLAM or CD46 is not

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new (Andres et al. 2003; Blixenkrone-Moller et al. 1998; Takeda et al. 2007), though the potential that such a receptor may be bound by MV F, rather than H, broadens this original hypothesis. As a result of this work, the current model for MV trans-synaptic spread is a microfusion event at the synapse, illustrated in Fig. 1.2. In this model, at least the MV RNP and F glycoprotein are actively transported to the synapse. F engagement of NK-1 may then allow a membrane fusion event to permit movement of the RNP from one neuron to the next. The neurotransmitter receptors neurokinin-2 and -3 (NK-2 and NK-3) bind to neurotransmitters of the same family as substance P and therefore may also play a role in MV trans-synaptic spread.

Effects of Immune Responses on MV Spread in Neurons It has long been known that multiple host factors contribute to the ability of a virus to replicate in a given cell. The literature contains several examples in which the elicitation or removal of a host immune response can alter MV replication and subsequent spread. While most of these studies have measured the consequences of ablation of a particular immune cell type (e.g., recombinase-activating gene (RAG) knockout (KO) mice that lack functional T and B cells), intrinsic responses of neurons can also influence the outcome of infection. For example, expression of the heat shock protein hsp72, which would be induced during a febrile illness, increased MV gene expression following infection of neonatal hsp72 transgenic mice. The increased MV RNA levels correlated with a higher mortality rate (Carsillo et al. 2006), providing an example wherein the cellular response to viral infection increases MV pathogenesis. Conversely, the removal of the adaptive immune response protein TAP1 (the transporter associated with antigen presentation) led to enhanced spread of MV into the brains of infected mice. The HNT strain of MV trafficked from the olfactory bulb into the limbic

Fig. 2.2 Model for MV-trans-synaptic spread. RNPs and F are transported to the synapse. The interaction of F with neurokinin-1 triggers a microfusion event, allowing the RNP to cross the synapse and infect the synaptically connected neuron. Neurokinin-2 and -3 may act as receptors for MV F, given their homology to neurokinin-1

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system of wild-type and TAP1–/– mice but showed a greater dissemination into the brains of TAP1–/– mice, as compared to that seen in wild-type mice (Urbanska et al. 1997). Finally, an association was found between promoter polymorphisms of the innate immunity protein MxA and the occurrence of SSPE in Japan. The most frequent polymorphisms caused the MxA promoter to be more responsive to type I interferon than the wild-type promoter sequence. Therefore, the increased activity of MxA in response to type I interferon positively correlated with SSPE occurrence, suggesting that MxA may play a role in the establishment of persistent MV infections in neurons (Torisu et al. 2004), though these studies have been somewhat controversial (Pipo-Deveza et al. 2006). The analysis of SSPE lesions of affected individuals revealed that the cells staining positive for MxA were mainly astrocytes located in a belt around the region of MV antigen-positive cells in affected areas of the brain. Similar to the conclusions drawn from the Japanese cases of SSPE, the authors suggested that MxA plays an important role in slowing down viral spread in SSPE and therefore may contribute to the persistent nature of this MV CNS infection (Ogata et al. 2004; Torisu et al. 2004).

Remaining Questions As this review has highlighted, the molecular mechanisms that govern MV transport in neurons are slowly coming into focus. Ehrengruber’s study on the spread of rMV-EGFP in cultured rat hippocampal slices found that MV spreads through the rat hippocampus in a retrograde direction, with the MV F, H, M, and P proteins localizing to the dendrite of infected neurons (Ehrengruber et al. 2002). Lawrence et al. demonstrated by electron microscopy the presence of MV nucleocapsids at the presynaptic membrane of infected primary hippocampal neurons, suggesting that MV may, in fact, spread in an anterograde direction, or from the axon of an infected neuron to the dendrite of a neighboring neuron (Lawrence et al. 2000). However, these studies could not distinguish between nucleocapsids that were exiting or nucleocapsids that were entering the neuron. Furthermore, there have been reports in support of both anterograde and retrograde trafficking of MV in neurons (McQuaid et al. 1998; Urbanska et al. 1997), which may not be unexpected as the virus must travel to the cell body to replicate and then back to the periphery of the neuron for egress, regardless of which neuronal process it uses (the dendrite or the axon) for entry and for exit. While a comprehensive view of how the unique environment of the neuron affects MV replication, spread and, ultimately, neuropathogenesis awaits further study, the tools and ideas are in place for exciting advances in the coming years. Acknowledgements We gratefully acknowledge the assistance of Christine Matullo and Lauren O’Donnell in the preparation of this review. V.A. Young is supported by T32 NS007180-25 from the University of Pennsylvania. G. R. is supported by NIH grants NS40500, MH56951, and CA006927, as well as generous support from the F.M. Kirby Foundation.

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Rietdorf J, Ploubidou A, Reckmann I, Holmstrom A, Frischknecht F, Zettl M, Zimmermann T, Way M (2001) Kinesin-dependent movement on microtubules precedes actin-based motility of vaccinia virus. Nat Cell Biol 3:992–1000 Rima BK, Duprex WP (2005) Molecular mechanisms of measles virus persistence. Virus Res 111:132–147 Rima BK, Earle JA, Baczko K, ter Meulen V, Liebert UG, Carstens C, Carabana J, Caballero M, Celma ML, Fernandez-Munoz R (1997) Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture. J Gen Virol 78:97–106 Roos RP, Griffin DE, Johnson RT (1978) Determinants of measles virus (hamster neurotropic strain) replication in mouse brain. J Infect Dis 137:722–727 Rudd PA, Cattaneo R, von Messling V (2006) Canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion. J Virol 80:9361–9370 Runkler N, Pohl C, Schneider-Schaulies S, Klenk HD, Maisner A (2007) Measles virus nucleocapsid transport to the plasma membrane requires stable expression and surface accumulation of the viral matrix protein. Cell Microbiol 9:1203–1214 Runkler N, Dietzel E, Moll M, Klenk HD, Maisner A (2008) Glycoprotein targeting signals influence the distribution of measles virus envelope proteins and virus spread in lymphocytes. J Gen Virol 89:687–696 Samuel MA, Wang H, Siddharthan V, Morrey JD, Diamond MS (2007) Axonal transport mediates West Nile virus entry into the central nervous system and induces acute flaccid paralysis. Proc Natl Acad Sci U S A 104:17140–17145 Schneider H, Spielhofer P, Kaelin K, Dotsch C, Radecke F, Sutter G, Billeter MA (1997) Rescue of measles virus using a replication-deficient vaccinia-T7 vector. J Virol Methods 64:57–64 Schneider-Schaulies J, ter Meulen V, Schneider-Schaulies S (2003) Measles infection of the central nervous system. J Neurovirol 9:247–252 Schnorr JJ, Seufert M, Schlender J, Borst J, Johnston IC, ter Meulen V, Schneider-Schaulies S (1997) Cell cycle arrest rather than apoptosis is associated with measles virus contact-mediated immunosuppression in vitro. J Gen Virol 78:3217–3226 Schubert S, Moller-Ehrlich K, Singethan K, Wiese S, Duprex WP, Rima BK, Niewiesk S, Schneider-Schaulies J (2006) A mouse model of persistent brain infection with recombinant measles virus. J Gen Virol 87:2011–2019 Sellin CI, Davoust N, Guillaume V, Baas D, Belin MF, Buckland R, Wild TF, Horvat B (2006) High pathogenicity of wild-type measles virus infection in CD150 (SLAM) transgenic mice. J Virol 80:6420–6429 Servet-Delprat C, Vidalain PO, Bausinger H, Manie S, Le Deist F, Azocar O, Hanau D, Fischer A, Rabourdin-Combe C (2000) Measles virus induces abnormal differentiation of CD40 ligand-activated human dendritic cells. J Immunol 164:1753–1760 Sheppard RD, Raine CS, Burnstein T, Bornstein MB, Feldman LA (1975) Cell-associated subacute sclerosing panencephalitis agent studied in organotypic central nervous system cultures: viral rescue attempts and morphology. Infect Immun 12:891–900 Shingai M, Inoue N, Okuno T, Okabe M, Akazawa T, Miyamoto Y, Ayata M, Honda K, KuritaTaniguchi M, Matsumoto M, Ogura H, Taniguchi T, Seya T (2005) Wild-type measles virus infection in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection. J Immunol 175:3252–3261 Sips GJ, Chesik D, Glazenburg L, Wilschut J, De Keyser J, Wilczak N (2007) Involvement of morbilliviruses in the pathogenesis of demyelinating disease. Rev Med Virol 17:223–244 Smith GA, Gross SP, Enquist LW (2001) Herpesviruses use bidirectional fast-axonal transport to spread in sensory neurons. Proc Natl Acad Sci U S A 98:3466–3470 Sodeik B, Ebersold MW, Helenius A (1997) Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol 136:1007–1021 Stallcup KC, Raine CS, Fields BN (1983) Cytochalasin B inhibits the maturation of measles virus. Virology 124:59–74 Steele MD, Giddens WE Jr, Valerio M, Sumi SM, Stetzer ER (1982) Spontaneous paramyxoviral encephalitis in nonhuman primates (Macaca mulatta and M. nemestrina). Vet Pathol 19:132–139 Sun X, Burns JB, Howell JM, Fujinami RS (1998) Suppression of antigen-specific T cell proliferation by measles virus infection: role of a soluble factor in suppression. Virology 246:24–33

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Takasu T, Mgone JM, Mgone CS, Miki K, Komase K, Namae H, Saito Y, Kokubun Y, Nishimura T, Kawanishi R, Mizutani T, Markus TJ, Kono J, Asuo PG, Alpers MP (2003) A continuing high incidence of subacute sclerosing panencephalitis (SSPE) in the Eastern Highlands of Papua New Guinea. Epidemiol Infect 131:887–898 Takeda M, Tahara M, Hashiguchi T, Sato TA, Jinnouchi F, Ueki S, Ohno S, Yanagi Y (2007) A human lung carcinoma cell line supports efficient measles virus growth and syncytium formation via a SLAM- and CD46-independent mechanism. J Virol 81:12091–12096 Tamashiro VG, Perez HH, Griffin DE (1987) Prospective study of the magnitude and duration of changes in tuberculin reactivity during uncomplicated and complicated measles. Pediatr Infect Dis J 6:451–454 Tatsuo H, Ono N, Tanaka K, Yanagi Y (2000) SLAM (CDw150) is a cellular receptor for measles virus. Nature 406:893–897 Thormar H, Mehta PD, Lin FH, Brown HR, Wisniewski HM (1983) Presence of oligoclonal immunoglobulin G bands and lack of matrix protein antibodies in cerebrospinal fluids and sera of ferrets with measles virus encephalitis. Infect Immun 41:1205–1211 Topp KS, Meade LB, LaVail JH (1994) Microtubule polarity in the peripheral processes of trigeminal ganglion cells: relevance for the retrograde transport of herpes simplex virus. J Neurosci 14:318–325 Torisu H, Kusuhara K, Kira R, Bassuny WM, Sakai Y, Sanefuji M, Takemoto M, Hara T (2004) Functional MxA promoter polymorphism associated with subacute sclerosing panencephalitis. Neurology 62:457–460 Tyrrell DLJ, Norrby E (1978) Structural polypeptides of measles virus. J Gen Virol 39:219–229 Urbanska EM, Chambers BJ, Ljunggren HG, Norrby E, Kristensson K (1997) Spread of measles virus through axonal pathways into limbic structures in the brain of TAP1–/– mice. J Med Virol 52:362–369 Van PC, Rammohan KW, McFarland HF, Dubois-Dalcq M (1979) Selective neuronal, dendritic, and postsynaptic localization of viral antigen in measles-infected mice. Lab Invest 40:99–108 Vanchiere JA, Bellini WJ, Moyer SA (1995) Hypermutation of the phosphoprotein and altered mRNA editing in the hamster neurotropic strain of measles virus. Virology 207:555–561 Verhey KJ, Lizotte DL, Abramson T, Barenboim L, Schnapp BJ, Rapoport TA (1998) Light chaindependent regulation of kinesin’s interaction with microtubules. J Cell Biol 143:1053–1066 Verhey KJ, Meyer D, Deehan R, Blenis J, Schnapp BJ, Rapoport TA, Margolis B (2001) Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules. J Cell Biol 152:959–970 Wang T, Town T, Alexopoulou L, Anderson JF, Fikrig E, Flavell RA (2004) Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis. Nat Med 10:1366–1373 Ward BM, Moss B (2004) Vaccinia virus A36R membrane protein provides a direct link between intracellular enveloped virions and the microtubule motor kinesin. J Virol 78:2486–2493 Welstead GG, Iorio C, Draker R, Bayani J, Squire J, Vongpunsawad S, Cattaneo R, Richardson CD (2005) Measles virus replication in lymphatic cells and organs of CD150 (SLAM) transgenic mice. Proc Natl Acad Sci U S A 102:16415–16420 Wyde PR, Ambrose MW, Voss TG, Meyer HL, Gilbert BE (1992) Measles virus replication in lungs of hispid cotton rats after intranasal inoculation. Proc Soc Exp Biol Med 201:80–87 Wyde PR, Moore-Poveda DK, Daley NJ, Oshitani H (1999) Replication of clinical measles virus strains in hispid cotton rats. Proc Soc Exp Biol Med 221:53–62 Yang WX, Terasaki T, Shiroki K, Ohka S, Aoki J, Tanabe S, Nomura T, Terada E, Sugiyama Y, Nomoto A (1997) Efficient delivery of circulating poliovirus to the central nervous system independently of poliovirus receptor. Virology 229:421–428 Zhai RG, Bellen HJ (2004) Hauling t-SNAREs on the microtubule highway. Nat Cell Biol 6:918–919

Chapter 2

Modeling Subacute Sclerosing Panencephalitis in a Transgenic Mouse System: Uncoding Pathogenesis of Disease and Illuminating Components of Immune Control M.B.A. Oldstone Contents Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction: Measles Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical Background: Subacute Sclerosing Panencephalitis. . . . . . . . . . . . . . . . . . . . . . . . . Transgenic Mouse Model of SSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contribution of the Hypermutated M Protein to the Chronic Progressive CNS Disease . . . . Hypothesis to Explain the Initiation and Pathogenesis of SSPE . . . . . . . . . . . . . . . . . . . . . . . Contribution of Biased Hypermutation Predominantly in the M Gene of Measles Virus to the Pathogenesis of SSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contribution of Antibody-Induced Modulation of Measles Virus Antigens to the Pathogenesis of SSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dual-Hit Hypothesis: Acute Immunosuppressive Event Preceding Measles Virus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of the Components of Measles Virus-Specific Immune Response Required for Clearing Measles Virus from the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future: Studies to Further Dissect the Molecular Pathogenesis of SSPE . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Subacute sclerosing panencephalitis (SSPE) is a chronic neurodegenerative disease of the central nervous system (CNS) that afflicts eight to 20 individuals per one million of those who become infected with measles virus (MV). The six cardinal elements of SSPE are: (1) progressive fatal CNS disease developing several years after MV infection begins; (2) replication of MV in neurons; (3) defective nonreplicating MV in the CNS that is recoverable by co-cultivation with permissive tissue culture cells; (4) biased hypermutation of the MV recovered from the CNS with massive A to G (U to C) base changes primarily in the M gene of the virus; (5) high titers of antibody to MV; and (6) infiltration of B and T cells into the CNS. All these parameters can be mimicked in a transgenic (tg) mouse model that expresses the MV receptor, thus enabling infection of a usually uninfectable mouse in which the immune system is or is not manipulated. Utilization and analysis of

M.B.A. Oldstone Viral-Immunobiology Laboratory, Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla CA, USA, e-mail: [email protected]

D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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such mice have illuminated how chronic measles virus infection of neurons can be initiated and maintained, leading to the SSPE phenotype. Further, an active role in prolonging MV replication while inhibiting its spread in the CNS can be mapped to a direct affect of the biased hypermutations (A to G changes) of the MV M gene in vivo.

Abbreviations SSPE CNS MV

Subacute sclerosing panencephalitis Central nervous system Measles virus

Introduction: Measles Virus Although diseases caused by viruses, including measles, were known and described in antiquity, viruses per se were not recognized as separate infectious agents until the late 1890s, over 110 years ago. Their recognition followed the pioneering work in bacteriology by Louis Pasteur and Robert Koch and their associates in the mid1800s. During that interval, the laboratory culturing process was developed, so it became possible to grow bacteria in culture for eventual placement on glass slides, staining, and observation under the microscope. Importantly, bacteria placed on Pasteur-Chamberland filters of varying pore sizes were retained upon vacuum pressure enabling the identification of specific bacteria, which when re-introduced into the appropriate host recaused the specific illness, thus linking the infectious agent with a particular disease state. It was on this framework that the first viruses were uncovered in 1898 (Loeffler and Frosch 1898; Beijerinck 1899; Ivanovski 1899). In contrast to the retention of bacteria on the Pasteur-Chamberland filters, viruses were characterized by their passage through the filters, their invisibility under light microscopy and their inability to grow in bacterial (cell-free) cultures. Thirteen years later, in 1911, Goldberger and Anderson (1911) showed that respiratory tract secretions from measles virus-infected patients were not retained but passed through the Pasteur-Chamberland-like filter, were invisible to light microscopy, and were unable to replicate in cell-free bacterial cultures. Moreover, macaque monkeys inoculated with material that passed through the filter developed the features of a measles virus-like disease. Another 43 years passed before MV was isolated and adapted to grow in cell cultures (Enders and Peebles 1954). This feat was accomplished by John Enders, to whom this volume is dedicated. His work formed the basis of subsequent extensive biological research on the pathogenesis of MV, methods for diagnosing MV, and eventually the development of the measles virus vaccine, again the achievement of Enders and his laboratory associates (Katz and Enders 1959; Enders et al. 1960; Katz 1965). Sam Katz, a protégé of Enders who was instrumental in the development and testing of the vaccine has recorded some of his personal observations with Enders in chapter 1 of volume 329.

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In addition to the usual transient CNS symptoms and signs that accompany acute measles virus infection, two serious and debilitating manifestations of CNS injury, although uncommon, are consequences of acute MV infection: postinfectious encephalitis and subacute sclerosing panencephalitis (SSPE). Measles virus infection can occasionally be complicated by sudden onset of encephalitis. The incidence of MV postinfection encephalitis is 1 per 1000 cases (Miller 1964; Scott 1967) and the clinical picture includes high fever, headache, vomiting, and convulsions, often leading to coma in an individual who has begun to recover from the initial acute infection. This manifestation usually occurs at 5 days, but in some instances 25 days after the measles virus rash appears. Mortality is approximately 10% with one-quarter of those who survive showing a permanent neurologic defect. Rarely is MV recovered from the patient’s cerebral spinal fluid or brain, and the pathogenesis of this disease is immunologic in nature. Infection by other viruses such as mumps, varicella, rubella, smallpox, and vaccinia may cause a similar postinfection encephalitis. In contrast to post-MV encephalitis, SSPE does not appear for several years after the primary MV infection, and no fever or other signs of acute CNS injury are present. SSPE presents with behavioral changes and mental deterioration followed by neurodegeneration of the pyramidal, extrapyramidal, and cerebellar systems, invariably leading to death. In this situation, MV genetic material is easily identified in the CNS. Despite its rarity, the lethal sequence of SSPE and its long period of latency have motivated the author’s investigation of this disease. This chapter discusses SSPE and recent observations from the author’s laboratory detailing the molecular pathogenesis of the disease.

Historical Background: Subacute Sclerosing Panencephalitis As early as the 1930s, the clinical picture of SSPE was suggestive of a potential infectious etiology. Neuropathologic evaluation (Dawson 1933; van Bogaert 1945) showed acidophilic intranuclear and cytoplasmic inclusion bodies in neurons and glia cells of the CNS. However, although infection was the suspected cause, attempts to implicate any microorganism failed. Indeed, evidence for a viral etiology required an additional 30 years. Not until the mid-1960s did Bouteille and his associates (1965) view by electron microscopy structures resembling nucleocapsids of a paramyxovirus within neurons from the brain material of a patient with SSPE. Two years later, Connolly and colleagues (1967) demonstrated that patients with SSPE had excessively high titers of antibodies to measles virus in their sera and cerebral spinal fluid. Further, by immunohistochemistry measles virus antigens were found in neurons and glia. The last and essential piece of evidence establishing an association with MV came independently from two laboratories, one at the NIH from Horta-Barbosa and colleagues (1969) and the second from a team at the University of Michigan lead by Francis Payne (Payne et al. 1969). Their work verified that the MV was defective in replication and that the recovery of virus required the assistance of a co-cultivation assay. Thus, viable brain tissue from SSPE

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patients when co-cultivated in tissue culture with cells permissive for MV lead to recovery of the virus. These results have now been confirmed multiple times and extended by direct cloning of measles virus from the brains of SSPE patients, thus firmly establishing the relationship between SSPE chronic progressive neurologic disease and MV infection. Thereafter, a plethora of papers appeared concerning measles virus and SSPE and occasionally also implicating mumps virus with SSPE (see PubMed). The fact that SSPE cases have decreased in parallel with the implementation of vaccination against MV clearly indicates that MV vaccination does not cause SSPE (Campbell et al. 2007). Noteworthy and seminal was the report concerning SSPE from the laboratories of Cattaneo, ter Meulen, and Billeter (Cattaneo et al. 1988), who performed direct cloning and molecular analysis of the MV genome recovered from brains of SSPE patients. These investigators noted biased hypermutation in the MV genome, most often affecting the matrix (M) gene, with conversion of U to C and A to G bases. The debate that followed centered on the question could such a defective MV cause or result from the persistent infection? The evidence on hand given later in this review indicates that the defective virus does not initiate infection but is a consequence of it. Further, the defective virus contributes to the ongoing pathogenesis of the disease. Currently, there is no reliable treatment that delays or aborts this chronic fatal neurodegenerative disease. Thus, investigation has centered first on establishing animal model(s) to mimic this disorder and second, on utilizing such models to understand how measles virus can establish, under rare conditions, a persistent infection and why the host cannot clear the infection. With the availability of such models, the pathogenesis of this disease can be decoded and therapeutic approaches designed and tested. An animal model that mimics SSPE must exhibit six cardinal elements: 1. Progressive fatal CNS disease; 2. Replication of measles virus in neurons; 3. Defective (nonreplicating) virus that can be recovered by co-cultivation with permissive tissue culture cells; 4. Dramatic biased hypermutation in the measles virus genome primarily involving one of the eight measles virus genes, the M gene, with conversion of U to C and A to G bases; 5. High titers of antibodies to measles virus; 6. Infiltration of B cells and T cells into the CNS. Several animal models discussed in this current volume of CTMI or the CTMI volume 191 on this subject satisfy one or more of these demands. However, the one described below fulfills all six criteria. The tg mouse model we use in which the MV receptor CD46 is transcriptionally expressed by its authentic CD46 promoter (YAC-CD46 tgs), placed on the C57Bl/6 background and then crossed onto the Rag1 genetically deleted (Rag1−/−) background provides all of the six necessary fingerprints cited above. First, such tg mice express CD46 on neurons as well as all nucleated cells. Crossing these tg mice on to the Rag1−/− background results in offspring whose B and T lymphocytes have been deleted. This maneuver removes the adaptive immune response and allows

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MV infection to persist. This approach was first successfully employed in the Rall laboratory where Rag−/− mice (Lawrence et al. 1999) were crossed with previously constructed tg mice that expressed the CD46 molecule exclusively on neurons by use of the neuron-specific enolase (NSE) promoter (Rall et al. 1997). A further benefit of using either the YAC-CD46 or NSE-CD46 mice is that reconstitution of purified and specific components of the host’s immune system can be adaptively transferred to the measles virus-infected host deleted of immune cells (YAC-CD46 × Rag−/− or NSE-CD46 × Rag−/− tg mice), thereby allowing insight into which the missing component(s) of the immune system is essential for the control and purging of measles virus from the CNS.

Transgenic Mouse Model of SSPE The primary host for measles virus is humans. Measles virus can infect monkeys but as far as we know, that occurs by transmission from humans to monkeys either by experimental inoculation or by air droplet spread to susceptible monkeys from humans infected with the virus. Earlier investigators utilized forced adaptation of measles virus by serial passage through brains of a variety of animals. Eventually, use of this method produced rodent adapted MV capable of infecting in the rodent host in which the virus had been passed. Although useful data were obtained from such experimental systems, the models were limited primarily by the disease phenotype produced, the age of the host required for infection, and the forced passage production of a mutated measles virus genome that was very distant from the sequence of the measles viruses isolated from humans and used for adaptation. Once the receptors (CD46, SLAM) for measles virus in humans were discovered, this limitation was overcome by expressing the authentic human virus receptor in mice using tg protocols. The transcriptional expression of CD46 or SLAM in mice, especially in C57Bl/6 (B6) mice, provided an easily manipulatable small-animal model in which the genetics were known and abundant markers were present to dissect the immunologic response to the virus by deletion and reconstitution of unique cells of the immune system. However, a limitation remained. Although the mouse and human genomes are over 98% identical, not all the host factors required for optimal measles virus replication and control of infection may be present or optimally functional in the rodent. Nevertheless, either tissue-specific promoters to transcriptionally express MV in cells of the immune system (e.g., CD11c for dendritic cells, LCK for T cells) in neurons in NSE for neurons or promoters to express receptors more generally have been used by a number of laboratories (Rall et al. 1997; Thorley et al. 1997; Blixenkrone-Moller et al. 1998; Mrkic et al. 1998; Oldstone et al. 1999; Hahm et al. 2003, 2004; Carsillo et al. 2006; Kurihara et al. 2006; Sellin et al. 2006; Ohno et al. 2007). Our tack and the tg model we utilized were the CD46 molecule expressed under control of its own CD46 promoter. This strategy allowed us to infect cells of the immune system and neurons in the brain. Since the CD46 genome was obtained from a yeast artificial chromosome (YAC) library (Yannoutsos et al. 1996), the

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resultant tg mice were termed YAC-CD46. Infection of YAC-CD46 mice led to virus replication in and recovery from cells of the animal immune system. This process was associated with suppression of primary and secondary humoral and cell-mediated immune responses (Oldstone et al. 1999). Further, the use of YACCD46 tg mice, measles virus infection, and challenge with a secondary bacterial infection, Listeria monocytogenes, demonstrated that measles virus suppressed both the innate and adoptive immune responses required to control the Listeria infection. Further, the molecules and immune cells involved in the process were mapped (Slifka et al. 2003). Pertinent for this chapter, infectious virus also replicated in and was recovered from neurons in the CNS. However, replication of MV in the CNS was markedly enhanced and prolonged when YAC-CD46 mice were placed on a Rag1−/− background. Rag1−/− mice have a deletion in B and T immune cells and thus cannot generate an immune response when challenged with an antigen. Thus, by avoiding a host-generated immune response to MV, adult YAC-CD46 × Rag1−/− mice, replicated and spread MV infections in their neurons. This allowed the infection to persist in the CNS for months. Figure 2.1 shows neurons of YAC-CD46 × Rag1−/− tg mice 60 days after the initiation of MV infection in adult mice. However, no MV could be

Fig. 2.1 Infection of neurons and accumulative mortality of YAC-CD46 × Rag1−/− tg mice infected with MV (Edmonston strain)

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recovered from brains of infected YAC-CD46 × Rag1−/− tg mice unless viable brain tissue was co-cultured with viable permissive tissue culture cells such as Vero cells. Electron microscopy performed by Samuel Dales, currently in Gunter Blobel’s laboratory at the Rockefeller University in New York, showed a picture in which structures of measles virus nucleocapsids were clearly present in the cytoplasm and nucleus of hippocampal neurons in MV-infected YAC-CD46 × Rag1−/− tg mice (Fig. 2.2). Further, direct cloning of the MV genome from brains of such tg mice by Lee Martin, when a postdoctoral fellow in my laboratory, revealed biased hypermutations in the M gene. Figure 2.3 displays the geography of A to G hypermutation in the M protein from one such mouse, #1919, in which a remarkable 35%–40% of the gene is mutated. Also listed in Fig. 2.3 is the A to G hypermutations in four other YAC-CD46 × Rag1−/− mice. Their clinical state at the time of sacrifice and the number of A to G mutations found are displayed. Hypermutation of the M gene commonly knocked out a unique Alu1 restriction site. This allowed Martin to devise a scheme for enrichment of additional hyperimmune M gene sequences (see Oldstone et al. 2005 for details). Cattaneo and Billeter (1988, 1992) had previously reported similar mutations and hypermutations in MV persisting in brain material from patients with SSPE. Thus the YAC-CD46 × Rag1−/− tg mouse infected with measles virus (Edmonston strain) mimicked the SSPE disease in humans by the following criteria. The first is a progressive fatal CNS disease with measles virus replication in neurons. Second, measles virus in the CNS was defective in that it was recoverable only by co-cultivation on permissive cells in culture. Third, analysis of the measles virus genome revealed biased hypermutation primarily in the M gene

Fig. 2.2 Electron micrographs of brains from YAC-CD46 × Rag1−/− tg mice infected with MV. Note presence of viral nucleocapsids in neurons. EM courtesy of Sam Dales

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Fig. 2.3 Biased hypermutations (A to G) in the M gene of MV from brains of YAC-CD46 × Rag1−/− tg mice persistently infected with MV. Each dot records A to G mutation

with A to G (U to C) changes. Further, classic SSPE-like nucleocapsid structures were seen by electron microscopic examination of infected neurons. In addition, when such YAC-CD46 × Rag1−/− tg mice were reconstituted with splenic lymphocytes and infected with measles virus, high titers of measles virus antibodies were recorded in the sera, presumably in response to repetitive stimulation of the immune system by measles virus (Oldstone et al. 2005; Tishon et al. 2006). The heightened anti-measles virus antibody response was accompanied by a modest infiltration of B and T cells in the CNS (Oldstone et al. 2005; Tishon et al. 2006).

Contribution of the Hypermutated M Protein to the Chronic Progressive CNS Disease Now that a reproducible small-animal model of SSPE was available, we could answer the question of whether or not the hypermutated A to G bases in the M gene of measles virus directly contributed to the chronic progressive CNS disease. This was accomplished by John Patterson, a former postdoctoral fellow in my laboratory, who used the reverse genetics system for MV developed by Martin Billeter and his colleagues in Zurich (Radecke et al. 1995). Patterson replaced the M gene

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of Edmonston strain measles virus with the M gene taken from Biken strain SSPE MV (Patterson et al. 2001a). The newly constructed recombinant virus was then injected into adult YAC-CD46 × Rag1−/− tg mice. Comparison of Edmonston measles virus (contains non-A to G mutated M gene) with recombinant Edmonston/M genome Biken (A to G mutated M gene) indicated that the Edmonston/M gene Biken recombinant was infectious and produced a protracted CNS disease in the range of 1–3 months longer than Edmonston alone. Both viruses caused death. Further, the recombinant virus appeared predominantly in clusters so that the infection of neurons was limited. That is, samples of multiple levels of brain tissues from tg mice infected with the recombinant Edmonston/Biken SSPE virus when compared to those infected with Edmonston virus showed less spread of the Edmonston/ Biken SSPE virus in neurons. The best interpretation of these results is that the biased hypermutations of the M gene most likely slowed the migration of the virus and thereby prolonged the infection. These data noted in vivo supported the earlier observations in vitro of Cathomen et al. (1998) that a functioning M gene is not required for measles virus replication or transcription. The other conclusion from this work is that mutations in the M gene do not harm the virus, as do some mutations to other MV genes required for replication, transcription, or viral assembly. Those detrimental mutations in other MV genes likely result in the virus’ elimination, whereas mutations in M are well tolerated and have little if any affect on virus survival.

Fig. 2.4 Evidence of a pathogenic role for the biased hypermutated M gene in the pathogenesis of SSPE. The M gene of Edmonston strain of MV was replaced by the hypermutated M gene from human SSPE MV (Biken isolate). The resultant recombinant MV when inoculated into YACCD46 × Rag1−/− tg mice significantly prolonged the time of the progressive CNS degenerative disease (left panel). Further, MV-infected neurons appeared as clumps, suggesting impairment of viral spread in vivo (compare infected neurons in Fig. 1 and Fig. 5, Edmonston MV infection) with neurons in right panels (recombinant Edmonston/Biken M gene MV). For details see Cattaneo et al. 1988; Wong et al. 1989; Patterson et al. 2001a

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Figure 2.4 illustrates the accumulated mortality curves of both groups and reveals the enhanced survival time of YAC-CD46 × Rag1−/− mice infected with the recombinant measles virus bearing the A to G hypermutated M gene. The insert shows clumping of MV-infected neurons as detected by immunochemistry.

Hypothesis to Explain the Initiation and Pathogenesis of SSPE Hypotheses regarding the initiation and pathogenesis of SSPE revolve around three clinical/pathological findings. The first concerns the issue of whether biased hypermutations in the M gene of measles virus contribute to the development of SSPE or if such mutations per se are not involved in either the initiation or maintenance of pathogenesis, but simply accumulate because the M gene is not necessary for the virus’s life cycle during CNS infection. For instance, it has been suggested that the measles virus causing SSPE may be a unique strain or recombinant virus. However, this explanation is unlikely because epidemiologic analysis of identical twins infected simultaneously, i.e., with the same measles virus, show discordance in that only one twin developed SSPE (Houff et al. 1979; Cianchetti et al. 1983; DhibJalbut and Haddad 1984). Thus, although it is unlikely that a unique strain of measles virus (carrying the biased hypermutation primarily of the M gene) is responsible for initiation of measles virus infection leading to SSPE, it is possible that such biased hypermutated viruses in the CNS, once formed, contribute to ongoing SSPE disease. Further, a suspected enzyme may be responsible for the U to C (A to G) M hypermutations and could be triggered by virus-induced cytokines in the CNS. Such cytokines would enhance the interferon-inducible double-stranded RNA adenosine deaminase (ADAR1). ADAR1 is presumed to propagate the biased hypermutations by adenosine to inosine conversion in double-stranded RNA intermediates (Cattaneo et al. 1988; Bass and Weintraub 1988; Bass et al. 1989; Baczko et al. 1993; Patterson and Samuel 1995; Patterson et al. 2001a, 2001b). Further, MVs, as they continue their infection of neurons, may alter these cells’ expression of selected genes and function without killing the infected cell. ADAR is associated with Ca++ flux and glutamate receptor editing. The second hypothesis focuses on the role of excessively high titers of antibody to measles virus in the blood and cerebral spinal fluid of patients with SSPE. This theory questions whether such antibody could participate in modulating MV gene products and thus alter the virus’s life cycle, favoring its ability to persist in infected cells. The basic idea here is that MV per se is not cytotoxic for cells but causes their injury by cell-to-cell fusions and syncytial formation, which depends on the viral glycoproteins (Joseph and Oldstone 1975; Oldstone and Tishon 1978; Fujinami and Oldstone 1979, 1980, 1983; Oldstone et al. 1980). Fusion/syncytial formation is the sine qua non of measles virus infection in tissue culture and during acute infection, i.e., syncytia of lymphoid cells in lymphoid organs, but is surprisingly absent or minimally present in neurons of the CNS in SSPE patients (Fenner 1974; Adams et al. 1984). The third hypothesis centers on the profound suppression of the immune system associated with measles virus infection. Indeed, among viruses, measles was the

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first to be recognized as causing a profound immunosuppression (Osler 1904; von Pirquet 1908; Wagner 1968), which was discovered several years before the virus was isolated (Goldberger and Anderson 1911). Measles virus-induced immunosuppression led to the activation of a latent tuberculosis, changing it to a miliary or systemic spread of tuberculin disease (von Pirquet 1908). Further, the strength of immune system suppression by MV is profound and, in fact, measles was used by physicians therapeutically before the discovery of corticosteroids to treat aggressive autoimmune nephritis (Blumberg and Cassady 1947). The activation of secondary microbial infections and other infectious agents was linked with MV well before MV was isolated and parallels the later observations with the human immunodeficiency (HIV, AIDS) virus (reviewed in McChesney and Oldstone 1989). The foundation of this hypothesis rests on three observations. The first is the study of identical twins infected simultaneously with MV (presumably the same strain) show a discordance because SSPE develops in only one twin (Houff et al. 1979; Cianchetti et al. 1983; Dhib-Jalbut and Haddad 1984). This observation suggests that an environmental effect, not a genetic predisposition, is important in initiating SSPE. This observation also disputes a component of the first hypothesis that a unique measles virus strain or altered measles virus is responsible for initiation of SSPE. The second arm of the immunosuppressive hypothesis is the clinical documentation that unimmunized patients who receive intensive immunosuppressive therapy are more vulnerable to SSPE than immunocompetent individuals infected with measles virus (Coulter et al. 1979; Fukuya et al. 1992). The third arm is that the majority of SSPE cases occur in children infected before the age of 2 years (Cattaneo et al. 1988; Griffin 2007) when the immune system is still immature. In the next section follows the experimental support for these various hypotheses, along with the possibility or realization that components of each and not necessarily only one may be involved in the initiation and pathogenesis of SSPE.

Contribution of Biased Hypermutation Predominantly in the M Gene of Measles Virus to the Pathogenesis of SSPE To determine whether a biased hypermutation of the M gene directly participates in SSPE pathogenesis we used reverse genetics to construct and rescue a recombinant measles virus containing the hypermutated M gene. The Edmonston strain of MV backbone was modified to contain the biased hypermutated A to G M gene of a human SSPE isolate Biken strain (Cattaneo et al. 1988; Wong et al. 1989). Once CNS infection was initiated in CD46 MV receptor expressing tg mice, this recombinant virus was found to be defective in budding from the plasma membranes of neurons and was recoverable only by co-cultivation of infected brains with tissue culture cells permissive for measles virus. The recombinant virus was expressed in neurons and the time course for the persistent infection was dramatically prolonged by months when compared to similar tg mice persistently infected with Edmonston virus alone. Further, recombinant Edmonston/Biken SSPE biased hypermutated

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M gene measles virus appeared predominantly in clusters and did not spread as widely through the CNS as did the Edmonston measles virus. Together these observations indicated that the biased hypermutations in the M gene likely slow the migration of the virus through the CNS and prolong the infectious cycle in the host. This outcome in an experimental animal model documented clearly and for the first time that the biased hypermutated M gene of SSPE virus per se contributes to the chronicity of SSPE disease. Probably, however, the M protein is not necessary for MV replication and transcription (Cathomen et al. 1998). Hence, mutations in the M gene are not lethal for the virus. Yet, the hypermutated M gene still allows the virus to replicate and spread. This conclusion amplifies that of Baczko et al. (1993) who recorded clonal expansion of a different hypermutated MV variant. Similarly, our use of Edmonston measles virus infection in YAC-CD46 × Rag1−/− tg mice resulted in long stretches of A to G hypermutations in the M gene and the COOH−-terminal end of the F gene (Oldstone et al. 2005). The preference for A to G and U to C mutations was dramatic accounting for over 95% of mutations in M and 73% for F genes, respectively. Further, all five mice studied showed clonal expansion of M genes with biased hypermutations. Each of the mice had Alu1 hypermutated M genes, thus mimicking the end stage found in brains of humans with SSPE (Cattaneo and Billeter 1992; Baczko et al. 1993; Radecke et al. 1995). Further, the high percentage of A to G and U to C mutations suggests the possible superimposition of mutations induced by a host enzyme such as adenosine deaminase (ADAR) (Cattaneo et al. 1988; Bass et al. 1989; Patterson and Samuel 1995). Accordingly, the hypothetical scenario is that cytokines made in the CNS during persistent MV infection enhances the interferon-inducible ADAR1 enzyme. ADAR1 would then propagate the biased hypermutation by adenosine to inosine conversion in viral double-stranded intermediates. Since the measles virus M gene is not essential to virus replication or transcription, it could absorb multiple mutations, whereas, for example, the virus polymerase–replication complex could not, resulting in the virus’s survival. Further, by bearing an increase of mutations in M, the infecting virus survives longer, thereby establishing a near continuous feedback loop cycle over a number of years in SSPE patients to favor persistence and continuous stimulation for the production of antibody to MV.

Contribution of Antibody-Induced Modulation of Measles Virus Antigens to the Pathogenesis of SSPE Patients with SSPE possess logarithmically higher titers of antibodies to measles virus than found in individuals following either acute measles virus infection or vaccination against measles. Presumably the cause is continuous immunogenic stimulation from the persisting MV. Over three decades ago, investigators recognized that MV-infected cells in culture bathed in antibodies to MV resulted in a redistribution of MV antigens on the cell surface and the suppression of both

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immune mediate injury and, more importantly, the infected cells continued to survive and switch into a state of persistent infection (Joseph and Oldstone 1974, 1975; Oldstone et al. 1980). Impressively, by removing (capping off) the MV glycoproteins the hemagglutinin (HA) and the fusion (F) protein, antibody to MV prevented cell-to-cell fusion and giant cell syncytial formation, essential components for MV-induced death. In addition, by antibody modulation therapy a time occurred when restoration of HA and F proteins (by removal of anti-measles virus antibody) no longer occurred on the surface of infected cells and, uniquely, measles virus nucleocapsids similar to those in SSPE patients appeared in the nucleus and cytoplasm of infected cells. Of interest here is that the prevention of cell-to-cell fusion by another means recapitulates these events. When MV-infected cells are actively maintained in suspension to prevent cell–cell contact, the cell lysis does not occur and persistent infection follows. Thus the abnormal nucleocapsid formation seen in patients with SSPE is also observed in either antibody-treated cells or in tg mice expressing the CD46 measles virus receptor and whose neurons are persistently infected with measles. At the molecular level, MV antibody-induced modulation occurs after using monoclonal antibodies to specific epitopes on the measles virus HA and results in a signal delivered to the plasma membrane of infected cells that alters the measles virus transcriptional complex, especially the measles virus phosphoprotein inside the cell (Fujinami and Oldstone 1979, 1980; Oldstone et al. 1980; Fujinami et al. 1984). These results from our experiments with antibodies to MV used to treat measles-infected HeLa cells were subsequently confirmed in ter Meulen’s laboratory in mouse neuroblastoma cells or rat glioma cells infected with MV (Schneider-Schaulies et al. 1992). Further, we noted that this antibody induced modulation of measles-infected cells was specific since antibodies to HeLa cellsurface antigens as well as antibodies to other viruses, i.e., influenza, failed to modulate MV-infected cells. Conversely, the antibodies to MV failed to modulate off-surface antigens of influenza or lymphocytic choriomeningitis virus on infected cells (Fujinami and Oldstone 1979, 1980; Oldstone et al. 1980; Fujinami et al. 1984; Go et al. 2006). Recently, we obtained RNA profiles and performed mass spectrometry on the antibody-modulated cells in order to identify the host genes or proteins either up- or downregulated. These gene products are still being characterized (Go et al. 2006). One gene product of interest is ADAR1. The role played by MV antibody-induced antigen modulation in vivo has been more difficult to define, although two observations are noteworthy. Wear and Rapp (1971) demonstrated that only MV (adapted to the hamster brain) infected newborn hamster pups suckled to mothers having antibodies to measles developed a persistent infection in their CNS. In contrast, similarly infected pups suckled to nonimmune mothers died of acute encephalitis. Later, working with rats and rat-adapted measles virus, ter Meulen’s laboratory (Liebert et al. 1990) found that passive transfer of neutralizing antibodies directed against selected MV HA determinants were able to modulate MV gene expression at the level of transcription and switched an expected acute necrotizing encephalitis in either Lewis rats or brown Norway rats into a persistent CNS infection.

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Dual-Hit Hypothesis: Acute Immunosuppressive Event Preceding Measles Virus Infection Clinical reports that SSPE was associated with measles virus infection occurring after drug-induced immunosuppression (Coulter et al. 1979; Fukuya et al. 1992) usually used in treatment of acute childhood leukemia, coupled with the discordance of SSPE in identical twins infected at the same time with measles virus, raised the possibility that an immunosuppressive event narrowly preceding acute MV infection might allow the virus to initially escape the host’s immune response that would otherwise terminate the acute virus infection. Persistent virus infection would then follow. Further, the timing of both events would be critical and thus account for the low frequency of SSPE. To test this hypothesis, we initially infected tg mice expressing the MV receptor with a virus, lymphocytic choriomeningitis virus (LCMV) Clone 13 (Cl 13), known to transiently suppress the mouse immune system yet itself be noncytopathic (Oldstone 2002). Infection with measles virus 10 days later, a time of maximal immunosuppression by LCMV Cl 13 in its natural murine host (Sevilla et al. 2000), resulted in persistent measles virus infection of neurons. Such mice then developed a chronic CNS disease leading to death. Analysis of their brains showed the biased A to G (U to C) hypermutations in the measles virus M gene. Further, the other cardinal manifestations of SSPE occurred in this dual-hit model: (1) generation of a defective measles virus in the CNS that could be recovered by co-cultivation with tissue culture cells permissive for measles virus; (2) high titers of antibodies to measles, and (3) infiltration of B and T cells into the CNS. The timing between initiation of the immunosuppressive event and contracting measles virus infection was crucial. LCMV Cl 13 infection of mice causes a generalized suppression of both T (cell-mediated) and B cell (humoral antibody) arms of the adoptive immune system (Sevilla et al. 2000; Oldstone 2002). Thus, antigenspecific T cell responses to several RNA and DNA viruses as well as antibody responses to soluble and particulate antigens are significantly dampened. The decreased immune response peaks at day 10–14 following initiation of infection, wanes after day 20, and by 60–90 days after LCMV Cl 13 infection, the virus is usually cleared from the mouse (except from neurons and glomeruli in which viral clearance is delayed until days 110–120) (see Oldstone et al. 1986; Tishon et al. 1993). Further, LCMV Cl 13, by virtue of its tropism for dendritic cells, also interferes with or suppresses the early innate immune response (Zuniga et al. 2008) as well as the later adoptive immune response (Sevilla et al. 2000). Thus, when MV infection occurred 3 days after LCMV Cl 13 infection, a time when there is no LCMV-induced immunosuppression, none of the inoculated mice developed persistent infection of neurons. When MV was administered 30 days after the initiation of LCMV Cl 13 infection, a time when immunosuppression has begun to wane, only a minority of mice had evidence of persistent MV infection in their neurons. In contrast, when LCMV Cl 13 was given initially followed by MV 10 days later, the time of maximal LCMV-induced immunosuppression, all mice developed

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a persistent measles virus infection of neurons (see Fig. 2.5). Cloning of virus from their brains revealed A to G (U to C) biased hypermutations prevalent in the M gene and electron microscopy revealed viral nucleocapsids in the cytoplasm and nuclei of infected neurons (Oldstone et al. 2005). Sera harvested from these mice had antibody titers that were 20- to 30-fold higher than observed in immunocompetent mice immunized with measles virus. Further, like human patients with SSPE, such mice displayed infiltration of predominantly CD4 T cells and B cells as well as CD8 T cells in their brains.

Fig. 2.5 Experimental evidence for dual-hit strategy to explain the initiation of persistent MV infection in CNS neurons. Lymphocytic choriomeningitis virus (LCMV) Clone (Cl) 13 induces a generalized immunosuppression in mice due to its replication in dendritic cells (DCs) and interferes with DC activation of T and B cells (see Sevilla et al. 2000 and Oldstone 2002 for details). Judged by results of mixed leukocyte reaction (MLR), there is negligible inhibition on MLR by day 3 but maximal inhibition at day 10 with modest inhibition at day 30 post-LCMV Cl 13 infection. The SSPE phenotype in YAC-CD46 mice infected first with LCMV and then with measles refers to: (1) persistent MV infection of neurons with chronic progressive neurologic disease; (2) presence of nucleocapsids in disarray in the cytoplasm and nuclei of infected neurons; (3) biased hypermutation in MV M genome cloned from CNS; (4) recovery of defective MV from the CNS only by co-cultivation of viable brains with MV permissive cultured cells; (5) markedly elevated amounts of antibodies to MV in the sera of infected mice (significantly higher than antibody titers in acutely infected or vaccinated mice); and (6) infiltration of T and B cells into the brains of infected mice. See Oldstone et al. 2005 for details

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Patients with clinical manifestations of SSPE when their disease emerges, several years after the initial MV infection, are not severely immunosuppressed. Our early studies with Rag1−/−- mice in which T and B cells are deleted, crossed to YACCD46 recapitulated all the findings of the LCMV Cl 13/measles virus dual-hit model (10-day interval before the second infection). The longer-lasting persistent MV infection observed with the dual-hit model then likely results from two separate factors. One is the ability of antibody to MV to prolong the lifespan of MV-infected cells (Joseph and Oldstone 1975; Oldstone et al. 1980). This observation and its consequence are discussed above in Sect. 5.2. The second factor is the enhanced A to G (U to C) mutation in the M gene in patients with SSPE (Cattaneo et al. 1988) and in the murine model outlined here (Patterson et al. 2001a; Oldstone et al. 2005). Indeed, mice given recombinant Edmonston/Biken M SSPE measles virus in which the M gene of the Biken strain from an SSPE patient replaced the normal nonmutated M gene in Edmonston measles virus (Patterson et al. 2001a) have a persistent infection lasting several months longer than mice given the Edmonston virus alone. Two essential points emerge from this model. First, a system is available to further dissect the molecular pathogenesis of SSPE and, second, an animal model of SSPE is available for testing and evaluating various protocols and therapies for treatment of this chronic neurologic disease. At present, therapy for SSPE remains controversial and overall has not worked. Drugs such as steroids, ribavirin, type I and II interferons, have been tried singularly or in combination but with either conflicting or negative results (reviewed in Gascon 2003).

Identification of the Components of Measles Virus-Specific Immune Response Required for Clearing Measles Virus from the CNS Measles virus infection is most often an acute self-limiting disease. Both humoral (antibody) and cell-mediated (T cell) immune responses act to restrict infection, although T cells appear to be the major players (Burnet 1968; Whitton and Oldstone 2001; Griffin 2007). However, clearance of persisting virus from the CNS may require a unique combination of immune cells. For example, CD8 T cells alone can overcome an acute LCMV infection, but the mix of CD4 T cells along with CD8 T cells is an absolute requirement to purge this virus from persistently infected neurons (Tishon et al. 1995). To determine the essential prerequisite for clearance of MV from neurons during a persistent MV infection, we took advantage of the YACCD46 tg mouse model and the technical ability to purify or delete individual cell constituents of the immune response and adaptively transfer them into the YACCD46 × Rag1−/− tg mice so infected. Measles virus fails to establish a persistent infection of neurons in adult YAC-CD46 tg mice because such mice have a competent and mature immune system. However, when the immune system is voided by crossing YAC-CD46 mice onto the Rag1−/− background, infection with MV then causes a persistent neuronal infection. Adaptive transfer of 5 × 107 or 1 × 107 splenic

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colleagues, we have received NOD-SCID mice implanted when 1-day-old with human neuronal stem cells. Accordingly, 1 × 105 stem cells were inoculated into six different CNS locations. These cells express the CD46 molecule and are permissive to MV infection in vivo. When the mice were 8 weeks old, they were inoculated with MV, which subsequently induced a persistent infection in the animals’ acquired human neurons. Since the MV was engineered to express GFP, infected neurons were easily identified. This technique allowed analysis of the MV genome biased mutations of A to G (U to C) and use of gene display to profile RNA expression of human neuronal genes during persistent MV infection. The MV-infected neurons in this stem cell model, as well as those in the YAC-CD46 × Rag1−/− model, mirror the situation of most infected neurons in brains of SSPE patients in that none of these neurons engage in fusion. Additionally, since MV per se is not cytotoxic for cells, neuronal apoptosis was not an issue. The hypothesis being tested is that, in SSPE, persistent MV infection of neurons affects the differentiation (luxury) function of neurons without killing them. As a result, the altered homeostasis of neuronal function leads to disease. This mechanism of disease, e.g., disturbed cell function in the absence of cell cytotoxicity, was first uncovered during study of persistent LCMV infection of several differentiated cell types in vivo – e.g., neurons, endocrine cells, immune cells – and has been extended to a variety of RNA and DNA viruses (reviewed in Oldstone 2002). A second ongoing study tests the hypothesis that cytokines released in the brain during persistent MV infection induce the dsRNA adenosine deaminase enzyme responsible for A to G (U to C) changes in the virus’s M gene (see Fig. 2.8). The resultant behavioral/neurodegenerative phenotype may likely be caused by faulty glutamate receptor editing in neurons.

Fig. 2.8 Illustrated hypothesis for the induction of cytokines inducing ADAR1 enzyme and its conversion of A to G base changes in the MV genome

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ADAR is an interesting candidate to evaluate for four reasons. First, the presence of ADAR could account for the molecular hallmark of prolonged MV infection of the CNS, the biased conversion of A to G (U to C) found in humans with SSPE (Cattaneo et al. 1988; Bass and Weintraub 1988; Bass et al. 1989; Baczko et al. 1993; Patterson and Samuel 1995; Patterson et al. 2001a, 2001b) and in the tg animal model of SSPE (Patterson et al. 2001a; Oldstone et al. 2005). Second, we found elevations of ADAR in brains of some measles virus-infected YAC-CD46 mice compared to uninfected YAC-CD46 controls. Third, ADAR is involved in neuronal glutamate receptor editing, defects that can cause neuronal dysfunction due to excess Ca++ flux (Seeburg et al. 1998; Liu and Samuel 1999). Fourth, ADAR is an interferon-inducible protein (Patterson and Samuel 1995; Patterson et al. 1995) and amounts of this cytokine are elevated in MV-infected brains. To explore the role of ADAR in the pathogenesis of SSPE disease, Bumsuk Hahm and Lee Martin, former postdoctoral fellows in my laboratory, in collaboration with Charles Samuel and Cyril George at the University of California, Santa Barbara, constructed an ADAR1a plasmid with a neo-insert for the purpose of producing ADAR1a knockout mice. At present, several potential ADAR1a knockout founders have been identified and are currently being bred to determine germline transmission of the gene. Once ADAR1a knockout lines have been confirmed, these mice will be used to generate triple tg mice: ADAR1a−/− × YAC-CD46 × Rag1−/−. Such mice, after infection with MV, will provide a valuable resource to determine the roles of ADAR in the generation of biased hypermutations A to G (U to C) and the role, if any, of glutamate receptor editing in the pathogenesis of chronic measles virus infection of neurons. Acknowledgements This is Pub. No. 19363 from the Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA. This work was supported in part by NIH grant AI036222 and NIH postdoctoral training fellowships to Glenn Rall, Lee Martin, and John Patterson. Bumsuk Hahm was supported by NIH grants AI036222 and AI074564.

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Oldstone MB, Tishon A (1978) Immunologic injury in measles virus infection. IV. Antigenic modulation and abrogation of lymphocyte lysis of virus-infected cells. Clin Immunol Immunopathol 9:55–62 Oldstone MB, Fujinami RS, Lampert PW (1980) Membrane and cytoplasmic changes in virusinfected cells induced by interactions of antiviral antibody with surface viral antigen. Prog Med Virol 26:45–93 Oldstone MBA, Blount P, Southern PJ, Lampert PW (1986) Cytoimmunotherapy for persistent virus infection: unique clearance pattern from the central nervous system. Nature 321:239–243 Oldstone MBA, Lewicki H, Thomas D, Tishon A, Dales S, Patterson J, Manchester M, Homann D, Naniche D, Holz A (1999) Measles virus infection in a transgenic model: virus-induced central nervous system disease and immunosuppression. Cell 98:629–640 Oldstone MBA, Dales S, Tishon A, Lewicki H, Martin L (2005) A role for dual viral hits in causation of subacute sclerosing panencephalitis. J Exp Med 202:1185–1190 Osler W (1904) The principles and practice of medicine. New York Patterson JB, Samuel CE (1995) Expression and regulation by interferon of a double-stranded RNA-specific adenosine deaminase from human cells: evidence for two forms of the deaminase. Mol Cell Biol 15:5376–5388 Patterson JB, Cornu TI, Redwine J, Dales S, Lewicki H, Holz A, Thomas D, Billeter MA, Oldstone MB (2001a) Evidence that the hypermutated M protein of a subacute sclerosing panencephalitis measles virus actively contributes to the chronic progressive CNS disease. Virology 291:215–225 Patterson JB, Manchester M, Oldstone MBA (2001b) Disease model: dissecting the pathogenesis of the measles virus. Trends Mol Med 7:85–88 Patterson JB, Thomis DC, Hans SL, Samuel CE (1995) Mechanism of interferon action: doublestranded RNA-specific adenosine deaminase from human cells is inducible by alpha and gamma interferons. Virology 210:508–511 Payne FE, Baublis JV, Habashi HH (1969) Isolation of measles virus from cell cultures of brain from a patient with subacute sclerosing panencephalitis. N Engl J Med 281:585–589 Radecke F, Spielhofer P, Schneider H, Kaelin K, Huber M, Dotsch C, Christiansen G, Billeter MA (1995) Rescue of measles viruses from cloned DNA. EMBO J 14:5773–5784 Rall GF, Manchester M, Daniels LR, Callahan EM, Belman AR, Oldstone MB (1997) A transgenic mouse model for measles virus infection of the brain. Proc Natl Acad Sci U S A 94:4659–4663 Schneider-Schaulies S, Liebert UG, Segev Y, Rager-Zisman B, Wolfson M, ter Meulen V (1992) Antibody-dependent transcriptional regulation of measles virus in persistently infected neural cells. J Virol 66:5534–5541 Scott T (1967) Postinfectious and vaccinial encephalitis. Med Clinics North Am 51:701–707 Seeburg PH, Higuchi M, Sprengel R (1998) RNA editing of brain glutamate receptor channels: mechanism and physiology. Brain Res Rev 26:217–229 Sellin CI, Davoust N, Guillaume V, Baas D, Belin MF, Buckland R, Wild TF, Horvat B (2006) High pathogenicity of wild-type measles virus infection in CD150 (SLAM) transgenic mice. J Virol 80:6420–6429 Sevilla N, Kunz S, Holz A, Lewicki H, Homann D, Yamada H, Campbell KP, de la Torre JC, Oldstone MBA (2000) Immunosuppression and resultant viral persistence by specific viral targeting of dendritic cells. J Exp Med 192:1249–1260 Slifka MK, Homann D, Tishon A, Pagarigan R, Oldstone MB (2003) Measles virus infection results in suppression of both innate and adaptive immune responses to secondary bacterial infection. J Clin Invest 111:805–810 Thorley BR, Milland J, Christiansen D, Lanteri MB, McInnes B, Moeller I, Rivailler P, Horvat B, Rabourdin-Combe C, Gerlier D, McKenzie IF, Loveland BE (1997) Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection. Eur J Immunol 27:726–734

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Tishon A, Eddleston M, de la Torre JC, Oldstone MB (1993) Cytotoxic T lymphocytes cleanse viral gene products from individually infected neurons and lymphocytes in mice persistently infected with lymphocytic choriomeningitis virus. Virology 197:463–467 Tishon A, Lewicki H, Rall G, von Herrath M, Oldstone MB (1995) An essential role for type 1 interferon-gamma in terminating persistent viral infection. Virology 212:244–250 Tishon A, Lewicki H, Andaya A, McGavern D, Martin L, Oldstone MBA (2006) CD4 T cell control primary measles virus infection of the CNS: regulation is dependent on combined activity with either CD8 T cells or with B cells: CD4, CD8 or B cells alone are ineffective. Virology 347:234–245 van Bogaert L (1945) Une leucoencéphalite sclérosante subaigüe. J Neurol, Neurosurg, Psych 8:101–120 von Pirquet C (1908) Das Verhalten del kutanen Tuberkulin-Reaktion Wahrend der Masern. Dtsch Med Wochenschr 34:1297 Wagner R (1968) Clements von Pirquet: his life and work. Baltimore, MD Wear DJ, Rapp F (1971) Latent measles virus infection of the hamster central nervous system. J Immunol 107:1593–1598 Whitton JL, Oldstone MBA (2001) The immune response to viruses. In: Knipe DM, Howley PM (eds) Fields virology, 4th edn. Lippincott Williams Wilkins, Philadelphia, pp 285–320 Wong TC, Ayata M, Hirano A, Yoshikawa Y, Tsuruoka H, Yamanouchi K (1989) Generalized and localized biased hypermutation affecting the matrix gene of a measles virus strain that causes subacute sclerosing panencephalitis. J Virol 63:5464–5468 Yannoutsos N, Ijzermans JN, Harkes C, Bonthuis F, Zhou CY, White D, Marquet RL, Grosveld F (1996) A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection. Genes Cells 1:409–419 Zuniga EI, Liou LY, Mack L, Oldstone MBA (2008) In vivo virus infection inhibits type 1 interferon production by plasmacytoid dendritic cells thereby facilitating opportunistic infections. Cell Host Microbe (in press)

Chapter 3

Measles Studies in the Macaque Model R.L. deSwart

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vaccination Studies in Macaques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Atypical Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Live-Attenuated Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New-Generation Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative Routes of Administration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis Studies in Macaques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Lines and Virus Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differences Between Macaque Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunity, Protection, and Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Infections with MV Expressing EGFP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Much of our current understanding of measles has come from experiments in non-human primates. In 1911, Goldberger and Anderson showed that macaques inoculated with filtered secretions from measles patients developed measles, thus demonstrating that the causative agent of this disease was a virus. Since then, different monkey species have been used for experimental measles virus infections. Moreover, infection studies in macaques demonstrated that serial passage of the virus in vivo and in vitro resulted in virus attenuation, providing the basis for all current live-attenuated measles vaccines. This chapter will review the macaque model for measles, with a focus on vaccination and immunopathogenesis studies conducted over the last 15 years. In addition, recent data are highlighted demonstrating that the application of a recombinant measles virus strain expressing enhanced green fluorescent protein dramatically increased the sensitivity of virus

R.L. deSwart Department of Virology, Erasmus MC, University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands, e-mail: [email protected]

D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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detection, both in living and sacrificed animals, allowing new approaches to old questions on measles vaccination and pathogenesis.

Introduction At the beginning of the twentieth century, it was demonstrated that measles virus (MV) could be transmitted from humans to non-human primates. One to 2 weeks after inoculation with blood collected from measles patients, macaques developed fever and skin rash (Anderson and Goldberger 1911). Disease transmission could also be achieved using filtered respiratory secretions, demonstrating that the causative agent of measles was a virus (Goldberger and Anderson 1911). A decade later, Blake and Trask followed up on these studies by experimentally infecting macaques with nasopharyngeal washings of measles patients, thereby successfully inducing measles in 16 animals (Blake and Trask 1921a). The virus could be passaged from monkey to monkey by intratracheal inoculation of tissue homogenates or by intravenous injection of citrated blood (Blake and Trask 1921a). The authors carefully documented incubation time, fever, leukopenia and pathology of enanthem and exanthem (Blake and Trask 1921b). In addition, they demonstrated that experimental MV infection resulted in complete immunity against reinfection (Blake and Trask 1921c). In the 1950s, it was demonstrated that experimental infections with MV isolated in cell culture also caused measles in macaques (Peebles et al. 1957). The authors were able to reisolate the virus and detect MV-specific serum antibody responses, thus bringing the model close to its current state. Although experimental MV infection of macaques had been demonstrated successfully, a number of other studies yielded negative results. Retrospectively, it can be concluded that in many of these cases animals were immune to measles due to prior exposure to the virus. Peebles et al. detected MV-specific serum antibodies in 22 out of 24 macaques tested from US laboratories, whereas animals screened immediately after capture all proved to be serum antibody-negative (Peebles et al. 1957). This illustrates that measles is a disease of humans and normally does not affect non-human primates unless they are brought in close contact with humans. Monkeys used as experimental animals were at that time in most cases wild-caught animals. Before and during transport they were housed under crowed conditions, and contacts with MV-infected humans could result in virus transmission (Meyer et al. 1962). On several occasions, measles outbreaks occurred at animal facilities after arrival of new animals, in some cases associated with significant morbidity (Potkay et al. 1966; Hime and Keymer 1975; Scott and Keymer 1975; Remfry 1976; MacArthur et al. 1979; Welshman 1989). Thus monkeys intended to be used for experimental MV infections needed to be transported using special precautions (Tauraso 1973). At present, all macaques used for experimental measles virus infections are purpose-bred animals, but prescreening for the absence of MV-specific antibodies remains imperative. The high susceptibility of non-human primates to MV infection provided the first animal model for measles. The two species used most often are the rhesus

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macaque (Macaca mulatta) and the cynomolgus macaque (Macaca fascicularis) (El Mubarak et al. 2007). As a result of the close similarity of clinical, virological, immunological and pathological parameters to those associated with measles in humans, the model continues to be used almost a century later. Although several rodent species and other small laboratory animals have been inoculated with MV, most of these do not or poorly replicate the virus (Stittelaar et al. 2002a; Pütz et al. 2003). Studies in cotton rats or transgenic animal species do reproduce certain aspects of measles, but none of these show the same level of similarity with the pathogenesis of measles in humans as the macaque model (see chapters 5 and 6). New world monkeys proved to be even more susceptible to MV infection than old world monkeys, but developed a disease with a different pathogenesis than that of measles in humans, which was associated with high mortality (Levy and Mirkovic 1971; Albrecht et al. 1980). Due to their high susceptibility, marmosets (Saguinus mystax) were later used for encephalogenicity studies, as reviewed elsewhere (Van Binnendijk et al. 1995). The current review will address macaque models of measles, with a focus on studies published after 1995.

Vaccination Studies in Macaques The first isolation of MV in cell culture (Enders and Peebles 1954) was immediately followed by attempts to develop a vaccine against measles. Two different strategies were pursued in parallel: inactivated vaccines and live-attenuated vaccines. Whereas the first category of vaccines proved to predispose for enhanced disease, the second was highly successful. In the 1990s studies were initiated to develop new-generation vaccines as potential successors of the current live-attenuated vaccines, some of which showed promise in preclinical studies in macaques. However, none of these candidate new measles vaccines have been pushed forward towards licensing for human use. Studies in the 1980s had already demonstrated that administration of the current MV vaccine by aerosol held great promise (Sabin et al. 1982). However, regulatory authorities consider a vaccine and its route of administration as one entity, which means that before the aerosol route for measles vaccination can be implemented licensure will be required, for which preclinical studies were conducted in macaques.

Atypical Measles In the 1960s, classical inactivated measles vaccines were manufactured by formalin inactivation of whole MV preparations, which were subsequently precipitated with aluminum salts (referred to as FI-MV). Initially, vaccination with FI-MV was shown to induce MV-specific serum antibody responses (Hilleman et al. 1962;

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Norrby et al. 1964; Foege et al. 1965), and the vaccine was used in large-scale clinical trials in humans. However, after a number of years it turned out that vaccination predisposed for enhanced disease upon natural MV infection, which was referred to as atypical measles (Fulginiti et al. 1967) (see also chapter 10). During the same period, vaccination trials with formalin-inactivated respiratory syncytial virus (FI-RSV), another member of the family Paramyxoviridae, also proved to predispose for enhanced disease upon natural infection (Fulginiti et al. 1969; Kapikian et al. 1969; Kim et al. 1969). Whereas availability of small animal models for RSV allowed extensive studies on the pathogenesis of this vaccinemediated enhanced disease, this remained difficult for measles. Because the pathogenesis remained subject to speculation, the risk of inducing atypical measles continued to form a major stumbling block for the development of new generation nonreplicating MV vaccines. In 1999, Polack and Griffin were successful in reproducing atypical measles in macaques (Polack et al. 1999), thus providing an opportunity to study the pathogenesis of the disease as well as a model to test candidate new MV vaccines for predisposition of similar aberrant responses upon challenge infection. They immunized macaques with the original FI-MV preparation that had been manufactured in the 1960s. When challenged with pathogenic wild-type MV, two out of five rhesus macaques vaccinated with FI-MV developed atypical measles, characterized by a petechial rash and pneumonitis associated with the presence of eosinophils and immune complexes in their lungs. The authors concluded that atypical measles “results from previous priming for a nonprotective type 2 CD4 T cell response rather than from lack of functional antibody against the fusion protein” (Polack et al. 1999). In follow-up studies, they further characterized immune responses in these animals, demonstrating in vivo impairment of interleukin (IL)-12 production and increased production of IL-4 by peripheral blood mononuclear cells (Polack et al. 2002). When characterizing the antibody responses, they found that these were not only transient but also lacked avidity maturation. Challenge infection resulted in anamnestic production of low avidity antibodies, which could explain the immune complex deposition in FI-MV-primed animals (Polack et al. 2003a). Before reproduction of atypical measles in the macaque model, it was speculated that inactivation with formalin had resulted in destruction of critical B cell epitopes on the fusion protein (Norrby et al. 1975). However, this hypothesis could be rejected on the basis of two macaque experiments: animals primed with FI-MV developed atypical measles in the presence of fusion-inhibiting antibodies (Polack et al. 1999), and macaques primed with a DNA vaccine encoding for the haemagglutinin gene that developed H- but not F-specific antibodies did not develop any of the clinical signs associated with atypical measles (Polack et al. 2000). Interestingly, vaccination of macaques with formalin-inactivated respiratory syncytial virus (RSV) or human metapneumovirus (hMPV) preparations also predisposed for hypersensitivity to challenge infection with the respective viruses, suggesting that these phenomena are characteristic for all members of the family Paramyxoviridae (De Swart et al. 2002, 2007c).

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Live-Attenuated Vaccines As mentioned above, the MV strain Edmonston isolated by Enders and colleagues in 1954 induced measles-like clinical signs in macaques (Peebles et al. 1957). Serial passage of this virus in vivo (in chicken embryos) and in vitro (in chicken embryo fibroblast [CEF] cells) resulted in virus attenuation: upon experimental infection of macaques, the virus still induced MV-specific immune responses but virtually no clinical signs (Enders et al. 1960). More than 30 years later, direct comparison of MV isolated in lymphoid cells (strain Bilthoven) with MV passaged in human and monkey kidney cells (strain Edmonston wild type) or live-attenuated MV passaged in CEF cells (strain Schwarz) demonstrated that these strains displayed high, intermediate and low pathogenicity in macaques, respectively (Van Binnendijk et al. 1994). Virus loads in peripheral blood mononuclear cells and in bronchoalveolar lavage cells were several log values lower in animals infected with MV strains of reduced pathogenicity. Whereas levels of MV-specific serum IgM antibodies seemed directly related to the magnitude of virus loads, levels of specific serum IgG and virus neutralizing (VN) antibodies induced by the three virus strains were on the same order of magnitude (Van Binnendijk et al. 1994). Macaques and other non-human primate species have been used to assess the levels of attenuation of different candidate vaccine virus strains. In most cases, this was done by studying pathological lesions induced by intracerebral MV infection (Buynak et al. 1962; Nii et al. 1964b; Albrecht et al. 1981; Sharova et al. 1984). At present, vaccine manufacturers still use this method to assess appropriate attenuation of new MV vaccine virus seed stocks.

New-Generation Vaccines Despite its documented safety and efficacy, live-attenuated MV vaccines also have drawbacks, most importantly their dependence on cold chain maintenance and their ineffectiveness in the presence of maternal antibodies (Stittelaar et al. 2002a; Pütz et al. 2003). Live-attenuated MV vaccines are ineffective when used before the age of 9 months, resulting in a window of susceptibility in young infants between waning of maternal immunity and acquisition of vaccine-induced immunity. Since the 1980s, new-generation candidate MV vaccines have been developed to address these issues, including subunit vaccines, vectored vaccines and nucleic acid vaccines (see chapter 10). In the macaque model, passive transfer of MV-specific VN antibodies inhibited effectiveness of the live-attenuated MV vaccine, thus providing a model to evaluate the potential of candidate new vaccines in the presence of maternal antibodies (Van Binnendijk et al. 1997). The scientific steering committee of the World Health Organization (WHO) adopted the macaque model for preclinical comparison of the different candidates. A strategy was developed in which vaccine candidates were first used to immunize juvenile animals, either in the absence or presence of passively transferred MV-specific antibodies. This would

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allow assessment of immunogenicity and longevity of the induced specific immune responses. Approximately 1 year after vaccination, animals were challenged with a pathogenic wild-type MV strain to assess levels of protection. Vaccine candidates that performed well in this evaluation were subsequently tested in infant macaques, to assess their potential effectiveness at an early age in the presence of true maternally derived VN antibodies. This strategy resulted in the identification of a number of promising candidate MV vaccines. However, further preclinical and clinical evaluation of a new MV vaccine will require huge financial investments. Moreover, improved coverage of the live-attenuated MV vaccine in recent years in combination with the implementation of a two-dose strategy has been highly successful in reducing measles mortality. As a result, it remains uncertain if any of the candidate new-generation MV vaccines will ever be licensed for human use.

Alternative Routes of Administration For regulatory purposes, a vaccine and the device used for administration form one integral entity: the current live-attenuated MV vaccines are licensed for injection only. However, alternative routes of administration have extensively been studied in humans. Whereas intradermal, conjunctival, oral or intranasal administration were not particularly successful, aerosol administration of nebulized MV vaccine proved highly effective (Cutts et al. 1997). The aerosol route closely mimics the natural route of MV infection and may result in both mucosal and systemic immunity (Valdespino-Gomez et al. 2006). The WHO, in partnership with the American Red Cross and the Centers for Disease Control and Prevention and with funding from the Bill and Melinda Gates Foundation, has initiated a Product Development Group (PDG) for measles aerosol vaccination. The ultimate objective of the PDG is to achieve licensure of a combination of measles vaccine and nebulizer, which is equally safe, effective and cheap as the currently licensed measles vaccines administered by injection, but is easier to administer, less invasive, and could be administered by nonmedical personnel. The PDG evaluated two alternative strategies for measles aerosol vaccination: nebulization of a reconstituted vaccine (Dilraj et al. 2000) or inhalation of a dry powder aerosol (LiCalsi et al. 2001). Both vaccination strategies were evaluated in immunocompetent and immunocompromised macaques and proved equally safe. However, vaccination with a nebulized aerosol proved more effective than inhalation of a dry powder vaccine (De Swart et al. 2006, 2007a). The animals used in these studies had body weights of 1.8–4.5 kg and consequently had much smaller tidal volumes than those of children. To mimic vaccination using a similar dose to that inhaled by a child in 30 s, the exposure time therefore had to be prolonged (De Swart et al. 2006). Before proceeding to clinical trials, a toxicology study was conducted using larger study groups than those used in the exploratory studies. These studies were also

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conducted in macaques, to allow MV vaccine replication in all exposed tissues. This study revealed no adverse events, and measles aerosol vaccination is currently under investigation in clinical trials. The PDG aims to achieve licensure for this vaccination route in 2009.

Pathogenesis Studies in Macaques Experimental MV infections of macaques have been crucial for our understanding of the pathogenesis of measles. Infections with MV isolated in cell culture demonstrated the importance of passage history of the virus: MV strains exclusively cultured in lymphoid cells retained pathogenicity in macaques, whereas passage in other cell lines often resulted in virus attenuation. However, nonattenuated MV strains were in some cases also associated with subclinical infections, which seemed to be related to species differences between rhesus and cynomolgus macaques rather than to virus differences. A large number of studies have focused on the development of MV-specific immune responses, and the role of different arms of the immune system in protection from measles. Measles is usually not recognized before onset of rash, approximately 2 weeks after MV infection, making studies on the early events following virus transmission in humans difficult to perform. Pathological studies of experimentally infected macaques have provided important insights in the tissue distribution and cell tropism of the virus. Recently, infections with recombinant MV expressing enhanced green fluorescent protein (EGFP) resulted in improved sensitivity of virus detection, and provided new insights in the role of different target cells. This new approach to an old animal model provides alternative possibilities to further unravel the pathogenesis of measles and measles-associated immunosuppression.

Cell Lines and Virus Strains The introduction of Epstein-Barr virus (EBV)-transformed marmoset B cell line B95a as a substrate for isolation of MV (Kobune et al. 1990) made it possible to isolate non-culture-adapted wild-type MV strains. Whereas isolation of wild-type MV from patient samples in Vero cells usually takes at least 2 weeks (and in many cases requires serial blind passage), virus isolation in B95a cells can be as rapid as 2–4 days (WHO 2007). Experimental infections with the Edmonston wild-type strain did not always result in detectable clinical signs (Enders et al. 1960; Hicks et al. 1977; Van Binnendijk et al. 1994), whereas non-cell culture-passaged virus usually induced more fulminant infections (Yamanouchi et al. 1973; Sakaguchi et al. 1986; McChesney et al. 1989). Kobune and colleagues demonstrated that MV isolated in B95a cells retained its pathogenicity in macaques, resulting in development of viremia, lymphopenia and rash (Kobune et al. 1996).

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Van Binnendijk et al. isolated wild-type MV in human EBV-transformed Blymphoblastic cell lines (BLCL) and demonstrated that this virus also retained pathogenicity (Van Binnendijk et al. 1994). These authors applied the infectious center assay to the model, thus quantifying the frequency of MV-infected cells during viremia. Intratracheal inoculation of macaques with a single infectious unit of wild-type MV strain Bilthoven could spark infection associated with similar viral load kinetics as infection with 104 infectious units, with the only difference that the peak of viremia shifted slightly backwards in time with decreasing infectious dosage (Van Binnendijk et al. 1994). McChesney and colleagues developed a measles model in macaques using a MV strain isolated during an outbreak of measles in a primate facility (McChesney et al. 1997). The virus was isolated in Raji cells, a human BLCL isolated from a patient with Burkitt’s lymphoma, a disease resulting from in vivo transformation of B lymphocytes by EBV infection. Subsequently, the virus was passaged in vivo in macaques, after which a challenge stock was produced in macaque mononuclear cells (McChesney et al. 1997). This virus was also fully pathogenic in macaques, as demonstrated by induction of clinical and pathological changes typical for measles (McChesney et al. 1997) and MV-specific immune responses (Zhu et al. 1997). Auwaerter and colleagues compared the pathogenicity in macaques of six different MV strains, demonstrating that the nonadapted Bilthoven strain was fully pathogenic while the other cell culture-adapted strains were not (Auwaerter et al. 1999). Interestingly, a recent study addressing genetic changes that affect the virulence of MV in macaques demonstrated that wild-type MV isolated in Vero cells expressing CD150 also retained pathogenicity in macaques and induced skin rash (Bankamp et al. 2008). In contrast, the same MV isolated and passaged in normal Vero cells or in CEF cells did not induce rash. This suggests that expression of CD150 on the cells used for virus isolation is crucial, and that the cells are not required to be of lymphoid origin. Cell culture adaptation does not necessarily result in adaptation to the use of CD46 as a receptor, as the adapted MV isolates in the above-mentioned study did not infect Chinese Hamster Ovary cells expressing CD46 (Bankamp et al. 2008).

Differences Between Macaque Species Although experimental MV infections have been conducted both in rhesus and cynomolgus macaques, clinical signs such as rash and conjunctivitis were especially reported in rhesus macaques (Blake and Trask 1921b; McChesney et al. 1997; Auwaerter et al. 1999). Although skin rash has also been reported in cynomolgus macaques (Kobune et al. 1996), this symptom seemed to be less prominent in this species. The first direct comparison of MV infection in these two macaque species by experimental infection with two different non-culture-adapted wild-type MV strains indeed seemed to confirm this assumption: although animals of both species displayed similar virus replication curves in peripheral blood and broncho-alveolar

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lavage cells as well as similar MV-specific immune responses, the appearance of skin rash was more prominent in rhesus macaques (El Mubarak et al. 2007). Infection studies with a recombinant MV strain expressing EGFP conducted in parallel in rhesus and cynomolgus macaques showed that in both animal species MV-infected cells were detected in many different tissues, including the skin (De Swart et al. 2007b). Also with respect to other virological and immunological parameters, including infection of specific lymphocyte subsets, MV infection followed a virtually identical course in both animal species, suggesting that both macaque species can be used for measles pathogenesis studies.

Immunity, Protection, and Immunosuppression Both in humans and macaques recovery from measles is accompanied by lifelong immunity (Blake and Trask 1921c). MV infection induces strong specific humoral and cellular immune responses, and many different assays have been developed to characterize these ex vivo in macaques. The most important serological parameter is the detection of VN antibodies in serum, of which levels above 0.1–0.2 IU/ml have been identified as a correlate of protection from measles in infants (Chen et al. 1990; Samb et al. 1995). In macaques, similar levels of passively transferred VN antibodies were shown to interfere with MV vaccination (Van Binnendijk et al. 1997). Measurement of specific cell-mediated immunity (CMI) is less well standardized, but several techniques including assessment of lymphoproliferation (Van Binnendijk et al. 1997; Pan et al. 2005), cytotoxicity (Van Binnendijk et al. 1997; Zhu et al. 1997, 2000), cytokine production (Auwaerter et al. 1999; Polack et al. 2002, 2003b; Stittelaar et al. 2002b; Premenko-Lanier et al. 2003; Pan et al. 2005; Pasetti et al. 2007) or flow cytometry-based stimulation assays (Stittelaar et al. 2000; Pahar et al. 2005; De Swart et al. 2006) have been employed as correlates of CMI. VN antibodies can confer complete protection from measles, as also illustrated by the fact that infants born of a mother with adequate MV-specific antibody titers are protected from measles during their first months of life. In contrast, clearance of an established MV infection is largely dependent on CMI. Agammaglobulinemic patients recover normally from measles, while individuals with impaired CMI may succumb to MV infection (Burnet 1968). The role of specific lymphocyte populations in clearance of MV was addressed in the macaque model by depleting single or multiple populations using monoclonal antibodies to CD20 and/or CD8. Macaques depleted of all T lymphocytes or of CD8+ T lymphocytes only at the moment of MV infection exhibited a more extensive rash, increased viral loads and delayed viral clearance (Hicks et al. 1977; Permar et al. 2003). In contrast, depletion of CD20+ B lymphocytes did not result in alterations of clinical signs or kinetics of MV clearance (Permar et al. 2004). These studies demonstrate that CMI (and more specifically CD8+ T lymphocyte responses), but not humoral immunity, plays a crucial role in MV clearance. These data also highlight a major strength of the

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macaque model, correlating specific immune responses to protection from or clearance of experimental MV infection. Measles is not only associated with the induction of strong MV-specific immune responses but also with a transient immunosuppression, leading to enhanced susceptibility to opportunistic infections (see also chapter 12). Many putative mechanisms have been suggested, but in vivo assessment of the relative importance of these hypotheses has remained difficult. MV infects lymphocytes in vitro and in vivo (Yamanouchi et al. 1973; McChesney et al. 1989), but may also alter functionality of noninfected lymphocytes (Okada et al. 2000) or antigen-presenting cells (Schneider-Schaulies et al. 2003). Furthermore, MV infection interferes with production of specific cytokines, thus changing the host response to invading pathogens (Griffin et al. 1994; Moss et al. 2002). Paradoxically, MV inhibits proliferation of lymphocytes in vitro (Hirsch et al. 1984), but recovery from measles is associated with extensive lymphocyte expansion in vivo (Mongkolsapaya et al. 1999). Due to difficulties in standardization of CMI assays, it has been difficult to evaluate mechanisms of immunosuppression in macaques. Lymphopenia and reduced responses to mitogen stimulation have been described extensively (McChesney et al. 1989; Kobune et al. 1996; Zhu et al. 1997; Auwaerter et al. 1999). MV infection of macaques specifically alters the production of IL-10 and IL-12, thus affecting the balance between phenotypically different T lymphocyte populations (Polack et al. 2002; Hoffman et al. 2003a, 2003b). Perhaps the most functional assessment of immunocompetence of macaques during measles is the assessment of responses to immunization with other antigens, e.g., tetanus toxoid, which has been applied both to assess recall of previously primed immune responses (Bankamp et al. 2008) or as primary immunization during measles (Premenko-Lanier et al. 2004). Flow cytometry studies on lymphocytes of macaques infected with MV-EGFP suggest that infection of specific memory T lymphocyte subsets may also play an important role in measles-associated immunosuppression.

Pathology Early studies on the pathology of measles in macaques demonstrated the remarkable similarity to measles in humans (Blake and Trask 1921b). However, experimental infections in an animal model offer the possibility to evaluate pathological changes during different phases of the pathogenesis, whereas human tissue samples collected during the prodromal phase of measles are relatively rare. The classical Warthin-Finkeldey-type syncytial cells observed in human lymphoid tissues during the prodromal phase (Warthin 1931; Finkeldey 1931) were also observed in macaque tissues (Nii et al. 1964a; Yamanouchi et al. 1970, 1973; Hall et al. 1971; Sakaguchi et al. 1986; McChesney et al. 1997). The major tissues affected by MV infection of macaques are the upper and lower respiratory tract, the gastrointestinal tract, the lymphoid system and the skin (Blake

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and Trask 1921b; Sergiev et al. 1960; Nii et al. 1964a; Hall et al. 1971; McChesney et al. 1997). However, the cellular origin of the lesions has been debated for decades (Nii et al. 1964a; De Swart et al. 2007b). Both in human and macaque tissues, MV antigen is commonly detected in association with epithelial tissues. In addition, culture-adapted MV strains grow very well in epithelial cells, which has led to the assumption that respiratory epithelial cells were a primary target for MV infection. However, epithelial cells do not express CD150 and are not easily infected with non-culture-adapted wild-type MV strains in vitro (Takeuchi et al. 2003). These observations warrant a reevaluation of the pathogenesis of measles, which may be facilitated by the combination of modern immunohistochemistry and flow cytometry techniques for characterizing cell subsets with experimental infections of macaques with MV strains expressing EGFP.

Infections with MV Expressing EGFP The development of reverse genetics techniques for non-culture-adapted MV strains resulted in the rescue of a MV strain from cloned cDNA that retained pathogenicity in macaques (Takeda et al. 2000). This molecular clone was used as a backbone for insertion of the gene encoding EGFP as an additional transcription unit upstream of the MV N gene. The resulting recombinant virus displayed similar in vitro replication characteristics as its parental strain, and infected cells produced high amounts of EGFP (Hashimoto et al. 2002). The MV-EGFP strain still proved to be virulent in macaques, and EGFP could be visualized macroscopically in both living and sacrificed animals, and microscopically by confocal microscopy and flow cytometry (De Swart et al. 2007b). As illustrated in Fig. 3.1, EGFP fluorescence was detected in skin, respiratory tract and digestive tract, but most intensely in lymphoid tissues. B and T lymphocytes expressing CD150 were the major target cells for MV infection. Highest percentages (up to more than 30%) of infected lymphocytes were detected in lymphoid tissues. In peripheral tissues, large numbers of MV-infected CD11c+ MHC class-II+ myeloid dendritic cells (DCs) were detected in conjunction with infected T lymphocytes, suggesting transmission of MV between these cell types. In the respiratory tract, the majority of MV-infected cells was detected in subepithelial tissues, but infected ciliated epithelial cells were also observed. In the T lymphocyte compartment, MV preferentially infected CD45RA− cells, which have a memory phenotype. This observation, which is in good accordance with recent data from MV infections in a human tonsillar explant model (Condack et al. 2007), pointed to a possible role for depletion of specific lymphocyte subsets in the transient disappearance of recall responses, as described in the early twentieth century (Von Pirquet 1908). By using cell-sorting techniques, the model allows both identification and functional assessment of MV-infected cell populations. The macaque model using the pathogenic autofluorescent wild-type MV strain IC323/EGFP clearly opens new possibilities for measles pathogenesis

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Fig. 3.1 A–M Imaging of MV-EGFP infection in macaques. A–G Macroscopic EGFP fluorescence in tissues of a cynomolgus macaque 9 days after experimental infection with MV-IC323-EGFP (close to the peak of virus replication): EGFP fluorescence in skin (A), gingiva and buccal mucosa (B), tongue and tonsils (C), inguinal lymph nodes (D), lungs with tracheobronchial lymph nodes

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studies, including identification of the first target cells infected upon transmission of MV to a naive host.

Concluding Remarks In conclusion, the macaque model has proven to be of crucial importance for development of measles vaccines, as well as for our understanding of measles pathogenesis. Vaccination and challenge studies in macaques have provided information on immunogenicity and protective capacity of both new vaccines and old vaccines given via alternative routes of administration. Modeling atypical measles has provided a tool for safety assessment of nonreplicating candidate measles vaccines. This important lesson from the past should help us avoid making similar mistakes in the future with other viral vaccines intended for priming immunity to respiratory viruses (Marshall and Enserink 2004; Ruat et al. 2008). In those cases, preclinical studies should not only evaluate acute toxicity responses to the vaccination, but also potential immunopathological responses to challenge infection. Further employment of the potential of reverse genetics holds the promise of new insights in measles pathogenesis in the coming years. Infection studies in macaques using pathogenic MV strains expressing EGFP may provide new insights in the pathogenesis of measles and measles-associated immunosuppression. In addition, by comparing pathogenic and vaccine strains expressing EGFP we may be able to learn more on the in vivo tropism of vaccine strains that can use either CD150 or CD46 as a receptor in vitro. Finally, manipulation of viruses and viral genes will enable direct assessment of the role of the different viral proteins in these processes. Fig. 3.1 (Continued) (E), stomach (left), spleen (upper left) and large intestine with gut-associated lymphoid tissue (GALT) (F), spleen (right) and large intestine with GALT (G). H, I cynomolgus macaque 13 days after MV-IC323-EGFP infection (late stage of the infection): skin rash shown under normal light (H) or by EGFP fluorescence (I). J Flow cytometric detection of EGFP+ cells in peripheral blood mononuclear cell (PBMC) subpopulations of a cynomolgus macaque at different time points after infection. Freshly isolated PBMCs were stained with monoclonal antibodies, and analyzed in a FACScalibur measuring approximately 500,000 events per sample to allow detection of low-frequent MV-infected cell populations. Results are shown as dot plots, with EGFP expression on the y-axis and CD150 expression on the x-axis. EGFP expression in CD3+CD4+ T lymphocytes is shown in red; in CD3+CD8+ T lymphocytes in green and in MHC class-II+CD20+ B lymphocytes in blue. K Confocal scanning laser microscopical image of EGFP+ cells in lymphoid follicles of the spleen of a rhesus macaque 9 days after infection with MVIC323-EGFP. MV infection was visualized in paraformaldehyde-fixed vibratome-cut tissue sections (100 µm) by direct detection of EGFP fluorescence. Propidium iodide (red) was used as a structural counter stain. L, M Confocal scanning laser microscopical image of EGFP+ cells in the trachea of a rhesus macaque 9 days after infection with MV-IC323-EGFP. MV infection was visualized in formalin-fixed microtome-cut tissue sections (6 µm) by staining with anti-EGFP antibodies. Propidium iodide (red) was used as a structural counter stain. A single MV-infected ciliated epithelial cell can be seen in the mucociliary epithelium (L), but the majority of infected cells is present in the lamina propria and submucosa of the epithelium. These include cells with the phenotype of lymphocytes and of dendritic cells. (From de Swart et al. 2007b)

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Acknowledgements I thank Albert Osterhaus, Rob van Binnendijk, Teunis Geijtenbeek and Lot de Witte for critical comments on the manuscript.

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with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies. J Virol 74:4236–4243 Stittelaar KJ, De Swart RL, Osterhaus ADME (2002a) Vaccination against measles: a neverending story. Expert Rev Vaccines 1:151–159 Stittelaar KJ, Vos HW, Van Amerongen G, Kersten GFA, Osterhaus ADME, De Swart RL (2002b) Longevity of neutralizing antibody levels in macaques vaccinated with Quil A-adjuvanted measles vaccine candidates. Vaccine 21:155–157 Takeda M, Takeuchi K, Miyajima N, Kobune F, Ami Y, Nagata N, Suzaki Y, Nagai Y, Tashiro M (2000) Recovery of pathogenic measles virus from cloned cDNA. J Virol 74:6643–6647 Takeuchi K, Miyajima N, Nagata N, Takeda M, Tashiro M (2003) Wild-type measles virus induces large syncytium formation in primary human small airway epithelial cells by a SLAM(CD150)independent mechanism. Virus Res 94:11–16 Tauraso NM (1973) Review of recent epizootics in nonhuman primate colonies and their relation to man. Lab Anim Sci 23:201–210 Valdespino-Gomez JL, De Lourdes Garcia-Garcia M, Fernandez de Castro J, Henao-Restrepo AM, Bennett J, Sepulveda-Amor J (2006) Measles aerosol vaccination. Curr Top Microbiol Immunol 304:165–193 Van Binnendijk RS, van der Heijden RWJ, Van Amerongen G, UytdeHaag FGCM, Osterhaus ADME (1994) Viral replication and development of specific immunity in macaques after infection with different measles virus strains. J Infect Dis 170:443–448 Van Binnendijk RS, van der Heijden RWJ, Osterhaus ADME (1995) Monkeys in measles research. Curr Top Microbiol Immunol 191:135–148 Van Binnendijk RS, Poelen MCM, Van Amerongen G, De Vries P, Osterhaus ADME (1997) Protective immunity in macaques vaccinated with live attenuated, recombinant, and subunit measles vaccines in the presence of passively acquired antibodies. J Infect Dis 175:524–532 Von Pirquet CE (1908) Das Verhalten der kutanen Tuberkulin-reaktion während der Masern. Dtsch Med Wochenschr 34:1297–1300 Warthin AS (1931) Occurrence of numerous large giant cells in the tonsils and pharyngeal mucosa in the prodromal stage of measles. Arch Pathol 11:864–874 Welshman MD (1989) Measles in the cynomolgus monkey (Macaca fascicularis). Vet Rec 124:184–186 WHO (2007) Manual for the laboratory diagnosis of measles and rubella virus infection. WHO/ IVB/07.01, Geneva Yamanouchi K, Egashira Y, Uchida N, Kodama H, Kobune F, Hayami M, Fukuda A, Shishido A (1970) Giant cell formation in lymphoid tissues of monkeys inoculated with various strains of measles virus. J Med Sci Biol 23:131–145 Yamanouchi K, Chino F, Kobune F, Kodama H, Tsuruhara T (1973) Growth of measles virus in the lymphoid tissues of monkeys. J Infect Dis 128:795–799 Zhu Y, Heath J, Collins J, Greene T, Antipa L, Rota PA, Bellini W, McChesney MB (1997) Experimental measles II. Infection and immunity in the rhesus macaque. Virology 233:85–92 Zhu Y-D, Rota P, Wyatt LS, Tamin A, Rozenblatt S, Lerche N, Moss B, Bellini W, McChesney M (2000) Evaluation of recombinant vaccinia virus – measles vaccines in infant rhesus macaques with preexisting measles antibody. Virology 276:202–213

Chapter 4

Ferrets as a Model for Morbillivirus Pathogenesis, Complications, and Vaccines S. Pillet, N. Svitek, and V. von Messling(*)

Contents Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ferret Husbandry and Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Availability and Development of Ferret-Specific Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . The Ferret as a Model for Measles Vaccine Development . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of Maternal Antibodies for Measles Vaccination Strategies . . . . . . . . . . . . . . . . Vaccination Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Ferret as Model for Measles-Induced Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . The Ferret as Model for Measles-Associated Neurological Complications . . . . . . . . . . . . . . Overview of Morbillivirus-Associated Central Nervous System Complications . . . . . . . . Subacute Sclerosing Panencephalitis in Ferrets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Morbillivirus Neuroinvasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract The ferret is a standard laboratory animal that can be accommodated in most animal facilities. While not susceptible to measles, ferrets are a natural host of canine distemper virus (CDV), the closely related carnivore morbillivirus. CDV infection in ferrets reproduces all clinical signs associated with measles in humans, including the typical rash, fever, general immunosuppression, gastrointestinal and respiratory involvement, and neurological complications. Due to this similarity, experimental CDV infection of ferrets is frequently used to assess the efficacy of novel vaccines, and to characterize pathogenesis mechanisms. In addition, direct intracranial inoculation of measles isolates from subacute sclerosing panencephalitis (SSPE) patients results in an SSPE-like disease in animals that survive the acute phase. Since the advent of reverse genetics systems that allow the targeted manipulation of viral genomes, the model has been used to evaluate the contribution of the accessory proteins C and V, and signalling lymphocyte activation molecule (SLAM)binding to immunosuppression and overall pathogenesis. Similarly produced green

V. von Messling INRS-Institut Armand-Frappier, University of Quebec, 531, boul. des Prairies, Laval, QC, H7V 1B7, Canada, e-mail: [email protected] D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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fluorescent protein-expressing derivatives that maintain parental virulence have been instrumental in the direct visualization of systemic dissemination and neuroinvasion. As more immunological tools become available for this model, its contribution to our understanding of morbillivirus–host interactions is expected to increase.

Abbreviations ADEM CAV2 CDV CNS eGFP F H IFN Ig IL MV MHC MIBE N P SLAM SSPE

Acute disseminated encephalomyelitis Canine adenovirus type 2 Canine distemper virus Central nervous system Enhanced green fluorescent protein Fusion Hemagglutinin Interferon Immunoglobulin Interleukin Measles virus Major histocompatibility complex Inclusion body encephalitis Nucleocapsid Phosphoprotein Signalling lymphocyte activation molecule Subacute sclerosing panencephalitis

Introduction Domestic ferrets (Mustela putorius furo) belong to the family Mustelidae, which also includes weasels, minks, martens, sables, skunks, otters, and badgers, in the order Carnivora (McKenna and Bell 1997). Due to their relatively small size and natural susceptibility to a broad range of respiratory viruses, ferrets are an attractive animal model for pathogenesis research and the development of new vaccines and treatment approaches (Hsu et al. 1994; Maher and DeStefano 2004; Osterhaus et al. 2004). While they do not develop signs of disease upon intranasal infection with measles virus (MV), intracranial inoculation of certain subacute sclerosing panencephalitis (SSPE) isolates results in a histopathologically similar subacute encephalitis (Thormar et al. 1985). In addition, ferrets are a natural host for canine distemper virus (CDV), a close relative of MV that infects a broad range of carnivores. They develop all signs of disease seen in MV-infected humans, including the characteristic rash, but usually succumb to the infection (von Messling et al. 2003). This clinical similarity combined with the high sensitivity make the study of CDV in ferrets an ideal model to characterize MV pathogenesis mechanisms.

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Ferret Husbandry and Physiology Ferrets have long tubular bodies with short legs, and they are very flexible. While most rabbit and cat cages correspond to regulatory requirements for the housing of ferrets in experimental settings, they require adaptation to prevent the animals’ escape through feeders or other small openings. Ferrets are generally friendly and display a broad range of behaviors. Whenever possible, they should be housed in groups and appropriate toys should be provided to avoid boredom. They can be fed ad libitum with commercially available cat food, and food should not be withheld for more than 6 h because of their rapid metabolism (Fox 1998; Moody et al. 1985). Ferrets reach sexual maturity around 6–8 months of age, with the weight of adult males reaching up to 2 kg, while females will weigh between 0.5 and 1 kg. Measurement of the rectal temperature and subcutaneous and intramuscular injections require only scruffing of the loose skin on the back of the neck, while intravenous injections and venipunctures are greatly facilitated by general anesthesia. This can be either achieved by intramuscular injection of a combination of ketamine and either midazolam, xylazine, or acepromazine, or by inhalation of isoflurane. Ferrets have a total blood volume of approximately 60 ml/kg, and 10% can be safely withdrawn once every 2 weeks. The most commonly used site for venipuncture is the jugular vein, but the anterior vena cava can also be used. For smaller volumes, the lateral saphenous or the cephalic vein and the tail artery are an alternative option. A detailed description of the techniques involved can be found in Quesenberry (1996).

Availability and Development of Ferret-Specific Reagents The limited availability of ferret-specific immunological reagents has been a major drawback of this animal model. Aside from secondary antisera to ferret IgG, IgM, IgA, an increasing number of cross-reacting antibodies against various epitopes have become commercially available (Table 4.1). In addition, the susceptibility of ferrets to influenza and SARS (Maher and DeStefano 2004; Osterhaus et al. 2004) has led to an increased effort to develop ferret-specific reagents. Several ferret cytokine and chemokine sequences have been published in GenBank, and PCRbased assays have been developed to assess gene expression (Danesh et al. 2008; Senchak et al. 2007; Svitek and von Messling 2007). While ferret-specific proteinbased assays are still rare (Ochi et al. 2008), the potential applicability of commercial canine or feline tests can now be evaluated based on sequence homology comparisons. The recent addition of the ferret genome to the sequencing targets approved by the NIH National Human Genome Research Institute will further accelerate the development of this model.

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Table 4.1 Noncomprehensive list of cross-reactive antibodies commercially available for ferrets Antibody

Clone

Company

Mouse monoclonal anti-CD79A

HM47

Mouse monoclonal anti-CD79α cy Mouse monoclonal anti-CD3

HM57 PC3/188A

Mouse monoclonal anti-CD3 Mouse monoclonal anti-CD14 Mouse monoclonal anti-CD172a Mouse monoclonal anti-CD18 Mouse monoclonal anti-CD21-like Mouse monoclonal anti-CD41/46 Mouse monoclonal anti-CD44 Mouse monoclonal anti-MHC I Mouse monoclonal anti-MHC II Mouse monoclonal anti-MHC II (HLA-DP) Mouse monoclonal anti-MHC II (HLA-DQ) Mouse monoclonal anti-MHC II (HLA-DRa) Mouse monoclonal anti-MHC II (HLA-DR) Mouse monoclonal anti-keratin Pan Ab-3 Rabbit polyclonal anti-glial fibrillary acidic protein Rabbit polyclonal anti-Von Willebrand Factor Goat polyclonal anti-ferret IgG H and l chains Goat polyclonal anti-ferret IgAa

F7.2.38 CAM36A DH59B BAQ30A F46A CL2A BAG40A H58A CAT82A H425A

Santa Cruz Biotechnology, CA DakoCytomation, CA Santa Cruz Biotechnology, CA DakoCytomation, CA VMRD, WA VMRD, WA VMRD, WA VMRD, WA VMRD, WA VMRD, WA VMRD, WA VMRD, WA VMRD, WA

TH81A5

VMRD, WA

TH14B

VMRD, WA

H34A

VMRD, WA

Lu-5

NeoMarkers, CA

–

DakoCytomation, CA

–

DakoCytomation, CA

–

Bethyl Laboratories, TX

–

Goat polyclonal anti-ferret IgM μ

–

Rockland Immunochemicals, PA Rockland Immunochemicals, PA

The Ferret as a Model for Measles Vaccine Development The Role of Maternal Antibodies for Measles Vaccination Strategies In addition to the immaturity of the infant’s immune system, the poor response to MV vaccination observed in infants under 1 year of age has also been linked to the presence of maternal antibodies (Gans et al. 1998; Schluederberg et al. 1973; Wilkins and Wehrle 1979). Thus, maternal antibodies are an important consideration for MV vaccination development and for the ongoing eradication

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campaign. Ferrets and dogs have been extensively used to characterize the interactions of live-attenuated morbillivirus vaccines with maternal antibodies. Since both species are highly susceptible to CDV, annual vaccination with a live-attenuated vaccine similar to that used in humans is recommended. Consequently, ferret kits and puppies are born with maternal antibodies that diminish as the animal ages. In ferret kits born to mothers vaccinated with a modified-live CDV vaccine of chicken tissue culture origin, virus-specific maternal antibodies have a half-life of approximately 9 days and reach levels below the detection limit after 12 weeks (Appel and Harris 1988), levels comparable to maternal canine parvovirus antibodies transferred to puppies (Pollock and Carmichael 1982). Despite this shorter half-life of maternal antibodies, ferrets and dogs thus recapitulate the overall aspects of maternally acquired anti-MV antibodies in human infants (Gans et al. 1998). In a study using a recombinant canarypox virus expressing the CDV fusion (F) and hemagglutinin (H) proteins, only 14% survival was seen in ferret kits with preexisting maternal immunity after mucosal or parenteral immunization with the recombinant candidate vaccines, despite high levels of neutralizing antibodies at the time of challenge. However, the percentage of survival increased to 33% when animals were inoculated by the combined parenteral/mucosal route (Welter et al. 2000), suggesting that multiple routes of inoculation may be advantageous. In puppies, maternal antibody interference can be efficiently overcome by the use of the same canarypox-based vaccine, a similarly constructed adenoviral vector, or DNA vaccination with a mixture of plasmids coding for the CDV nucleocapsid (N), phosphoprotein (P), and H genes (Fischer et al. 2002, 2003; Griot et al. 2004; Pardo et al. 2007). In all cases, the vaccination elicited an increase in neutralizing antibodies, and all animals were protected from subsequent challenge. This difference in vaccine efficacy observed between dogs and ferrets is likely due to differences in experimental setup and readout, but may also reflect the higher sensitivity of the latter to CDV infection. Taken together, these studies provide valuable insights for the development of MV vaccination strategies that retain the effectiveness in the presence of maternal antibodies.

Vaccination Studies Modified-Live and Inactivated Vaccines Ferrets and dogs have also been used to assess the efficacy of new CDV candidate vaccines and novel vaccination strategies. Early studies demonstrated the efficacy of the live-attenuated CDV vaccines upon subcutaneous and intramuscular inoculation, but also raised concerns because of the residual immunosuppressive properties and the occasional anaphylactic reactions observed (Cabasso et al. 1953; Gorham et al. 1954; Greenacre 2003; Hoover et al. 1989; Morris et al. 1954; Rockborn et al. 1965; Shen et al. 1984; Stephensen et al. 1997; Williams et al. 1996; Wimsatt et al. 2001). To circumvent this problem, the cross-protective

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properties of the live-attenuated MV vaccine were evaluated in puppies that had no maternal antibodies. While none of the adverse effects associated with the CDV vaccine were observed, the level of protection conferred was variable (Appel et al. 1984; Chalmers and Baxendale 1994; Strating 1975). A comparative study of a beta-propiolactone-inactivated virus and the live-attenuated CDV vaccine revealed that both vaccines conferred protective immunity, even though neutralizing antibody levels were lower in the group receiving the inactivated product. However, animals receiving the live-attenuated vaccine developed a transient lymphopenia after vaccination, demonstrating again that the live-attenuated vaccine virus had retained some immunosuppressive properties (Williams et al. 1996). This residual virulence of the commercially available live-attenuated vaccines is particularly pertinent for the development of vaccination strategies for CDV-susceptible wildlife species in zoos or in conservation programs. Many of these species may develop severe disease or even die from inoculation with these vaccines. The near extinction of black-footed ferrets caused by a vaccination campaign is the best known example (Carpenter et al. 1976; Pearson 1977), but there are similar reports for several other species, including domestic ferrets (Bush et al. 1976; Carpenter et al. 1976; Ek-Kommonen et al. 2003; Gill et al. 1988; Greenacre 2003). It was initially recommended to use either an inactivated vaccine or the live-attenuated MV vaccine for highly sensitive or endangered species, possibly followed by inoculation with the live-attenuated CDV vaccine after verification of antibody titers (Loeffler et al. 2007; Qin et al. 2007; Wimsatt et al. 2003). However, since its approval in 1997 (Pardo et al. 1997), the recombinant canarypox vaccine expressing the CDV envelope glycoproteins has replaced these approaches. In addition to providing insight into the risks and limitations of liveattenuated vaccines, these studies constitute a framework for the development of vaccination strategies for immunosuppressed individuals.

Novel Vaccine Candidates The main disadvantages of the currently commercially available live-attenuated morbillivirus vaccines lie in their temperature sensitivity and the need for intramuscular injection. Among the novel vaccine candidates brought forward to address these issues, recombinant poxviruses expressing the CDV envelope glycoproteins have been evaluated in ferrets (Stephensen et al. 1997). While the traditionally vaccinated group still experienced weight loss and developed erythematous rash and leukopenia, animals receiving the recombinant vaccine mounted a strong humoral immune response and were completely protected from clinical disease. This study provided the first proof-of-principle that recombinant poxviruses expressing morbillivirus F and H proteins elicit protective immunity. The same group subsequently demonstrated that these poxviral candidate vaccines were similarly efficacious upon intranasal inoculation while intraduodenal infection conferred only partial protection (Welter et al. 1999). In ferrets less than 12 weeks of age, vaccine efficacy was dependent on the absence of maternal antibodies (Welter et al. 2000). A similar

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vaccination strategy using two replication-competent canine adenovirus type 2 (CAV2) that expressed the CDV F and H proteins, respectively, was equally efficient in protecting CAV2 seronegative dogs from lethal challenge after intranasal inoculation (Fischer et al. 2002). In the presence of anti-CAV2 antibodies, this vaccine was only effective when inoculated via the subcutaneous route. DNA-based vaccines have not yet been evaluated in ferrets; however, studies conducted in dogs and minks (Dahl et al. 2004) have provided the proof-of-concept for this type of vaccination. Dogs immunized with a combination of plasmids expressing the CDV N, F, H proteins mounted a neutralizing immune response and were protected from challenge (Cherpillod et al. 2000). However, antibody titers markedly increased only after the third DNA immunization and reached high neutralizing values only after challenge. The formulation of plasmids encoding the CDV F and H proteins with the cationic lipid DMRIE-DOPE resulted in significant seroconversion and protection from lethal intracranial challenge after the second immunization (Fischer et al. 2003). These studies demonstrate that several alternatives to the currently used live-attenuated vaccines are available and suggest that similar approaches may be applicable to MV.

The Ferret as Model for Measles-Induced Immunosuppression Since the host range of MV is limited to humans and certain non-human primates, animal models using other morbilliviruses have been developed to characterize the mechanisms underlying the long-lasting immunosuppression associated with MV infection. The clinical and immunological similarities of the diseases caused by MV and CDV in their respective hosts, the fact that ferrets and dogs are common laboratory animals, and the lack of regulatory limitations restricting the work with ungulate morbilliviruses have resulted in preferential use of CDV-based models (Kauffman et al. 1982; Krakowka 1982; Krakowka et al. 1987; McCullough et al. 1974; von Messling et al. 2003, 2004). Ferrets or dogs infected with wild-type CDV strains develop the characteristic clinical signs of morbillivirus disease, including the typical rash, fever, and gastrointestinal and respiratory involvement. In addition, severe leukopenia, inhibition of lymphocyte proliferation upon nonspecific stimulation, and reduction or loss of delayed-type hypersensitivity reactions are observed within 1 week after infection (Griffin 2007; Kauffman et al. 1982; Krakowka et al. 1975; von Messling et al. 2003). This immunosuppression is sustained throughout the acute phase of the disease and may persist in dogs for weeks after virus clearance (McCullough et al. 1974; Schobesberger et al. 2005). The ability to generate genetically modified wild-type viruses by reverse genetics and the introduction of an additional transcriptional unit encoding the enhanced green fluorescent protein (eGFP) in the viral genome have proven an invaluable tool for the characterization of virus–host interactions on the macroscopic and microscopic level (von Messling et al. 2004). Time course studies showed that the infection is initially limited to lymphatic tissues, where T and B cells are the primary targets. Spread to epithelia occurs only after massive infection of the immune

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system and coincided with the onset of clinical signs, followed by gradual neuroinvasion (Rudd et al. 2006). The extent of the lymphotropism observed, where over 70% of T and B cells were eGFP-positive within the 1st week of a wild type infection, illustrated the direct impact of the infection on the immune system (von Messling et al. 2004). The importance of immune cell infection for viral pathogenesis was further demonstrated by the inability of a signalling lymphocyte activation molecule (SLAM)-blind virus to spread and cause disease (von Messling et al. 2006). Since ferrets usually succumb to the infection with wild-type CDV strains, the model is well suited for the characterization of genetic factors of immune evasion. A comparative study involving viruses that lacked the accessory proteins V and C either individually or in combination demonstrated the essential role of V, while indicating that C had a more subtle function (von Messling et al. 2006). Confirming findings from in vitro and murine studies with the corresponding recombinant MVs, a V-mediated block in cytokine induction was observed (Devaux et al. 2007; Fontana et al. 2008; Ohno et al. 2004; Palosaari et al. 2003). The subsequent analysis of cytokine profiles from animals that survived the infection revealed a response similar to that seen in patients with naturally acquired MV infection (Atabani et al. 2001; Moss et al. 2002; Svitek and von Messling 2007; Tetteh et al. 2003; Zilliox et al. 2007). The response was characterized by a strong interleukin (IL)-10 response as early as 3 days after infection, and sustained induction of cytokines of the proinflamatory or the TH1 and TH2 pathways such as IL-6, IL-2, IL-12p40, IL-4 and interferon (IFN)-gamma (Svitek and von Messling 2007). Taken together, these studies demonstrate the value of CDV-based models for the characterization of morbillivirus immunosuppression and highlight the complementarities of different experimental systems.

The Ferret as Model for Measles-Associated Neurological Complications Overview of Morbillivirus-Associated Central Nervous System Complications MV can result in early or late central nervous system (CNS) involvement. Based on onset, virus presence in the brain, and mitigating host factors, three distinct forms are recognized: acute disseminated encephalomyelitis (ADEM), which is thought to be mainly immune-mediated and occurs within 2–4 weeks after infection; inclusion body encephalitis (MIBE), a rare complication prevalent in immunocompromised patients, which develops within weeks after recovery and is associated with high amounts of replicating virus in the CNS; and SSPE, which manifests months to years after the initial infection and is characterized by the development and subsequent persistence of a highly cell-associated non-productive form of the virus (Griffin 2007; Sips et al. 2007).

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CDV is associated with one of the highest incidences of CNS complications among morbilliviruses. Up to 30% of dogs exhibit signs of neurologic involvement during or after CDV infection, and most wild carnivores that succumb to CDV display some evidence of CNS involvement (Appel and Summers 1995; Confer et al. 1975; Summers et al. 1984; van Moll et al. 1995). Similarly to MV, CDVinduced CNS complications can be categorized into acute encephalopathy, inclusion body polioencephalitis, and subacute to chronic demyelinating encephalitis (Krakowka et al. 1985). The Snyder Hill strain, which was selected for increased neurovirulence by multiple passages in dogs via intracerebral inoculation, induces acute encephalitis with high mortality that mimics the MV encephalitis seen in patients with acquired immune deficiency syndrome (Gillespie and Rickard 1956; McQuaid et al. 1998; Summers et al. 1984). In contrast, typical wild-type strains mainly induce demyelinating SSPE-like lesions in dogs. These lesions are first observed around 3 weeks postinfection and can progress even after the virus has been cleared from the periphery and the animal has recovered from the acute disease (Vandevelde et al. 1982; Vandevelde and Zurbriggen 2005). This chronic demyelinating encephalitis is characterized by perivascular cuffing, intracerebral infiltration of immune cells, MHC II upregulation, and increased levels of antiCDV antibodies in the cerebrospinal fluid, all hallmarks of SSPE (Alldinger et al. 1996; Dorries and Ter Meulen 1984; Nagano et al. 1991; Schneider-Schaulies et al. 1999; Vandevelde et al. 1986).

Subacute Sclerosing Panencephalitis in Ferrets Forty years ago, Katz et al. (1968) inoculated ferrets intracerebrally with brain cell cultures from SSPE patients, producing acute encephalitis in these animals. In contrast to typical cell-associated nonproductive SSPE strains, wild-type MV strains and the efficiently replicating SSPE isolates did not cause detectable encephalitis in young ferrets. Subsequent studies showed that the course of disease depends on the respective cell-associated SSPE strain and the type of cell culture used for virus propagation (Thormar et al. 1985). However, despite severe clinical signs and large amounts of virus in the brain, only minor lesions and no humoral immune response or inflammatory changes were observed in the CNS, in contrast to human SSPE tissues (Brown et al. 1985). In an attempt to reproduce SSPE more accurately, a nonproductive SSPE strain was inoculated into ferrets previously immunized with the live-attenuated MV vaccine. Among the 50% of survivors, some developed subacute encephalitis weeks or months after inoculation (Mehta and Thormar 1979). The neurological signs observed in these ferrets had certain similarities to those seen in SSPE patients, leading to coma and death. Cell-associated nonproductive MV was always present in the brain and sometimes in the spinal cord, and widespread inflammatory lesions were observed in both the white and gray matter. Ferrets developing subacute encephalitis furthermore exhibited increased IgG concentrations in the cerebrospinal fluid, another hallmark of SSPE (Mehta and Thormar 1979; Thormar

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et al. 1983). The lack of antibodies against the viral matrix protein despite high titers against other structural proteins was also observed (Thormar et al. 1985). In a follow-up study, cell-associated nonproductive SSPE viruses were inoculated intracardially to elucidate the route of CNS invasion. Although infectious virus was not detectable in the blood of ferrets tested daily following inoculation, clinical signs and inflammatory lesions as well as the presence of viral antigens in the brain demonstrate the ability of the virus to spread to the CNS and cause encephalitis within 5–7 days (Thormar et al. 1988). By reproducing key features of the disease, inoculation of ferrets with human isolates provides unique insights in SSPE pathogenesis. The findings emphasize the importance of the host’s immune response, especially the humoral response, in the establishment of a persistent morbillivirus infection in the CNS. Even though the intracerebral route of inoculation constitutes a major drawback of this model, it merits further development as more ferret-specific reagents for the assessment of the immune response become available.

Morbillivirus Neuroinvasion Due to the high incidence of CNS involvement associated with CDV infections, morbillivirus neuroinvasion has been extensively studied in dogs and more recently in ferrets (Axthelm and Krakowka 1987; Bonami et al. 2007; Rudd et al. 2006; Vandevelde and Zurbriggen 2005). In dogs experimentally infected with a neurovirulent strain, the outcome is determined by the individual’s ability to mount a rapid and specific immune response. Compared to complete recovery without CNS involvement, persistent CNS infection was associated with a delayed and reduced immune response, while animals that died during the acute disease phase were unable to mount any response (Appel et al. 1982). The regular presence of extravascular infected lymphocytes in the white matter and the choroid plexus of CDVinfected dogs during the acute disease phase was indicative of neurovasion via the hematogenous route (Summers et al. 1979), which may be facilitated by alterations of the blood–brain barrier resulting from direct infection of endothelial cells (Axthelm and Krakowka 1987). Using an eGFP-expressing derivative of A75/17 that retained parental virulence and tropism in the ferret model, it was shown that neuroinvasion occurred not only hematogenously through infected circulating lymphocytes, but also anterogradely via the olfactory signaling route (Rudd et al. 2006). In fact, the earliest macroscopically detectable infected foci in the brain were located in the olfactory bulb, emphasizing the importance of this route of entry for CNS dissemination. CNS invasion only occurred after extensive infection of immune and epithelial tissues, when the animal was already highly immunosuppressed, indicating that morbillivirus neuroinvasion is a late event and requires an inefficient or absent immune response. In a follow-up study, it was shown that the duration of the infection in the absence of an immune response, and not differences in attachment protein-mediated tropism, determines the ability of a CDV strain to invade the CNS (Bonami et al. 2007).

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Conclusion and Perspectives The recognition that CDV causes several pathologies in its natural hosts that are similar to MV-associated disease syndromes in humans has promoted the study of host–CDV interactions as a model for measles. Compared to non-human primates or even dogs, ferrets are relatively inexpensive small animals that do not require costly facilities and are easy to house and manipulate. CDV in ferrets recapitulates the clinical signs of measles in humans including the typical rash, fever, severe leukopenia, and general immunosuppression as well as gastrointestinal and respiratory involvement. The study of CDV infection in ferrets is therefore increasingly used as a model for pathogenesis studies and vaccine development. The ongoing effort to develop ferret-specific immunological tools and the advances in the genetic manipulation of morbilliviruses will further increase the attractivity of this model for morbillivirus research.

References Alldinger S, Wunschmann A, Baumgartner W, Voss C, Kremmer E (1996) Up-regulation of major histocompatibility complex class II antigen expression in the central nervous system of dogs with spontaneous canine distemper virus encephalitis. Acta Neuropathol 92:273–280 Appel MJ, Harris WV (1988) Antibody titers in domestic ferret jills and their kits to canine distemper virus vaccine. J Am Vet Med Assoc 193:332–333 Appel MJ, Shek WR, Summers BA (1982) Lymphocyte-mediated immune cytotoxicity in dogs infected with virulent canine distemper virus. Infect Immun 37:592–600 Appel MJ, Shek WR, Shesberadaran H, Norrby E (1984) Measles virus and inactivated canine distemper virus induce incomplete immunity to canine distemper. Arch Virol 82:73–82 Appel MJ, Summers BA (1995) Pathogenicity of morbilliviruses for terrestrial carnivores. Vet Microbiol 44:187–191 Atabani SF, Byrnes AA, Jaye A, Kidd IM, Magnusen AF, Whittle H, Karp CL (2001) Natural measles causes prolonged suppression of interleukin-12 production. J Infect Dis 184:1–9 Axthelm MK, Krakowka S (1987) Canine distemper virus: the early blood-brain barrier lesion. Acta Neuropathol 75:27–33 Bonami F, Rudd PA, von Messling V (2007) Disease duration determines canine distemper virus neurovirulence. J Virol 81:12066–12070 Brown HR, Thormar H, Barshatzky M, Wisniewski HM (1985) Localization of measles virus antigens in subacute sclerosing panencephalitis in ferrets. Lab Anim Sci 35:233–237 Bush M, Montali RJ, Brownstein D, James AE Jr, Appel MJ (1976) Vaccine-induced canine distemper in a lesser panda. J Am Vet Med Assoc 169:959–960 Cabasso VJ, Stebbins MR, Cox HR (1953) Active immunization of ferrets by simultaneous injections of avianized canine distemper vaccine and anticanine distemper hyperimmune serum. Cornell Vet 43:179–183 Carpenter JW, Appel MJ, Erickson RC, Novilla MN (1976) Fatal vaccine-induced canine distemper virus infection in black-footed ferrets. J Am Vet Med Assoc 169:961–964 Chalmers WS, Baxendale W (1994) A comparison of canine distemper vaccine and measles vaccine for the prevention of canine distemper in young puppies. Vet Rec 135:349–353 Cherpillod P, Tipold A, Griot-Wenk M, Cardozo C, Schmid I, Fatzer R, Schobesberger M, Zurbriggen R, Bruckner L, Roch F et al (2000) DNA vaccine encoding nucleocapsid and surface proteins of wild type canine distemper virus protects its natural host against distemper. Vaccine 18:2927–2936

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von Messling V, Svitek N, Cattaneo R (2006) Receptor (SLAM [CD150]) recognition and the V protein sustain swift lymphocyte-based invasion of mucosal tissue and lymphatic organs by a morbillivirus. J Virol 80:6084–6092 Welter J, Taylor J, Tartaglia J, Paoletti E, Stephensen CB (1999) Mucosal vaccination with recombinant poxvirus vaccines protects ferrets against symptomatic CDV infection. Vaccine 17:308–318 Welter J, Taylor J, Tartaglia J, Paoletti E, Stephensen CB (2000) Vaccination against canine distemper virus infection in infant ferrets with and without maternal antibody protection, using recombinant attenuated poxvirus vaccines. J Virol 74:6358–6367 Wilkins J, Wehrle PF (1979) Additional evidence against measles vaccine administration to infants less than 12 months of age: altered immune response following active/passive immunization. J Pediatr 94:865–869 Williams ES, Anderson SL, Cavender J, Lynn C, List K, Hearn C, Appel MJ (1996) Vaccination of black-footed ferret (Mustela nigripes) x Siberian polecat (M. eversmanni) hybrids and domestic ferrets (M. putorius furo)against canine distemper. J Wildl Dis 32:417–423 Wimsatt J, Jay MT, Innes KE, Jessen M, Collins JK (2001) Serologic evaluation, efficacy, and safety of a commercial modified-live canine distemper vaccine in domestic ferrets. Am J Vet Res 62:736–740 Wimsatt J, Biggins D, Innes K, Taylor B, Garell D (2003) Evaluation of oral and subcutaneous delivery of an experimental canarypox recombinant canine distemper vaccine in the Siberian polecat (Mustela eversmanni) J Zoo Wildl Med 34:25–35 Zilliox MJ, Moss WJ, Griffin DE (2007) Gene expression changes in peripheral blood mononuclear cells during measles virus infection. Clin Vaccine Immunol 14:918–923

Chapter 5

Current Animal Models: Cotton Rat Animal Model Cotton Rat S. Niewiesk

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reagents and Methods for the Analysis of Infectious Diseases in Cotton Rats . . . . . . . . . . . Growth Behavior of Measles Wild Type, Vaccine, and Recombinant Viruses in Cotton Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viral Replication and Spread of Measles Virus in Cotton Rats . . . . . . . . . . . . . . . . . . . . . . Role of Cytoplasmic Tails of MV Glycoproteins in Cell-to-Cell Transfer . . . . . . . . . . . . . Role of Measles Virus V Protein for Virus Replication. . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction of Heat Shock Protein 72 with Measles Virus Nucleocapsid . . . . . . . . . . . . . . Vector Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Virus-Induced Immune Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Inhibits T Cell Rather than B Cell Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Suppresses the CD8 T Cell Response In Vivo Using the DTH Model of Epicutaneous DNFB Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Glycoproteins Are Sufficient and Necessary to Suppress Proliferation. . . . . . . . . . . . . . MV Induces Cell Cycle Retardation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Testing of Antiviral Substances and Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evaluating Antivirals in Cotton Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immune Response Against Measles Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Testing of Measles Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of Seroconversion in the Presence of Maternal Antibodies . . . . . . . . . . . . . . . . Experimental Vector Systems Do Not Induce Protection in the Presence of MV-Specific (Maternal) Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . Failure of Experimental Vaccines to Immunize in the Presence of Maternal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract The cotton rat (Sigmodon hispidus) model has proven to be a suitable small animal model for measles virus pathogenesis to fill the niche between tissue culture and studies in macaques. Similar to mice, inbred cotton rats are available in a microbiologically defined quality with an ever-increasing arsenal of reagents and S. Niewiesk College of Veterinary Medicine, Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210, USA, e-mail: [email protected] D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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methods available for the study of infectious diseases. Cotton rats replicate measles virus in the respiratory tract and (depending on virus strain) in lymphoid organs. They can be infected with vaccine, wild-type, and recombinant measles viruses and have been used to study viruses with genetic modifications. Other areas of study include efficacy testing of antivirals and vaccines. The cotton rat also has been an informative animal model to investigate measles virus-induced immune suppression and suppression of vaccination by maternal antibodies. In addition, the cotton rat promises to be a useful model for the study of polymicrobial disease (interaction between measles virus and secondary pathogens).

Introduction The value of the cotton rat (Sigmodon hispidus) for the investigation of measles vaccination and pathogenesis is its natural susceptibility toward measles virus (MV) infection after intranasal inoculation. Virus replicates mostly in the respiratory tract, causing interstitial pneumonia without overt clinical signs of disease. The cotton rat is semipermissive for measles virus infection, which means that the amount of virus obtained from the animal is proportionate to the amount of virus inoculated. In contrast, in a fully permissive animal, only viral kinetics and not the level of virus replication are determined by the viral inoculum. The first report of the use of cotton rats as laboratory animals stems from 1939 when it was found that poliovirus induced paralysis in cotton rats (Armstrong 1939). Since then, it has been shown that cotton rats can be infected with a variety of human viral, bacterial, and parasitic pathogens: human immunodeficiency virus (Rytik et al. 1995; Langley et al. 1998); herpes simplex virus type 1 (Lewandowski et al. 2002); herpes simplex virus type 2 (Yim et al. 2005); adenovirus (Pacini et al. 1984); measles virus (Wyde et al. 1992, 1999); respiratory syncytial virus (Dreizin et al. 1971; Murphy et al. 1990); human metapneumovirus (Wyde et al. 2005; Steinbach and Duca 1940; Williams et al. 2005; Hamelin et al. 2005); parainfluenza virus (Murphy et al. 1981); influenza A virus (Brydak 1990; Ottolini et al. 2003); Helicobacter pylori (Mähler et al. 2002); Staphylococcus aureus (Weidenmaier et al. 2004); Mycobacterium tuberculosis (Elwood et al. 2007); Borrelia burgdorferi (Burgdorfer and Gage 1987; Oliver et al. 1995); Francisella tularensis (Lowery 1981); Toxoplasma gondii (Clark 1984); Leishmania donovani (Azazy et al. 1994); and Echinococcus multilocularis (Kroeze and Tanner 1985); Dipetalonema vitae (Bayer and Wenk 1988). The high susceptibility to productive infection might be linked to similarities between cotton rats and humans in their antipathogen response, which differs from mice (Mestas and Hughes 2004). Examples of these similarities are the low production of nitric oxide by macrophages (Carsillo et al. 2008) and the presence of functional Mx proteins in cotton rats (Stertz et al. 2007). The additional value of the cotton rat is its well-defined genetics and an increasing pool of reagents, thus providing a small animal model for measles virus research (for review Niewiesk and Prince 2002). The cotton rat has been used as a pathogenesis model, a tool

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to investigate the effect of modifications in measles virus on in vivo replication and a model for screening purposes to test antivirals and vaccines.

Genetics Cotton rats are new world rodents that have their natural habitat in the south of the United States of America, Central America, and northern regions of South America. In contrast to the more often used members of the Muridae family, mice (Mus musculus) and rats (Rattus norwegicus), cotton rats belong to the Sigmodontinae family. Originally, wild hispid cotton rats were captured to set up an outbred colony at the National Institutes of Health (NIH), Bethesda, MD, USA, Veterinary Resources Program. By caesarean section and weaning onto rat (Rattus norvegicus) foster mothers, a specific pathogen-free colony was rederived. By serial brother–sister mating, an inbred strain was established (SIG/N). Offspring from these animals are marketed worldwide by various commercial breeders in a microbiologically defined quality. Currently, some 300 genes of cotton rats have been sequenced, and sequence comparisons demonstrate a 75%– 95% homology to mice and rats and approximately 50% homology to humans.

Reagents and Methods for the Analysis of Infectious Diseases in Cotton Rats The use of cotton rats has been supported by the availability of inbred animals and the development of reagents and methods for the analysis of immune responses against infectious diseases in cotton rats. These include standard immunological methods for antibody detection such as ELISA and neutralization assays, and methods for T cell analysis such as T cell proliferation and cytotoxic T cell assays. In addition, antibodies against cell surface markers have been produced and detection systems for various chemokines and cytokines have been established (for details see Tables 5.1 and 5.2). For cotton rats, permanent cell lines are available and tissue culture methods for primary cell culture have been established (Niewiesk and Prince 2002; Blanco et al. 2004).

Growth Behavior of Measles Wild Type, Vaccine, and Recombinant Viruses in Cotton Rats The kinetics and extent of measles virus replication has been defined for vaccine and wild-type viruses. Based on this knowledge, recombinant viruses with specific mutations have been used to address the question of tropism and to compare in vitro with in vivo consequences of these mutants.

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Table 5.1 Antibodies for the analysis of immune responses in cotton rats Target molecule Antibody Reference CD3 Polyclonal Elwood et al. 2007 CD4 mAB Richter et al. 2005 CD8alpha mAB Streif et al. 2004 CD18 Polyclonal Carsillo et al. 2008 CD57 mAB (mouse, clone NK1.1) Eichelberger et al. 2006 CD79a mAB (human, clone HM57) Elwood et al. 2007 MHC I mAB (human, clone W6/32) Carsillo et al. 2008 MHC II mAB (mouse, clone 13/4) Pueschel et al. 2007 CD68 mAB (rat, clone ED1) Schachtner et al. 1995 IgA mAB Schlereth et al. 2003 IgG Polyclonal Schlereth et al. 2003 Mx 1 and 2 mAB (human, clone M143) Pletneva et al. 2008 Various published antibodies specific for cotton rat antigens are listed in the table. If the antibodies are cross-reactive with cotton rats, the original species and the clone name are given in brackets. mAB monoclonal antibody Table 5.2 Chemokine/cytokine detection systems in the cotton rat model Chemokine/cytokine Gene sequence Assay system Reference Interferon-alpha AF421386 ELISA, PCR Blanco et al. 2002 Interferon-gamma AF167349 ELISA, PCR Blanco et al. 2002 Tumor necrosis factor alpha AF421388 ELISA, PCR Blanco et al. 2002 Interleukin-1-alpha AF398548 ELISA Interleukin-1-beta AF421387 ELISA, PCR Blanco et al. 2002 Interleukin-2 AF398549 ELISA, PCR Boukhvalova et al. 2006 Interleukin-4 AF421390 ELISA, PCR Ottolini et al. 2005 Interleukin-5 AF148211 PCR Boukhvalova et al. 2006 Interleukin-6 AF421389 ELISA, PCR Blanco et al. 2002 Interleukin-9 AY327895 PCR Boukhvalova et al. 2006 Interleukin-10 AF398550 ELISA, PCR Blanco et al. 2002 Interleukin-12 p35 AF421396 Real-time PCR Carsillo et al. 2008 Interleukin-12/23 p40 AF421395 PCR Ottolini et al. 2005 Interleukin-13 AY327896 PCR Hassantoufighi et al. 2007 Interleukin-18 AY059406 Real-time PCR Carsillo et al. 2008 CCL2/MCP-1 AY165953 ELISA, PCR Boukhvalova et al. 2006 CCL3/MIP1-alpha AY059407 ELISA CCL4/MIP1-beta AF421392 NT, WB, PCR Blanco et al. 2002 CCL5/RANTES AF421391 ELISA, PCR Blanco et al. 2002 MIP2 AY059408 PCR CXCL10/IP-10/CRG-2 AF421394 ELISA, PCR Blanco et al. 2002 Growth-regulated protein AF421393 ELISA, PCR Blanco et al. 2002 GM-CSF AY065645 TGF beta AF480858 PCR Carsillo et al. 2008 Lymphotactin DQ136045 PCR Boukhvalova et al. 2006 Published gene sequences and detection systems are listed for cotton rat chemokines and cytokines. Antibody-based detection systems are commercially available from R&D Systems

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Viral Replication and Spread of Measles Virus in Cotton Rats Philip Wyde made the seminal observation that cotton rats can be infected with both vaccine and wild-type measles virus without adaptation (Wyde et al. 1992, 1999). In subsequent studies, it was determined that recombinant MV viruses also replicate well in cotton rat lung tissue (Tober et al. 1998; Moll et al. 2004). After infection, vaccine viruses replicate in the upper and lower respiratory tract but rarely are found in the lung-draining lymph node (Pfeuffer et al. 2003). In contrast, wild-type viruses spread to the mediastinal lymph nodes and (depending on viral strain) also to the spleen (Pfeuffer et al. 2003; Wyde et al. 1999). In tissue culture, tracheal epithelial cells can be infected, and in cotton rats MV infection was detected in lung epithelial cells (Moll et al. 2004). In addition, macrophages isolated by bronchoalveolar lavage from intranasally infected animals carried infectious virus (Wyde et al. 1999; Pfeuffer et al. 2003). After intraperitoneal inoculation of virus, infected macrophages could be obtained by lavage from the peritoneal cavity (Wyde et al. 1999). When virus was injected intramuscularly into the limb muscle of cotton rats, virus replication was detected in inguinal lymph nodes by GFP-expressing recombinant MV (Haga et al. 2008). Currently, the most likely carrier of virus seems to be the macrophage. However, dendritic cells are another potential candidate as carrier cells (de Swart et al. 2007) and need to be analyzed. An interesting finding is the fact that peripheral blood lymphocytes are positive for MV RNA by PCR, but no infectious virus could be obtained by co-cultivation (Niewiesk et al. 1997). The differences in virus spread between vaccine and wild-type strains are independent of the strain of virus used (Pfeuffer et al. 2003). This might be related to differences in receptor usage between vaccine and wild-type measles viruses (see the chapters by Y. Yanagi et al. and C. Kemper and J.P. Atkinson, This Volume). Whereas wildtype viruses use human CD150, vaccine viruses are able to use both human CD46 and CD150 as receptor molecules. CD150 is expressed on activated lymphoid cells. Since vaccine viruses were developed by passage on fibroblast cell lines (no expression of CD150 but expression of CD46), they are thought to have mutated their receptor usage in adaptation to the fibroblast as growth substrate. Interestingly, MV vaccine virus also replicates in CCRT, a cotton rat osteosarcoma cell line, whereas wild-type virus does not (S. Niewiesk, unpublished data). To investigate the relevance of receptor usage in cotton rats, recombinant viruses (on the vaccine strain Edmonston backbone) expressing the receptor-binding protein hemagglutinin (H) from wild-type virus WTF (uses human CD150) or vaccine strain Edmonston (uses both human CD150 and CD46) were generated. In cotton rats, recombinant viruses expressing the hemagglutinin (H) of wild-type strain WTF spread to mediastinal lymph nodes (MDLN) in similar fashion to wild-type strain WTF (Pfeuffer et al. 2003). In contrast, viruses expressing Edmonston-H did not spread to MDLN. These results were independent of the fusion (F) protein expressed by the respective virus. If a point mutation at amino acid 481 [asparagine (N) to tyrosine (Y)] was introduced in the H of WTF, the receptor usage switched to both human CD46 and CD150 (like vaccine virus) and virus replication was restricted to the respiratory

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tract. These data indicate that measles virus also uses two receptors in cotton rats that presumably are homologous to the human molecules.

Role of Cytoplasmic Tails of MV Glycoproteins in Cell-to-Cell Transfer In tissue culture, single tyrosine residues in the cytoplasmic tail of the measles virus glycoproteins H and F are critical for basolateral targeting of these proteins in polarized epithelial cells (Moll et al. 2001). After infection with virus mutants in which the basal lateral targeting signal of either one or both glycoproteins was destroyed due to substitutions of the tyrosine residues, H and F were expressed predominantly on the apical membrane of polarized Madin-Darby canine kidney cells. In contrast to parental measles virus, none of these virus mutants was able to spread by syncytia formation in polarized cells demonstrating that the presence of both glycoproteins at the basolateral cell surface is required for cell-to-cell fusion in vitro (Moll et al. 2004). To test the pathogenicity of these mutants in vivo, cotton rats were infected intranasally. After 4 days, virus titers in lung lavage cells and lung tissue of infected cotton rats were clearly lower than those found in animals infected with the parental virus. Thus, measles virus mutants encoding glycoproteins with altered basolateral targeting signals clearly showed reduced replication in the cotton rat respiratory tract. When animals infected with mutant virus were compared with animals infected with the parental virus by histology, in animals infected with the parental virus, small foci of infected epithelial cells were found in the nose and lung. In contrast, in animals infected with mutant virus, only individually infected epithelial cells were found in nose and lung. These data indicate that basolateral expression of MV glycoproteins is required for cell-to-cell spread in vivo.

Role of Measles Virus V Protein for Virus Replication The cotton rat model also has been used to evaluate the importance of MV V protein for replication in vivo. The V protein is translated from an edited P protein mRNA. V protein is not associated with intracellular or released viral particles. Recombinant MV strains genetically engineered to be deficient for V protein (EDV−) or to overexpress V protein (ED-V+) do not replicate as well as the parental strain (ED-tag) in tissue culture (Tober et al. 1998). In the absence of V protein, both measles virus-specific protein and RNA accumulated to levels higher than those in the parental measles virus derived from the molecular clone. Furthermore, infection resulted in similar viral titers 24 h after tissue culture infection and slightly reduced titers (0.5 log10) after 48 h. After overexpression of the V protein, measles virus gene expression, as well as viral titers, were strongly reduced

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in tissue culture (reduction of 1–1.5 log10). In cotton rats, ED-V - replication was reduced by 1 log10 TCID50 in lung tissue days 4 and 5 after infection (Tober et al. 1998). By comparison, ED-V+ replication was reduced by 2.5 log10 TCID50 in lung tissue on day 4 and no virus was found on day 5. This study indicated that replication characteristics of measles virus in tissue culture are recapitulated (and even more pronounced) in cotton rats thus making it an excellent model to study questions of how viral replication is controlled in vivo.

Interaction of Heat Shock Protein 72 with Measles Virus Nucleocapsid Heat shock protein 72 is a cellular factor that increases transcription of measles vaccine virus. This is achieved by complex formation with the nucleocapsid (N) of MV vaccine virus. The carboxy-terminus of N (N-tail) has two contact sites for HSP 72, the low-affinity binding site Box 2 and the high-affinity binding site Box 3. In contrast, wild-type viruses in their overwhelming majority interact with HSP72 through Box 2 only and do not change replication characteristics in response to heat shock. To test the relative merit of one versus two contact sites, the molecular MV Edmonston clone (which has two contact sites) was mutated at amino acid position 522 from asparagine (N) to aspartic acid (D). This change abolishes the HSP72 interaction with Box 3. When both viruses were compared in vitro they replicated to comparable titers. After infection of cotton rats, no difference in viral titers was detected after 5 days. Since the N522D mutation occurs in nearly all wild-type viruses, it was hypothesized that this mutant should be able to outcompete the virus derived from the vaccine-based parental clone. However, after co-infection of cotton rats with both viruses, the parental clone Edmonston clearly had a growth advantage over N522D. This in vivo experiment indicated that the difference in interaction with HSP72 between wild-type and vaccine viruses is not simply dependent on one amino acid change but might depend on the overall protein context.

Vector Development For the studies discussed so far, the molecular clone of the Edmonston B strain (ED-tag) (and derivatives thereof) that was produced by Martin Billeter’s group was used (Radecke et al. 1995). Based on the availability of molecularly cloned measles virus, it has been proposed to use recombinant measles viruses expressing additional genes either as vaccines (see the chapter by M.A. Billeter et al., This volume) and tools for oncolytic cancer therapy (see the chapter by S.J. Russell and K.W. Peng, this volume). For this purpose, a molecular clone of the temperature sensitive AIK-C virus, a vaccine derivative from Edmonston strain, was produced. When

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cotton rats were infected with this virus, it did not replicate in lung tissue but induced an immune response after intramuscular inoculation (Haga et al. 2008). This would indicate that AIK-C could be used as a very safe and immunogenic vector system. Other established molecular clones of measles virus include one of the Schwarz vaccine strains (Combredet et al. 2003) and the IBC wild-type strain (Takeda et al. 2000). Both molecularly cloned viruses have not been tested in cotton rats but the parental viruses replicated well in cotton rats (Pfeuffer et al. 2003; S. Niewiesk, unpublished data).

Measles Virus Induced Immune Suppression Children with acute measles often contract secondary infections that can be lethal. The increased susceptibility to opportunistic infections has been thought to be due to severe suppression of the immune system by measles virus infection itself (see the chapter by S. Schneider-Schaulies and J. Schneider-Schaulies, this volume). In contrast to infection with the wild-type virus, vaccination with the live-attenuated vaccine at the current dosage does not lead to clinical symptoms of immune suppression. However, the use of high titer vaccines (105 pfu) has been correlated with increased mortality, presumably related to immune suppression, which led to discontinuation of this immunization regimen (Aaby et al. 1988; Halsey 1993). Laboratory-based methods to determine immune suppression have included the measurement of the suppression of antigen-specific T cell proliferation (which measures CD4 T cells) and of mitogen-stimulated B cell and T cell responses. In tissue culture, the suppressive effect of measles virus on proliferation of peripheral blood lymphocytes could be demonstrated by infection with live virus or incubation with UV-inactivated virus. In the cotton rat model, it also was found that measles virus induces immune suppression. After infection, suppression of proliferation of keyhole limpet hemocyanin (KLH)-specific T cells and mitogenstimulated (Concanavalin A; ConA) proliferation of spleen cells was detected ex vivo (Niewiesk et al. 1997, 2000). Vaccine and wild-type viruses differ in their suppressive effect. Whereas wild-type virus suppresses immune responses at all titers tested (Pfeuffer et al. 2003), only high titers of vaccine virus (≥106 pfu) are suppressive (Niewiesk et al. 1997). After inoculation with 105 TCID50 wild-type virus (strain WTF), the ConA-stimulated proliferation of cotton rat spleen cells was suppressed for up to 20 days, whereas inoculation with 106 pfu vaccine strain (strain Edmonston B) suppressed proliferation for up to 10 days.

MV Inhibits T Cell Rather than B Cell Responses In order to investigate whether B cells or T cells were inhibited by MV infection, spleen cells from infected animals were stimulated with B cell and/or T cell mitogens. Ex vivo, both B cell and T cell proliferation were inhibited by MV

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(Niewiesk et al. 2000). This is consistent with findings in patients where infection with wild-type measles virus leads to reduction of B cell and T cell stimulation by mitogens ex vivo. To further investigate how the antigen-specific B cell and T cell response are affected by MV infection, cotton rats were injected with various antigens and immune responses measured ex vivo. After application of keyhole limpet hemocyanin coupled to dinitrophenyl groups (KLH-DNP), the antibody response measured from serum was impaired in infected animals after 2 weeks but was back to normal 1 week later (Niewiesk et al. 2000). During this time, there was no significant difference in the number of antigen-specific B cells (measured by Elispot) secreting either IgM or IgG specific for KLH-DNP so that the overall effect of MV infection on the B cell response was small. In contrast, T cell responses were severely affected. Antigen-specific T cell proliferation was reduced by MV infection in a mixed lymphocyte reaction after primary and secondary stimulation and in a KLH-specific proliferation assay. Similar to proliferation of T helper cells, the number of cytotoxic vaccinia virus-specific T cells generated ex vivo was markedly reduced in spleen cells from MV-infected animals. However, on a per cell basis, cytotoxicity was not reduced. Overall, the proliferation of T cells was severely inhibited, whereas B cell help and cytotoxicity did not seem to be affected. With only semiquantitative bioassays available at the time, the reduced secretion of IL2 and interferon gamma by antigen-specific T cells was not statistically significant. The difference in suppression of the B cell and T cell response measured in vivo and ex vivo might be due to differences in activation of these cells. Ex vivo, both cell populations were driven to proliferate for 3 days by mitogen. In vivo, the generation of antigen-specific T cell responses also occurred within a few days in a socalled clonal burst (Hou et al. 1996); immune suppression resulted in a reduced expansion and subsequently lower numbers of memory T cells. In contrast, B cells proliferated in vivo more slowly over weeks and therefore might have been less susceptible. In contrast, T cells might not only be affected by the above-mentioned cell cycle arrest, but also by a defect in antigen presentation.

MV Suppresses the CD8 T Cell Response In Vivo Using the DTH Model of Epicutaneous DNFB Application In order to test the suppression of both CD4 and CD8 T cells directly in vivo by MV, a delayed-type hypersensitivity model of epicutaneous sensitization (ECS) by 2,4 dinitrofluorobenzene (DNFB) to the ear was used (Niewiesk et al. 2000). After application of DNFB, dendritic cells (DCs) in the skin take up the antigen and transport it to draining mandibular lymph nodes to stimulate T cells (Bour et al. 1995). These lymph node cells can be analyzed by flow cytometry and will incorporate 3H-thymidine after overnight culture, thus giving a measure of induction and proliferation of T cells. After DNFB application to the ear of cotton rats, the number of lymphocytes was markedly increased (fivefold). The ratio of CD4 T cells to CD8 T cells changed from 1.5:1 in nonactivated lymph nodes to 1:1.7 in activated lymph nodes, indicating a strong stimulation of CD8 T cells (similar to

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the mouse model; Bour et al. 1995). T cell proliferation of lymph node cells from MV-infected animals was strongly reduced in comparison to control animals (Streif et al. 2004), whereas the CD4/CD8 ratio remained unchanged (as seen in humans). If DNFB is applied to the flank of mice, ear painting 10 days later will result in ear swelling due to the recall CD8 T cell response. In mice, CD8 cytotoxic T cells are the effector cells in ear swelling (Bour et al. 1995), whereas CD4 T cells can (down)regulate the activity of CD8 T cells (Dubois et al. 2003). To investigate which T cell subset is the effector subset in cotton rats, monoclonal antibodies specific for cotton rat CD4 and CD8 were used. When CD8 T cells were depleted, a marked reduction in ear swelling was observed, indicating that CD8 T cells are the main effector T cells in this model (Streif et al. 2004). In contrast, the depletion of CD4 T cells did not significantly influence ear swelling. These data suggest that in cotton rats CD4 T cells do not regulate the DNFB response and that the reduction in proliferation of DNFB-specific CD8 T cells is not due to regulatory CD4 T cells.

MV Glycoproteins are Sufficient and Necessary to Suppress Proliferation In vitro, a cotton rat T cell line infected with either vaccine or wild-type virus was UV-inactivated and tested for expression of equal amounts of MV glycoproteins. After contact with these cells, mitogen-dependent proliferation of spleen cells from naive animals was reduced (Pfeuffer et al. 2003). However, in contrast to in vivo findings and similar to in vitro data with human cells, no difference was seen between vaccine and wild-type virus. The only viral proteins to contact spleen cells in this assay are the MV glycoproteins fusion protein (F) and hemagglutinin (H). Therefore, it was investigated whether this contact alone is sufficient to suppress proliferation. 293T fibroblast cells transfected with both H and F suppressed proliferation of lymphocytes in vitro (Niewiesk et al. 1997). In addition, 293T cells transfected with both H and F suppressed proliferation of spleen cells 4 days after intraperitoneal injection. In contrast, a recombinant MV, where H and F were replaced by the G protein of vesicular stomatitis virus (MGV), did not suppress proliferation in cotton rats. In order to investigate the effect of the different glycoproteins in the context of measles virus in vivo, recombinant viruses (based on the molecular Edmonston clone) expressing either alone or in combination the H and the F of wild-type virus WTF and/or vaccine strain Edmonston were used. In cotton rats, recombinant viruses expressing the WTF-H spread to mediastinal lymph nodes (MDLN) and induced proliferation inhibition like wild-type WTF (Pfeuffer et al. 2003). In contrast, viruses expressing Edmonston-H did not spread to MDLN and did not suppress immunity at titers of up to 105 pfu. These results were independent of the F protein expressed by the respective viruses. In summary, these data show that both H and F proteins are necessary and sufficient to suppress proliferation. In the context

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of the whole virus and in vivo, however, H seems to be more important in explaining differences seen between vaccine and wild-type virus. In aggregate with the results that implicate that the dual receptor usage by MV in human cells is reflected in cotton rats, these data suggest that receptor usage is important for viral spread and immune suppression.

MV Induces Cell Cycle Retardation In vitro, suppression of lymphocyte proliferation has been investigated using live virus or inactivated virus. It was found that mechanisms such as apoptosis, fusion of cells, and unresponsiveness to IL-2 do not contribute to suppress proliferation (McChesney et al. 1987, 1988; Naniche et al. 1999; Schnorr et al. 1997) but that contact with MV is able to downregulate AKT, which results in cell cycle arrest (see the chapter by S. Schneider-Schaulies and J. Schneider-Schaulies, this volume). These results were confirmed in cotton rats in that only the retardation of cell cycle progression was found to be responsible for suppression (Niewiesk et al. 1999). Using fluorescent dyes (CFSE), it was found that all lymphocytes progress through the cell cycle, but preferentially gather in the G1/S phase. In agreement, the cell cycle-dependent kinases (CDK) 2 and 4, which propel progression through the cell cycle, were reduced in MV-infected animals (S. Niewiesk, unpublished data). Furthermore, the activity of AKT was severely reduced (Avota et al. 2001). When the kinetics of AKT regulation and T cell proliferation were investigated in the DNFB model, it was found that the reduction of AKT activity preceded a reduction of DNFB-specific T cell proliferation by 4 days, which is consistent with the notion that a change in signal transduction is responsible for reduced proliferation. These data indicate that the suppression of T cell proliferation is a major part of the immune suppression found after measles virus infection and that B cells are not affected. However, B cells and T cells are not the only effector cells of the immune system. Additional studies will be needed to investigate the effect on innate immune responses (see the chapter by B. Hahm, this volume) and the effect of infection on antigen-presenting cells like macrophages and dendritic cells and their role in T cell suppression.

Testing of Antiviral Substances and Vaccines The safety and efficacy of antivirals and vaccines against measles virus can be tested in Rhesus macaques, which develop a disease very similar to acute measles in children (see the chapter by R.L. de Swart, this volume). However, it is difficult and costly to use these animals to determine a number of basic questions such as evaluation of routes of administration, drug and vaccine doses, schedules, and regimens. For this reason, the use of cotton rats as a screen can be useful for primary evaluation of vaccines and drugs.

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Evaluating Antivirals in Cotton Rats Early during the drug screening process, antiviral substances found in tissue culture must be tested in a small animal model. In an example of how cotton rats can be utilized for drug testing of anti-measles virus substances, three compounds shown to be active against measles virus were tested in vivo (Wyde et al. 2000a). These compounds were ribavirin, 5-ethyl-1-β-D-ribo-furanosylimidazole-4-carboamide (EICAR) and poly(acryl-amidomethyl propanesulfonate) (PAMPS). When the drugs were administered twice daily i.p. and virus titers analyzed 4 days after infection, ribavarin (360 mg/kg/day) and EICAR (120 mg/kg/day) were shown to reduce virus titers by 1 log10 TCID50/g lung tissue. In contrast, PAMPS inhibited virus infection only when directly applied intranasally after virus infection. Later application of PAMPS was not effective, indicating that PAMPS acts extracellularly during viral binding and uptake. These data indicate that cotton rats may be a useful rodent model for testing antiviral substances against measles.

Immune Response Against Measles Virus In cotton rats, measles virus infection is overcome within 10 days. The decline in virus titers correlates with the development of measles virus-specific CD4 T cells. The T cell response is first detectable on day 5, reaches its peak on days 7–8 and remains detectable for at least 3 months (Pueschel et al. 2007). CD4 T cells recognize two epitopes (amino acid 261–285 and 348–372) on the nucleocapsid protein, one epitope on the hemagglutinin (amino acid 553–567) and one on the fusion protein (amino acid 347–372) (Pueschel et al. 2007). However, these CD4 T cells do not control viral replication since neither immunization with the hemagglutinin epitope nor depletion of CD4 T cells influences viral titers. In contrast to T cells, antibodies specific for measles develop later during infection. By ELISA, antibodies can be detected 6 days after infection and by neutralization assay antibodies can be found 12 days after infection (Niewiesk and Germann 2000). Although neutralizing antibodies develop too late during primary infection to protect against challenge, they protect against secondary infection. As in humans, passive transfer of MV neutralizing antibodies protects against infection (Schlereth et al. 2000b).

Testing of Measles Vaccines Acute measles can be prevented by immunization of seronegative children with the live attenuated measles virus vaccine (see the chapter by D.E. Griffin and C.-H. Pan, this volume). Protection against the acute disease is correlated with the titer of neutralizing antibodies (Chen et al. 1990). Although CD8 and CD4 T cells are activated during the acute disease in humans (van Binnendijk et al. 1990)

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and CD8 T cells seem to play a role in clearance in the late phase of infection in monkeys (Permar et al. 2003, 2004), stimulation of the T cell response by experimental vaccine vectors only confers limited protection (Fennelly et al. 1995; Schlereth et al. 2000a). Furthermore, a case report describing persistent MV infection in T cell-deficient patients supports the notion that T cells are required to clear infection but do not provide protection (Nahmias et al. 1967). This is in agreement with data from animal models (cotton rat, rhesus macaque) (see Schlereth et al. 2003 and references therein), where the only immune correlate consistently linked to protection against measles virus infection in animal models is the titer of neutralizing antibodies. To compare the efficacy of different routes of immunization with MV vaccine virus (Edmonston strain), cotton rats were immunized subcutaneously, intranasally, or intraperitoneally with different doses of virus. Similar to humans (Wong-Chew et al. 2004), immunization efficiency was twice as high after s.q. immunization than after i.n. immunization (studies in humans with aerosols show superior results to intranasal droplet immunization but have not been repeated in cotton rats). Immunization efficiency after i.p. immunization, which is not used in humans, was fourfold higher than after s.q. immunization (see Table 5.3). As in humans, cotton rats were effectively protected against challenge after vaccination. The only difference from humans is that for successful immunization of cotton rats, roughly tenfold more virus is necessary (105 pfu instead of 104 pfu s.q.). Based on the principle that the induction of neutralizing antibodies is protective, a number of alternate vaccine vectors have been produced that express the hemagglutinin and/or the fusion protein of measles virus, since these proteins are Table 5.3 Measles virus immunization in the presence and absence of passively transferred (maternal) antibodies Route of Dose of Amount of passively NT titer (±SD) immunization immunization (pfu) transferred antibodies (NT titer) i.n.

104 5 × 105

0 <10 0 30 + 20 320 <10 320 <10 2 × 107 0 20 + 10 s.q. 104 320 <10 0 60 + 20 5 × 105 320 <10 0 85 ± 60 i.p. 104 320 <10 0 185 + 60 105 320 0 Cotton rats were immunized either intranasally (i.n.), subcutaneously (s.q.), or intraperitoneally (i.p.) with different doses of Edmonston vaccine virus in the absence or presence (1 mL antiserum of NT titer 320) of maternal antibodies. Neutralizing antibodies were determined 6 weeks after immunization.

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the target of neutralizing antibodies. The advantage of the cotton rat model is that both markers of successful immunization (level of neutralizing antibodies as well as CD4 T cell responses) and markers of protection (level of neutralizing antibodies, reduction of viral titers after challenge infection and histological changes in lung tissue) can be measured and experiments can be conducted in group sizes large enough for statistical analysis in a relatively cost-efficient manner. For the measurement of neutralizing antibodies, the plaque reduction neutralization assay (PRNT) and the neutralization assay (NT) are utilized, which differ tenfold in titer (PRNT titer of 100 correlates with NT titer of 10). It is also easy to compare neutralizing antibody titers from animal experiments with studies in humans. In cotton rats, the titer of the virus inoculum determines whether it is possible to obtain sterile immunity (complete virus clearance) after immunization. If a low titer inoculum (≤104 pfu or TCID50) is used, virus usually is cleared completely. If higher inocula are used, it is not possible to achieve sterile immunity by day 5. A number of vector systems that have been shown to induce good immune responses in other laboratory animals have been used in cotton rats expressing the H, F, and/or N protein. Basically, all vectors were successful in inducing protective immunity, with the exception of plasmids expressing the N protein. This particular immunization induced good T cell and non-neutralizing antibody responses but no protective neutralizing antibodies (Schlereth et al. 2000a). After repeated immunization, all vector systems induced protective levels of neutralizing antibodies: plasmid immunization (Schlereth et al. 2000a), Salmonella and Shigella-mediated plasmid transfer (Pasetti et al. 2003), modified virus Ankara (MVA) (Weidinger et al. 2001) and canarypox virus vectors (ALVAC) (Wyde et al. 2000b), adenovirus (Fooks et al. 1998), immune stimulating complexes (ISCOM) (Wyde et al. 2000b) and TLR-2 specific adjuvants with measles vaccine virus (Luhrman et al. 2005). Probably the most remarkable results were obtained with recombinant vesicular stomatitis virus, which induced high titers of neutralizing antibodies after a single intranasal immunization. The reasons for this efficient induction are not understood fully but it might be due to increased immunogenicity of the rigid highly repetitive envelope of VSV-H (see Schlereth et al. 2003 and references therein) or to the interaction and subsequent stimulation of immune responses by VSV with TLR-7 (Lund et al. 2004).

Inhibition of Seroconversion in the Presence of Maternal Antibodies Although vaccination of seronegative children is hugely successful with the liveattenuated measles virus, vaccination of children with maternal antibodies is notoriously difficult. Maternal antibodies protect children after birth but are metabolized over time. Eventually, low antibody titers no longer protect but still are able to interfere with immunization, which leads to the phenomenon that vaccination in the presence of maternal antibodies does not induce seroconversion. To test the

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inhibitory activity of maternal antibodies in young cotton rats, dams were immunized with measles virus before they gave birth and the level of MV-specific antibodies transmitted was measured in pups after birth (Schlereth et al. 2000b). Neutralizing antibodies declined with an approximate half-life of 7 days. After i.p. immunization with the MV vaccine strain, maternal antibodies inhibited vaccination in cotton rats in the same way as in humans. However, the amount of maternal antibody transferred to individual pups varied (probably according to their pecking order). To better standardize the experimental system, human MV-specific antibodies were transferred into cotton rats (Schlereth et al. 2000b). By injection of various amounts of human MV-specific antibodies, it is possible to quantify the level of passively transferred MV-specific antibodies. After vaccination, passively transferred human MV-specific antibodies can be distinguished from actively induced cotton rat antibodies using ELISA. In cotton rats, the half-life of human MV-specific antibodies is reduced twofold in comparison to cotton rat antibodies, thus reducing the time required per experiment. Immunization 1 day after serum transfer with 105 pfu vaccine virus i.p. failed to induce neutralizing antibody responses or protection against intranasal challenge with MV. It also has been tested whether different immunization routes, doses, or repeated administration could induce neutralizing antibodies in the presence of MV-specific antibodies. The results from the various immunization regimens are summarized in Table 5.3. Immunization with MV in the presence of MV-specific antibodies was never successful. In these studies, a human serum sample was used as a source of human MVspecific antibodies that contained 16 IU/ml as measured by ELISA against a reference serum and had a neutralization titer (NT) of 320. After injection of 1 ml of human serum (NT 320), antibodies declined with a half-life of roughly 3.5 days and neutralizing antibodies were detectable for 3 weeks (Schlereth et al. 2000b). Similar to humans, the presence of neutralizing antibodies was predictive of protection against infection. In children, neutralization titers of 120 or more measured in a plaque reduction neutralization (PRNT) assay were shown to protect against reinfection (Chen et al. 1990). In cotton rats, the same titers were protective [if tenfold less sensitive neutralization (NT) assay was used, this correlates with titers ≥12]). In the presence of neutralizing antibodies after serum transfer, virus titers in lung tissue 5 days after challenge were reduced by 1.5–2 log10 (Schlereth et al. 2000b). This level of reduction is comparable to that seen after s.q. immunization of cotton rats. To evaluate how strongly B cell and T cell responses were affected and how much MV-specific (maternal) antibody was needed to suppress immune responses, cotton rats were inoculated with the equivalent of 1 ml of human MV-specific antiserum with neutralization titers of 320, 160, and 80, respectively (Pueschel et al. 2007). One day later, this transfer resulted in neutralization titers in serum of 60, 30, and 15/ml blood, respectively. At this point, cotton rats were immunized i.p. with 105 pfu MV vaccine virus. After 9 weeks, animals were challenged with MV intranasally and T cell proliferation, neutralizing antibodies and virus titers in lung tissue were measured. Immunization in the presence of high levels of maternal antibodies (NT 320 and NT 160) reduced (although did not abolish) the T cell

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response, whereas low levels had no effect. In contrast, the neutralizing antibody response was blocked completely after immunization in the presence of NT 320 and NT 160, and only a low level NT titer of 12.5 was generated after immunization in the presence of NT 80. The lack of NT antibody generation correlated with a lack of protection after viral challenge (after immunization in the presence of NT 320 and NT 160). Generation of a low NT response resulted in partial protection (after immunization in the presence of NT 80) (Pueschel et al. 2007). These data demonstrated that the generation of neutralizing antibodies is much more sensitive than T cell responses to the presence of passively transferred antibody at the time of immunization. They also showed that no protection was achieved in the absence of neutralizing antibodies, although T cell responses were detectable.

Experimental Vector Systems Do Not Induce Protection in the Presence of MV-Specific (Maternal) Antibodies The lack of protection by MV-specific T cells also was observed in our vaccination studies using plasmids (Schlereth et al. 2000a). To find potential alternative vaccine vector systems that might induce protection in the presence of maternal antibodies, plasmids expressing the MV nucleocapsid, the fusion protein and hemagglutinin were tested in seronegative cotton rats. Immunization with the nucleocapsid induced high antibody titers (which did not neutralize!) and T cell responses. However, no protection was conferred by T cells in the absence of neutralizing antibodies. In contrast, plasmids expressing the fusion protein and hemagglutinin induced both neutralizing antibodies and effector T cells and successfully protected against infection (Schlereth et al. 2000a). However, in the presence of passively transferred antibodies, immunization with both plasmids failed to induce neutralizing antibodies and did not protect against infection. The same was true for a modified vaccinia virus Ankara (MVA-H) expressing MV hemagglutinin (Weidinger et al. 2001). In contrast, a vesicular stomatitis virus vector expressing MV-H (VSV-H) was partially effective, where intranasal immunization with a single dose of VSV-H led to the generation of neutralizing antibodies and protection against viral challenge (Schlereth et al. 2000b, 2003). After immunization with VSV-H in the presence of passively transferred antibodies, MV neutralizing antibody titers were reduced tenfold in comparison to immunization in the absence of passively transferred antibodies (NT titers 60 instead of 600). However, neutralizing antibody titers against VSV remained the same, indicating a specific suppression of MV-H-specific B cells. Comparison of all three vector systems indicated that the route of immunization does not seem to be decisive because in contrast to VSV-H, both plasmid and MVA-H (and measles vaccine virus) did not induce protection after intranasal administration. Even though the hemagglutinin was a passenger protein not required for vector replication in all three systems, B cell responses were inhibited, indicating that neutralization by passively transferred antibodies is not important for inhibition.

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Failure of Experimental Vaccines to Immunize in the Presence of Maternal Antibodies In the cotton rat and the rhesus/cynomolgous macaque models of measles virus infection, two vaccine candidates (plasmid immunization and modified vaccinia virus Ankara (MVA), which are used in clinical trials for HIV) have been tested in the presence of maternal antibodies (Schlereth et al. 2000a; Weidinger et al. 2001; van Binnendijk et al. 1997; Stittelaar et al. 2000; Premenko-Lanier et al. 2003; Zhu et al. 2000). Although the results, at first glance, seem to be contradictory in nature, a common theme emerges when taking into account the level of maternal antibodies present at the time of immunization, the number of immunizations, induction of immune responses (T cell or B cell response), and level of protection (reduced viral titers or reduced histologically detectable tissue damage). High antibody titers inhibit vaccination more strongly than low titers and repeated immunizations improve vaccination success in the face of waning antibody titers. It is easier to detect T cell proliferation than neutralizing antibodies and to find reduced tissue damage by histology than reduction of virus titers. Given these caveats, neither plasmid nor MVA immunization have proven to be effective by strict standards (i.e., single immunization in the presence of high titers of maternal antibody, induction of neutralizing antibodies, and reduction of viral titers as measure of protection).

Summary and Outlook The cotton rat has proven that it can fill the void left by the lack of a mouse model for measles virus pathogenesis research as a small rodent model. By now, this animal model is well defined because of the commercial availability of microbiologically defined inbred cotton rats. The on-going development of genetic sequences, reagents, and methods increasingly allows investigators to address mechanistic questions in this animal model. An added advantage is the susceptibility of cotton rats to other human pathogens, potentially making it a good animal model to study polymicrobial disease (e.g., tuberculosis and measles virus infection).

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Azazy AA, Devaney E, Chance ML (1994) A PEG-ELISA for the detection of Leishmania donovani antigen in circulating immune complexes. Trans Royal Soc Trop Med Hyg 88:62–66 Bayer M, Wenk P (1988) Homologous and crossreacting immune response of the jird and cotton rat against microfilariae of Dipetalomena vitae and Litosomoides carinii (Nematoda Filaroidea). Trop Med Parasit 39:304–308 Blanco JC, Richardson JY, Darnell ME, Rowzee A, Pletneva L, Porter DD, Prince GA (2002) Cytokine and chemokine gene expression after primary and secondary respiratory syncytial virus infection in cotton rats. J Infect Dis 185:1780–1785 Blanco JC, Pletneva L, Boukhvalova M, Richardson JY, Harris KA, Prince G (2004) The cotton rat: an underutilized animal model for human infectious diseases can now be exploited using specific reagents to cytokines, chemokines, and interferons. J Interferon Cytokine Res 24:21–28 Boukhvalova MS, Prince GA, Soroush L, Harrigan DC, Vogel SN, Blanco JC (2006) The TLR4 agonist, monophosphoryl lipid A, attenuates the cytokine storm associated with respiratory syncytial virus vaccine-enhanced disease. Vaccine 24:5027–5035 Bour H, Peyron E, Gaucherand M, Garrigue JL, Desvignes C, Kaiserlian D, Revillard JP, Nicolas JF (1995) Major histocompatibility complex class I-restricted CD8+ T cells and class IIrestricted CD4+ T cells, respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene. Eur J Immunol 25:3006–3010 Brydak L (1990) Studies on adaptation of influenza virus replicated at low temperature. IV. Sensitivity of neuraminidase and hemagglutinin to some proteolytic enzymes, detergents and chemical agents. Acta Microbiol Pol 39:137–147 Burgdorfer W, Gage KL (1987) Susceptibility of the hispid cotton rat (Sigmodon hispidus) to the Lyme disease spirochete (Borrelia burgdorferi). Am J Trop Med Hyg 37:624–628 Chen RT, Markowitz LE, Albrecht P, Stewart JA, Mofenson LM, Preblud SR, Orenstein WA (1990) Measles antibody: reevalution of protective titers. J Inf Dis 162:1036–1042 Clark JD (1984) Biology and diseases of other rodents. In: Fox JG, Cohen BJ, Loew FM (eds) Laboratory animal medicine. Academic, Orlando, pp 183–206 Combredet C, Labrousse V, Mollet L, Lorin C, Delebecque F, Hurtrel B, McClure H, Feinberg MB, Brahic M, Tangy F (2003) A molecularly cloned Schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice. J Virol 77:11546–11554 de Swart RL, Ludlow M, de Witte L, Yanagi Y, van Amerongen G, McQuaid S, Yuksel S, Geijtenbeek TB, Duprex WP, Osterhaus AD (2007) Predominant infection of CD150+ lymphocytes and dendritic cells during measles virus infection of macaques. PLoS Pathog 3:e178 Dreizin RS, Vyshnevetskaia LO, Bagdamian EE, Iankevich OD, Tarasova LB (1971) Experimental RS virus infection of cotton rats. A viral and immunofluorescent study(in Russian). Vopr Virusol 16:670–676 Dubois B, Chapat L, Goubier A, Papiernik M, Nicolas JF, Kaiserlian D (2003) Innate CD4+CD25+ regulatory T cells are required for oral tolerance and inhibition of CD8+ T cells mediating skin inflammation. Blood 102:3295–3301 Eichelberger MC, Bauchiero S, Point D, Richter BW, Prince GA, Schuman R (2006) Distinct cellular immune responses following primary and secondary influenza virus challenge in cotton rats. Cell Immunol 243:67–74 Elwood RL, Wilson S, Blanco JC, Yim K, Pletneva L, Nikonenko B, Samala R, Joshi S, Hemming VG, Trucksis M (2007) The American cotton rat: a novel model for pulmonary tuberculosis. Tuberculosis 87:145–154 Fennelly GJ, Flynn JAL, ter Meulen V, Liebert UG, Bloom BR (1995) Recombinant Bacille Calmette Guérin priming against measles. J Infect Dis 172:698–705 Fooks AR, Jeevarajah D, Lee J, Warnes A, Niewiesk S, ter Meulen V, Stephensom JR, Clegg JCS (1998) Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus proteins: protective immune responses in rodents. J Gen Virol 79:1027–1031 Haga T, Murayama N, Shimizu Y, Saito A, Sakamoto T, Morita T, Komase K, Nakayama T, Uchida K, Katayama T, Shinohara A, Koshimoto C, Sato H, Miyata H, Katahira K, Goto Y

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(2008) Analysis of antibody response by temperature-sensitive measles vaccine strain in the cotton rat model. Comp Immunol Microbiol Infect Dis (in press) Halsey N (1993) Increased mortality following high titer measles vaccines: too much good thing. J Pediatr Infect Dis 12:462–465 Hamelin ME, Yim K, Kuhn KH, Cragin RP, Boukhvalova M, Blanco JC, Prince GA, Boivin G (2005) Pathogenesis of human metapneumovirus lung infection in BALB/c mice and cotton rats. J Virol 79:8894–8903 Hassantoufighi A, Oglesbee M, Richter BW, Prince GA, Hemming V, Niewiesk S, Eichelberger MC (2007) Respiratory syncytial virus replication is prolonged by a concomitant allergic response. Clin Exp Immunol 148:218–229 Hou S, Hyland L, Ryan KW, Portner A, Doherty PC (1996) Virus-specific CD8+ T-cell memory determined by clonal burst size. Nature 369:652–654 Kroeze WK, Tanner CE (1985) Echinococcus multilocularis: responses to infection in cotton rats (Sigmodon hispidus). Int. J Parasitol 15:233–238 Langley RJ, Prince GA, Ginsberg HS (1998) HIV type-1 infection of the cotton rat (Sigmodon fulviventer and S. hispidus). Proc Natl Acad Sci U S A 95:14355–14360 Lewandowski G, Zimmerman MN, Denk LL, Porter DD, Prince GA (2002) Herpes simplex type 1 infects and establishes latency in the brain and trigeminal ganglia during primary infection of the lip in cotton rats and mice. Arch Virol 147:167–179 Lowery GH (1981) The mammals of Louisiana and its adjacent waters. Louisiana State University Press, Baton Rouge Luhrman A, Tschernig T, Pabst R, Niewiesk S (2005) Improved intranasal immunization with live-attenuated measles virus after co-inoculation of the lipopeptide MALP-2. Vaccine 23:4721–4726 Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, Iwasaki A, Flavell RA (2004) Recognition of single-stranded RNA viruses by Toll-like receptor 7. PNAS 101:5598–5603 Mähler M, Heidtmann W, Hedrich HJ, Beil W, Gruber A, Niewiesk S, Wagner S (2002) Helicobacter pylori infection induces chronic active gastritis in cotton rats. Gastroenterology 122:A532–A533 McChesney MB, Kehrl JH, Valsamakis A, Fauci AS, Oldstone MBA (1987) Measles virus infection of B lymphocytes permits cellular activation but blocks progression through the cell cycle. J Virol 61:3441–3447 McChesney MB, Altmann A, Oldstone MBA (1988) Suppression of T lymphocyte function by measles virus is due to cell cycle arrest in G1. J Virol 140:1269–1273 Mestas J, Hughes CC (2004) Of mice and not men: differences between mouse and human immunology. J Immunol 172:2731–2738 Moll M, Klenk HD, Herrler G, Maisner A (2001) A single amino acid change in the cytoplasmic domains of measles virus glycoproteins H and F alters targeting, endocytosis, and cell fusion in polarized Madin-Darby canine kidney cells. J Biol Chem 276:17887–17894 Moll M, Pfeuffer J, Klenk H-D, Niewiesk S, Maisner A (2004) Polarized glycoprotein targeting affects spread of measles virus in vitro and in vivo. J Gen Virol 85:1019–1027 Murphy BR, Sotnikov AV, Lawrence LA, Banks SM, Prince GA (1990) Enhanced pulmonary histopathology is observed in cotton rats immunized with formalin-inactivated respiratory syncytial virus (RSV) or purified F glycoprotein and challenged with RSV 3–6 months after immunization. Vaccine 8:497–502 Murphy TF, Dubovi EJ, Clyde WA (1981) The common cotton rat as an experimental model of human parainfluenza virus type 3 disease. Exp Lung Res 2:97–109 Nahmias AJ, Griffith D, Salsbury C, Yoshida K (1967) Thymic aplasia with lymphopenia, plasma cells, and normal immunoglobulins. JAMA 201:729–734 Naniche D, Reed SI, Oldstone MBA (1999) Cell cycle arrest during measles virus infection: a G0like block leads to suppression of retinoblastoma protein expression. J Virol 73:1894–1901 Niewiesk S, Germann P-G (2000) Development of neutralizing antibodies correlates with resolution of interstitial pneumonia after measles virus infection in cotton rats. J Exp Anim Sci 40:201–210

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Niewiesk S, Prince GA (2002) Diversifying animal models: the use of hispid cotton rats (Sigmodon hispidus) in infectious diseases. Lab Anim 36:357–372 Niewiesk S, Eisenhuth I, Fooks A, Clegg JCS, Schnorr J-J, Schneider-Schaulies S, ter Meulen V (1997) Measles virus-induced immune suppression in the cotton rat (Sigmodon hispidus) model depends on viral glycoproteins. J Virol 71:7214–7219 Niewiesk S, Ohnimus H, Schnorr J-J, Götzelmann M, Schneider-Schaulies S, Jassoy C, ter Meulen V (1999) Measles virus-induced immunosuppression in cotton rats is associated with cell cycle retardation in uninfected lymphocytes. J Gen Virol 80:2023–2029 Niewiesk S, Götzelmann M, ter Meulen V (2000) Selective in vivo suppression of T lymphocyte responses in experimental measles virus infection. Proc Natl Acad Sci U S A 74:4652–4657 Oliver JH, Chandler FW, James AM, Sanders FH, Hutcheson HJ, Huey LO, McGuire BS, Lane RS (1995) Natural occurrence and characterization of the Lyme spirochete, Borrelia burgdorferi, in cotton rats (Sigmodon hispidus) from Georgia and Florida. J Parasitol 81:30–36 Ottolini M, Blanco J, Porter D, Peterson L, Curtis S, Prince G (2003) Combination anti-inflammatory and antiviral therapy of influenza in a cotton rat model. Pediatr Pulmonol 36:290–294 Ottolini MG, Blanco JC, Eichelberger MC, Porter DD, Pletneva L, Richardson JY, Prince GA (2005) The cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis. J Gen Virol 86:2823–2830 Pacini DL, Dubovi EJ, Clyde WA (1984) A new animal model for human respiratory tract disease due to adenovirus. J Infect Dis 150:92–97 Pasetti MF, Barry EM, Losonsky G, Singh M, Medina-Moreno SM, Polo JM, Ulmer J, Robinson H, Sztein MB, Levine MM (2003) Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin, inducing specific immune responses and protection in cotton rats. J Virol 77:5209–5217 Permar SR, Klumpp SA, Mansfield KG, Kim WK, Gorgone DA, Lifton MA, Williams KC, Schmitz JE, Reimann KA, Axthelm MK, Polack FP, Griffin DE, Letvin NL (2003) Role of CD8(+) lymphocytes in control and clearance of measles virus infection of rhesus monkeys. J Virol 77:4396–4400 Permar SR, Klumpp SA, Mansfield KG, Carville AA, Gorgone DA, Lifton MA, Schmitz JE, Reimann KA, Polack FP, Griffin DE, Letvin NL (2004) Limited contribution of humoral immunity to the clearance of measles viremia in rhesus monkeys. J Infect Dis 190:998–1005 Pfeuffer J, Püschel K, ter Meulen V, Schneider-Schaulies J, Niewiesk S (2003) Extent of measles virus spread and immune suppression differentiates between wildtype and vaccine strains in the cotton rat model (Sigmodon hispidus). J Virol 77:150–158 Pletneva LM, Haller O, Porter DD, Prince GA, Blanco JC (2008) Induction of type I interferons and interferon-inducible Mx genes during respiratory syncytial virus infection and reinfection in cotton rats. J Gen Virol 89:261–270 Premenko-Lanier M, Rota PA, Rhodes G, Verhoeven D, Barouch DH, Lerche NW, Letvin NL, Bellini WJ, McChesney MB (2003) DNA vaccination of infants in the presence of maternal antibody: a measles model in the primate. Virology 307:67–75 Pueschel K, Tietz A, Carsillo M, Steward M, Niewiesk S (2007) Measles virus-specific CD4 Tcell activity does not correlate with protection against lung infection or viral clearance. J Virol 81:8571–8578 Radecke F, Spielhofer P, Schneider H, Kaelin K, Huber M, Dötsch C, Christiansen G, Billeter MA (1995) Rescue of measles virus from cloned DNA. EMBO J 14:5773–5784 Richter BW, Onuska JM, Niewiesk S, Prince GA, Eichelberger MC (2005) Antigen-dependent proliferation and cytokine induction in respiratory syncytial virus-infected cotton rats reflect the presence of effector-memory T cells. Virology 337:102–110 Rytik PG, Kucherov II, Muller WE, Podol´skaia IA, Kruzo M, Duboiskaia GP, Poleshchuk NN (1995) The use of the polymerase chain reaction in modelling HIV infection in animals (in Russian). Zh Mikrobiol Epidemiol Immunobiol 8:86–89 Schachtner SK, Rome JJ, Hoyt RF Jr, Newman KD, Virmani R, Dichek DA (1995) In vivo adenovirus-mediated gene transfer via the pulmonary artery of rats. Circ Res 76:701–709

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Schlereth B, Germann P-G, ter Meulen V, Niewiesk S (2000a) DNA vaccination with the hemagglutinin and the fusion proteins, but not the nucleocapsid protein protects against experimental measles virus infection. J Gen Virol 81:1321–1325 Schlereth B, Rose KJ, Buonocore L, ter Meulen V, Niewiesk S (2000b) Successful vaccineinduced seroconversion by single dose immunization in the presence of measles virus specific maternal antibodies. J Virol 74:4652–4657 Schlereth B, Buonocore L, Tietz A, ter Meulen V, Rose JK, Niewiesk S (2003) Successful mucosal immunization of cotton rats in the presence of measles virus-specific antibodies depends on degree of attenuation of vaccine vector and virus dose. J Gen Virol 84:2145–2151 Schnorr J-J, Seufert M, Schlender J, Borst J, Johnson ICD, ter Meulen V, Schneider-Schaulies S (1997) Cell cycle arrest rather than apoptosis is associated with measles virus contact-mediated immunosuppression in vitro. J Gen Virol 78:3217–3226 Steinbach MM, Duca CJ (1940) Experimental tuberculosis in the cotton rat (Sigmodon hispidus littoralis). Proc Soc Exp Biol Med 44:288–290 Stertz S, Dittmann J, Blanco JC, Pletneva LM, Haller O, Kochs G (2007) The antiviral potential of interferon-induced cotton rat mx proteins against orthomyxovirus (influenza), rhabdovirus, and bunyavirus. J Interferon Cytokine Res 27:847–855 Stittelaar K, Wyatt L, de Swart R, Vos H, Groen J, van Amerongen G, van Binnendijk R, Rozenblatt S, Moss B, Osterhaus A (2000) Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies. J Virol 74:4236–4243 Streif S, Pueschel K, Tietz A, Blanco J, Meulen VT, Niewiesk S (2004) Effector CD8+ T cells are suppressed by measles virus infection during delayed type hypersensitivity reaction. Viral Immunol 17:604–608 Takeda M, Takeuchi K, Miyajima N, Kobune F, Ami Y, Nagata N, Suzaki Y, Nagai Y, Tashiro M (2000) Recovery of pathogenic measles virus from cloned cDNA. J Virol 74:6643–6647 Tober C, Seufert M, Schneider H, Billeter MA, Johnston ICD, Niewiesk S, ter Meulen V, Schneider-Schaulies S (1998) Expression of measles virus V protein is associated with transcriptional control and pathogenicity. J Virol 72:8124–8132 van Binnendijk RS, Poelen MCM, Kuijpers KC, Osterhaus ADME, Uytdehaag FGCM (1990) The predominance of CD8 T cells after infection with measles virus suggests a role for CD8 class I MHC-restricted cytotoxic T lymphocytes (CTL) in recovery from measles. J Immunol 144:2394–2399 van Binnendijk RS, Poelen MCM, van Amerongen G, de Vries P, Osterhaus ADME (1997) Protective immunity in macaques vaccinated with live attenuated, recombinant, and subunit measles vaccines in the presence of passively acquired antibodies. J Infect Dis 175:524–532 Weidenmaier C, Kokai-Kun JF, Kristian SA, Chanturiya T, Kalbacher H, Gross M, Nicholson G, Neumeister B, Mond JJ, Peschel A (2004) Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections. Nat Med 10:243–245 Weidinger G, Ohlmann M, Schlereth B, Sutter G, Niewiesk S (2001) Vaccination with recombinant modified vaccinia virus Ankara protects against measles virus infection in the mouse and cotton rat model. Vaccine 19:2764–2768 Williams JV, Tollefson SJ, Johnson JE, Crowe JE Jr (2005) The cotton rat (Sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity. J Virol 79:10944–10951 Wong-Chew RM, Islas-Romero R, Garcia-Garcia ML, Beeler JA, Audet S, Santos-Preciado JI, Gans H, Lew-Yasukawa L, Maldonado YA, Arvin AM, Valdespino-Gomez JL (2004) Induction of cellular and humoral immunity after aerosol or subcutaneous administration of Edmonston-Zagreb measles vaccine as a primary dose to 12-month-old children. J Infect Dis 189:254–257 Wyde PR, Ambrosi MW, Voss TG, Meyer HL, Gilbert BF (1992) Measles virus replication in lungs of hispid cotton rats after intranasal inoculation. Proc Soc Exp Biol Med 201:80–87 Wyde PR, Moore-Poveda DK, Daley NJ, Oshitani H (1999) Replication of clinical measles virus strains in hispid cotton rats. Proc Soc Exp Biol Med 221:53–62

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Wyde PR, Moore-Poveda DK, De Clercq E, Neyts J, Matsuda A, Minakawa N, Guzman E, Gilbert BE (2000a) Use of cotton rats to evaluate the efficacy of antivirals in treatment of measles virus infections. Antimicrob Agents Chemother 44:1146–1152 Wyde PR, Stittelaar KJ, Osterhaus ADME, Guzman E, Gilbert BE (2000b) Use of cotton rats for preclinical evaluation of measles vaccines. Vaccine 19:42–53 Wyde PR, Chetty SN, Jewell AM, Schoonover SL, Piedra PA (2005) Development of a cotton rat-human metapneumovirus (hMPV) model for identifying and evaluating potential hMPV antivirals and vaccines. Antiviral Res 2005:57–66 Yim KC, Carroll CJ, Tuyama A, Cheshenko N, Carlucci MJ, Porter DD, Prince GA, Herold BC (2005) The cotton rat provides a novel model to study genital herpes infection and to evaluate preventive strategies. J Virol 79:14632–14639 Zhu Y, Rota P, Wyatt L, Tamin A, Rozenblatt S, Lerche N, Moss B, Bellini W, McChesney M (2000) Evaluation of recombinant vaccinia virus—measles vaccines in infant rhesus macaques with preexisting measles antibody. Virology 276:202–213

Chapter 6 Current Animal Models: Transgenic Animal Models for the Study of Measles Pathogenesis C.I. Sellin and B. Horvat(*)

Contents Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mice Transgenic for CD46 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HMGCR-CD46 Mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NSE-CD46 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genomic CD46 Transgenic Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD150 (SLAM) Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lck-CD150 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD11c-CD150 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD150Ge Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Knockin CD150 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HMGCR-CD150 Mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD46 × CD150 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Animal models are highly important to understand the pathologic mechanisms of viral diseases. Therefore, the lack of a suitable animal model has greatly hindered the research into the pathogenesis of measles. Identification of two human receptors for measles virus, CD46 and CD150 (SLAM) has opened new perspectives in this field. During the last decade, numerous transgenic animal models have been developed in order to humanize mice and use them to study measles infection and virus–host interactions. Despite their limitations, these models have provided remarkable insights in different aspects of measles infection, providing a better understanding of virus-induced neuropathology, immunosuppression, mechanisms of virus virulence, and contribution of innate and adaptive immune response in viral clearance. They should certainly continue to help in studies of the host and viral factors that are important in measles infection and in developing of new antiviral agents

B. Horvat U758-ENS Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France, e-mail: branka.horvat @inserm.fr

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and measles virus-based vaccines. In addition, as CD46 serves as a receptor for two other human viruses, some of these models may also find an important application in the study of adenovirus and herpesvirus 6 infection. In this review, we describe different CD46 and CD150 transgenic models and detail their utilization in the study of various aspects of measles pathogenesis.

Abbreviations CNS DCs HMGCR i.c. IFN IFNAR i.n. i.p. i.v. M MV NSE RAG TLR SLAM SSPE STAT YAC

Central nervous system Dendritic cells Hydroxymethylglutaryl coenzyme A reductase Intracranial Interferon Interferon alpha/beta receptor Intranasal Intraperitoneal Intravenous Matrix Measles virus Neuron-specific enolase Recombinase-activating gene Toll-like receptor Signaling lymphocytic activation molecule Subacute sclerosing encephalitis Signal transducer and activator of transcription Yeast artificial chromosome.

Introduction Infectious diseases remain a major cause of mortality in the world today, particularly among children in developing countries, where they are the first cause of death (WHO 2002). The pathogenesis of many viral diseases that pose a significant public health problem cannot be easily studied because of the lack of an adequate small-animal model. Although monkeys could often be used to study infection by human viruses, more practical and less expensive animal models are highly desirable. As mice remain the most convenient and well-characterized animal model, a great effort has been made to find the best way to use them to study the pathogenesis of different viral diseases, including measles. The natural resistance of mice to measles virus infection has forced different laboratories to search for a means to overcome this obstacle. As viral tropism is determined by the pattern of expression of virus-specific cellular receptor(s): these molecules are the key player in infection. A lack of human cell receptors on small animals therefore presents a major barrier to successful

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infection. Development of transgenic technology has helped enormously in this field to generate animals expressing receptors for several human viruses. Therefore, the identification of two human cell receptors for measles virus (MV)—CD46 (Dorig et al. 1993; Naniche et al. 1993) and CD150 or SLAM (signaling lymphocytic activation molecule) (Tatsuo et al. 2000) (Fig. 6.1)—has made it possible to create transgenic animals that express these receptors, with the aim of establishing mice that are susceptible to MV infection. Since 1996, numerous transgenic animal lines have been engineered to study MV pathogenesis. The choice of the promoter, composition and integration site of the transgenic construct, differentially engineered by various laboratories, is responsible for the principal differences between the models obtained. These transgenic lines express one or both human MV receptors on different cell types and some of them have been crossed in a particular genetic background, usually allowing a higher susceptibility to the MV infection. They have been intensively used during the last decade to study different aspects of MV infection and provide new insights into MV–host interaction. This review summarizes different characteristics of these transgenic models and their application to the study of MV pathogenesis.

Mice Transgenic for CD46 CD46 is a ubiquitously expressed human transmembrane protein, whose primary function is to regulate complement activation by binding and inactivating the C3b and C4b component of the complement (Fig. 6.1). During the last decade, CD46

CD150

CD46 NH2

NH2

1 MV 2

Short Consensus Repeat

V C4b

3 4 B C

STP

C3b

C Extracellular domains

Cyt1 or Cyt2 COOH

MV

Ig-like domains

ITSM Intracellular domains

ITSM COOH

Fig. 6.1 Schematic representation of two identified human MV receptors CD46 and CD150 (SLAM) expressed in different transgenic animal lines. STP serine threonine and proline rich region, Cyt cytoplasmic tail, ITSM immuno-tyrosine based switch motif

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has been also identified as a receptor for several different human viruses, including laboratory strains of measles virus (Dorig et al. 1993; Naniche et al. 1993), herpesvirus 6 (Santoro et al. 1999), and groups B and D of adenovirus (Gaggar et al. 2003). As mice express a CD46 murine counterpart only in testis, numerous transgenic mouse lines have been produced to obtain human CD46 expression in mice. These lines were generated using different promoters and genetic backgrounds and they showed differential susceptibility to MV infection (Table 6.1).

HMGCR-CD46 Mice The first CD46 transgenic mice were generated using the HMGCR promoter, which drives the expression of an enzyme, hydroxymethylglutaryl coenzyme A reductase, involved in cholesterol metabolism (Horvat et al. 1996; Evlashev et al. 2000). These mice thus express CD46 ubiquitously with one of two different cytoplasmic domains either Cyt1 or Cyt2. Permissibility of transgenic lymphocytes to MV infection in vitro is much lower than human lymphocytes, suggesting the existence Table 6.1 Main characteristics of mouse lines transgenic for human CD46, used as models of measles Name of the transgenic model

Pattern of expression and immunological status

Major clinical signs and pathology following MV infection

HMGCR-CD46 (Horvat et al. 1996)

Ubiquitous expression Immuno-competent

NSE-CD46 (Rall et al. 1997)

Neuron-restricted Immunocompetent

NSE-CD46 X RAG–2 (Lawrence et al. 1999) YAC-CD46 (Oldstone et al. 1999)

Neuron-restricted Immunodeficient

YAC-CD46

Ubiquitous expression similar to humans Immunodeficient

i.c. injection in neonates → neurological syndrome, death: age-dependent susceptibility i.c. injection in neonates → neurological syndrome, death: age-dependent susceptibility i.c. injection in neonates and adults → neurological syndrome, death i.c. injection in neonates → neurological syndrome, death/i.p. and i.v. injection on adults → replication in lymphoid cells + immunosuppression i.c. injection in adults → neurological syndrome, death

X RAG-1 (Patterson et al. 2001) CD46Ge X IFNAR-KO (Mrkic et al. 2000)

Ubiquitous expression similar to humans Immunocompetent

Ubiquitous expression similar to humans Immunodeficient

i.c. and i.p. injection in adults → replication in PBMC and lymphoid organs + MV systemic propagation

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of intracellular factors in murine cells, which limit MV replication (Evlashev et al. 2001). However, intracranial infection of newborn transgenic mice with laboratory strain of MV, Edmonston, induces a severe neurological disease, followed with a lack of mobility, weight loss, ataxia, and death (Evlashev et al. 2000). Measles virus replication in these mice is followed with the production of infectious virus particles and associated with widely spread neuronal lesions and apoptosis. The pathology observed is similar to what is seen in progressive infectious measles encephalitis in immunocompromised patients. In addition to being used to analyze MV-induced neuropathology, CD46 transgenic mice expressing Cyt1 domain (Evlashev et al. 2000; Thorley et al. 1997) have been used to study the most important pathology associated with measles: immunosuppression. These studies demonstrated that MV proteins in the absence of viral replication could induce an inhibition of inflammatory responses known to be altered in measles patients (Marie et al. 2001). UV-inactivated MV injection in CD46 transgenic mice resulted in a decrease in hypersensitivity responses, associated with an alteration in antigen-presenting cell functions, a decrease in the number of dendritic cells producing IL-12, and an abrogation of antigen specific proliferation of T cells. Two distinct mechanisms have been implicated: the interaction between CD46 and MV envelope glycoproteins and MV nucleoprotein binding to Fcγ receptor, both with the important role in the generation of MV-induced immunosuppression. HMGCR transgenic mice wee used to analyze the role of CD46 in the generation of anti-MV responses. It has been demonstrated that the interaction between MV hemagglutinin and CD46 increases the uptake of MV particles and leads to the enhanced MHC-class II presentation MV antigens in vivo (Rivailler et al. 1998). Finally, these mouse lines were of critical importance to demonstrate a differential effect CD46 cytoplasmic domains in the regulation of the immune response, following the CD46 engagement by MV hemagglutinin (Marie et al. 2002). Mice expressing Cyt-1 generate decreased inflammatory responses, whereas mice expressing the Cyt-2 isoform develop increased inflammation following CD46 engagement. This differential effect was associated with the modulation of Vav phosphorylation in lymphocytes, thus affecting their cytotoxic activity, T cell proliferation, IL-2 and IL-10 production. These findings underline the importance of CD46 receptor in the regulation of the immune response, which could be subverted by pathogens capable of binding to this molecule.

NSE-CD46 Mice To study pathogenesis of MV-induced persistent brain infection, another model was generated: a transgenic mouse line expressing human CD46 under the control of a neuron-specific promoter (NSE-CD46) (Rall et al. 1997), therefore restricting the expression of CD46 to the brain. An intracranial injection of MV Edmonston into NSE-CD46 neonates resulted in the development of clinical neurological signs of infection and consecutive death of infected animals. Similarly to what has been

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observed with HMGCR-CD46 mice, MV susceptibility depends on the age at the time of infection, as adult CD46 mice were resistant to infection. However, viral particles could not be isolated from injected mouse brains, resembling persistent MV infection in patients with subacute sclerosing encephalitis (SSPE). Although NSE-CD46 mice succumb to MV infection, they developed a substantial inflammatory response during MV-induced encephalitis, similarly to what has been observed in human cases of SSPE. Infiltrates of T and B lymphocytes and macrophages were found in the brain, associated with upregulation of MHC class I and II expression, reactive astrocytosis and microgliosis, upregulation of RANTES, IP-10 (IFNγ-induced 10-kD protein), IL-6, TNFα and IL-1β, and an increase in neuronal apoptosis (Manchester et al. 1999). IP-10 and RANTES were shown to be secreted by infected neurons and to attract lymphocytes in the brain (Patterson et al. 2003). To assess the role and the importance of the different immune cell subsets in the regulation of MV infection, NSE-CD46 mice have been crossed on RAG-KO immunodeficient background, lacking both B and T lymphocytes. In contrast to adult single transgenic mice, NSE-CD46 × RAG-2 adult mice are susceptible to MV brain infection (Lawrence et al. 1999). These results suggested that the adaptive immune response had a protective role against MV infection, indicting that the major cause of MV-induced neurological complications is the viral replication rather then the antiviral response in infected brains. In addition, NSE-CD46 mice have then been crossed on other backgrounds in order to measure the role of different cell subsets, deficient in CD4, β2-microglobulin, perforin or IFNγ. Results obtained with these transgenic models suggested that IFNγ plays a crucial role in viral protection, but that this protection is multifactorial. Moreover, CD4+ and CD8+ T cells are implicated in the local antiviral response and are both required for complete antiviral protection. Finally, these models suggested that anti-MV immune response favors viral clearance without concomitant neuronal loss, probably via cytokine secretion (Patterson et al. 2002). Primary neuronal cultures were obtained from NSE-CD46 mice to study the spread of MV between neurons (Lawrence et al. 2000). These experiments suggested that CD46 expression, syncytia formation or viral budding are not required for viral propagation in the brain. CD46 remains required for initial infection, but cellular contact is necessary for the virus to spread, suggesting an alternative viral propagation, potentially via synapses, specific of the neuronal environment. The mechanism of viral spread in the central nervous system (CNS) was further analyzed by studying the role of MV fusion protein (Makhortova et al. 2007). Viral spread and infection were inhibited by a tri-peptide, known to have a strong antifusogenic activity, showing that fusion played a role in MV infection and propagation in neurons. Substance P, a neurotransmitter, with the same active site as the tri-peptide, also blocked neuronal MV spread. Finally both genetic deletion and pharmacological inhibition of substance P receptor, neurokinin 1, reduced infection of susceptible mice. These data indicated a role for neurokinin 1 in MV CNS infection and spread, perhaps serving as an MV fusion receptor or co-receptor in neurons. This study was conducted using MV Edmonston strain and should therefore be confirmed with wild-type MV strains.

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Genomic CD46 Transgenic Animals In order to mimic the cellular distribution and amount of CD46 found in humans as closely as possible, several transgenic lines have been generated using genomic clones carrying the complete CD46 gene. However, the first transgenic models generated using this approach were not susceptible to MV infection in vivo. Both CD46 transgenic rats (Niewiesk et al. 1997) and mice (Blixenkrone-Moller et al. 1998) expressed widely transgenic human CD46 receptor and murine transgenic cell cultures were permissive to MV infection in vitro, although both models were resistant to the infection in vivo. Nevertheless, the race to obtain better CD46 transgenic models continued. Utilization of the yeast artificial chromosome (YAC) libraries with a full-length CD46 genomic clone (>400 kb) allowed generation of several lines of CD46 transgenic mice expressing all major isoforms of CD46, with a pattern of expression that is very similar to humans, except a CD46 expression on mouse erythrocytes (Kemper et al. 2001). Two of them, YAC-CD46 and CD46ge, have been crossed in different genetic backgrounds and intensively characterized regarding MV infection

YAC-CD46 Mice The first transgenic line that contained YAC with the complete human CD46encoding gene, expressed comparably to what is observed in human tissues, was generated to study the role of CD46 in the regulation of complement activation in transplantation (Yannoutsos et al. 1996). This animal model was then successfully used to analyze different aspects of MV pathogenesis (Oldstone et al. 1999). Intracranial infection of YAC-CD46 newborns with MV Edmonston is lethal, associated with an intensive MV replication in neurons, where viral nucleocapsids accumulate without budding. MV is able to replicate in cells of the immune system as well, although at a low level, and viral RNA and proteins are detectable in lymphoid cells from peripheral blood, spleen, and lymph nodes. Injection of MV into adult YAC-CD46 mice induced a suppression of humoral and cellular responses to secondary antigens, suggesting that this model may be used to study MV-induced immunopathology. Indeed, a few years later it was demonstrated that MV-infected YAC-CD46 mice are much more susceptible then noninfected mice to the secondary infection with Listeria monocytogenes, due to the inhibition of both innate and adaptive immunity by MV (Slifka et al. 2003). This transgenic line has been further used to study the mechanisms initiating SSPE. YAC-CD46 adult mice were initially infected with lymphocytic choriomeningitis virus, which transiently suppresses the murine immune system. Ten days later, infection by MV Edmonston resulted in persistent MV infection of neurons and infiltration of T and B cells in the brain, MV presenting numerous mutations in the M gene. Furthermore, similarly to what is observed in SSPE, mice produced a high titer of anti-MV antibodies (Oldstone et al. 2005). This interesting experimental setup made it possible to suggest the importance of the immunological status of patients with

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measles, where existence of a stage of immunosuppression before infection may allow the generation of the persistent brain infection afterwards, therefore shedding new light on the immunopathogenesis of SSPE. YAC-CD46 mice were crossed into RAG-1-deficient background to obtain a model where adult mice are more susceptible to infection and then used to analyze MV brain infection. Using a MV reverse genetics system, an infectious MV virus was generated, in which the matrix (M) gene was replaced with the M gene of a SSPE MV strain (Patterson et al. 2001). As frequent mutations in the M gene, which are observed in viruses recovered from SSPE patients, could result in defective virus, the pathogenicity of this SSPE M-MV was tested in YAC-CD46 × RAG-1 adult mice. Results showed that this virus is infectious in vivo but produces a protracted progressive infection with a death of animals delayed compared to Edmonston strain. Studies were extended to primary neurons and showed that SSPE M-MV initiated infection only through the CD46 receptor. However, after initial entry, viral spread among neurons did not appear to require CD46, similarly to what has been shown in another study using NSE-CD46 mice (Lawrence et al. 2000). Finally, in order to measure the contribution of different immune cell subsets, YAC-CD46 mice were crossed on immunodeficient backgrounds, as this has been done with NSE-CD46 mice. Different cell subpopulations were tested with RAG1-KO, CD4-KO, CD8-KO, lymphocyte B-KO, Perforin-KO, TNF-α KO and IFN-γ KO backgrounds (Tishon et al. 2006). This study confirmed that IFN-γ was necessary for viral clearance, and that its absence results in persistent infection. B, CD4+ T or CD8+ T cells by themselves are not sufficient to control the infection. However, a combination of B and CD4+ T cells or CD4+ and CD8+ cells leads to viral clearance. Although B cells did not play a major role in the resolution of primary infection, it has been shown in HMGCR CD46 mice that immunized mothers can transfer immunity to their pups, probably via transplacental transfer of maternal antibodies (Evlashev et al. 2000). These different transgenic models help to obtain more insight into the complex interaction between MV and innate and adaptive responses and the role of different components of the immune system in viral clearance.

CD46Ge × IFNAR-KO Mice Another line generated using a full-length CD46 genomic clone was back crossed on the IFNAR-KO background, lacking alpha/beta interferon receptor and therefore deficient in the innate immune response (Mrkic et al. 1998). In contrast to the other models described, this transgenic line, CD46Ge × IFNAR-KO, was susceptible to developing MV infection by natural route of MV infection: intranasal inoculation. The absence of type I IFN signaling allowed an enhanced MV spread to the lungs and more prominent inflammatory responses. Virus replication was also detected in peripheral blood mononuclear cells, the spleen, and the liver, showing a systemic viral propagation in this transgenic model. Cells of the monocyte/macrophage lineage appeared to be early MV targets but also contributed to protect other immune cells from infection (Mrkic et al. 2000; Roscic-Mrkic et al. 2001).

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This model has also been used to assess the pathogenicity of different recombinant MV viruses. Viruses lacking nonstructural MV C or V proteins are less effective in spreading through the lymphatic system and were not detected in the liver (Mrkic et al. 2000). After intracerebral inoculation, recombinant viruses caused lethal disease less often and induced distinctive patterns of gliosis and inflammation. So V and C proteins seem to act as virulence factors, similarly to what has been demonstrated in parallel in YAC-CD46 mice (Patterson et al. 2000). Two other recombinant viruses were generated to mimic SSPE variants: MV deficient in matrix protein M (MV-Δ M) and a virus bearing glycoproteins with shortened cytoplasmic tails (MV-Δ tails). Both lost acute pathogenicity in CD46Ge × IFNAR-KO mice but penetrated more deeply into the brain parenchyma than standard MV (Cathomen et al. 1998). These different CD46 mouse models helped study the pathogenesis of infection with MV laboratory strains and reproduced many aspects of MV pathology, which were observed in monkeys or in naturally infected humans. In addition, they have an important application in the study of vaccination approaches, based on the recombinant MV vaccines (Combredet et al. 2003; Despres et al. 2005; Lorin et al. 2004), in MV-based oncolytic therapy (Peng et al. 2002), as well as in testing new antiviral concepts (Christiansen et al. 2000). However, all CD46 transgenic mice were resistant to wild-type MV strains, emphasizing that any direct extrapolation of results to human physiopathology may be hazardous. Furthermore, the obligation to eliminate either the adaptive (RAG KO) or innate immune system (Interferon type 1 receptor KO) to obtain susceptibility to MV in adult mice limited their application in studies where these immune responses are desirable to obtain relevant conclusions.

CD150 (SLAM) Transgenic Mice The identification of CD150 or SLAM as a receptor both for wild-type and laboratory MV strains (Tatsuo et al. 2000) (Fig. 6.1) opened up new perspectives in the generation of transgenic mouse models for infection with clinical MV isolates. Therefore, the race to generate better models to study MV pathogenesis has continued. As murine CD150 can not serve as a receptor for MV, over the last several years, numerous mouse lines that are transgenic for human CD150 have been developed. They differ in the promoter used to drive CD150 expression and therefore in the cell type expressing it, as well as in susceptibility to MV infection (Table 6.2).

Lck-CD150 Mice The first reported model transgenic for CD150 was engineered using the lck promoter, specific for T lymphocytes (Hahm et al. 2003). CD150 was thus restricted to immature and mature lymphocytes, in spleen, thymus, and blood. As in human

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Table 6.2 Main characteristics of mouse lines transgenic for human CD150, used as models of measles Name of the transgenic model

Pattern of expression and immunological status

Major clinical signs and pathology following MV infection

Lck-CD150

Expression restricted to T lymphocytes Immunocompetent

i.p. injection in neonates → infection of thymocytes ex vivo infection → inhibition of T cell proliferation i.v. injection in adults → infection of 2%–5% of dendritic cells/ex vivo infection of dendritic cells → immunosuppression i.n. injection in adults → transient infection of nasal lymph nodes i.n. and i.p. injection in adults → infection of the thymus spleen and nasal mesenteric and femoral lymph nodes + splenomegaly ex vivo infection of splenocytes i.n. and i.p. injection in adults → infection of spleen and lymph nodes + immuno suppression i.n. and i.c. injection in suckling mice → neurological syndrome, death i.n. and i.c. injection in suckling mice and adults → neurological syndrome, death i.n. Injection in adults → infection of dendritic cells in lymph nodes

(Hahm et al. 2003) CD11c-CD150 (Hahm et al. 2004)

Expression restricted to CD11c+ dendritic cells Immunocompetent

CD150Ge (Welstead et al. 2005)

Human-like expression Immunocompetent

CD150Ge X STAT1-KO (Welstead et al. 2005)

Human-like expression Immunodeficient

Knockin CD150 mouse C + human V (Ohno et al. 2007) Knockin CD150 X IFNAR-KO (Ohno et al. 2007)

Human-like expression Immunocompetent Human-like expression Immunodeficient

HMGCR-CD150 (Sellin et al. 2006)

Ubiquitous expression Immunocompetent

HMGCR-CD150 X IFNAR-KO (Druelle et al. 2008) CD46Ge X CD150Ge X IFNAR-KO (Shingai et al. 2005)

Ubiquitous expression Immunodeficient Human-like expression Immunodeficient

T cells, splenic transgenic lymphocytes were susceptible to in vitro infection with wild-type and vaccine MV strains, resulting in an inhibition of cell proliferation. Following infection or contact with an infected cell, downregulation of CD150 cell surface expression was observed. In addition, in neonate animals, Lck CD150 thymocytes expressed MV antigens at the surface 2 days after i.p. injection of MV, suggesting either their infection or binding of the residual MV to the cell receptor. Although no clinical signs of infection are observed in this model, inhibited capacity of infected lymphocytes to proliferate suggested MV-induced immunosuppression in these mice.

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CD11c-CD150 Mice Several in vitro studies have suggested that MV infection of human dendritic cells (DCs) may play an important role in MV-induced immunosuppression (Kerdiles et al. 2006). To test this hypothesis in vivo and analyze the mechanisms implicated, mice transgenic for CD150 were generated using a CD11c-specific promoter, thus allowing expression of MV receptor on splenic and bone-marrow-derived DCs (Hahm et al. 2004). Although in vitro infection of splenic DCs gave approximately 35% cells positive for MV, followed by a downregulation of co-stimulatory molecules (CD80, CD86, CD40) and MHC class I and II molecules, only 2%–5% of splenic DCs were infected upon i.v. MV injection. In vitro infected transgenic DCs fail to stimulate alloreactive responses and efficiently inhibit T cell proliferation, independently of IFN-α/β, TNF-α or lymphotoxin α or β. Results from these studies demonstrated that CD150 expression on T cells is not required for the inhibition of T cell functions by DCs and characterized the role of DCs in MV-induced immunosuppression. The same transgenic model was used few years later to study the role of Tolllike receptors (TLRs) in MV-induced immunosuppression (Hahm et al. 2007). Transgenic bone-marrow-derived DCs were infected with a wild-type MV, then stimulated with different TLR ligands, and the production of several cytokines was measured. The results showed that transgenic infected DCs were defective in the selective synthesis of IL-12 in response to stimulation of TLR4 signaling, but not to engagements of TLR2, 3, 7, or 9. Nonstructural MV V and C proteins were not responsible for this inhibition, and interaction of MV hemagglutinin with CD150 facilitated the suppression. These results suggested that MV, by altering DC function, renders them unresponsive to secondary pathogens via TLRR4. As virus virulence is often related to the ability of viruses to interfere with the host interferon system, the role of IFN α/β in MV infection was studied using CD11c-CD150 mice (Hahm et al. 2005). It was shown that MV infection interfered with development and expansion of DC both in vivo and in vitro, through generation of IFN α/β by DCs. Type I IFN acted via a signal transducer and activator of transcription 2 (STAT-2)-dependent but STAT-1-independent pathway, suggesting a new mechanism in the abrogating the function of DCs and generation of virusinduced immunosuppression.

CD150Ge Mice In 2005, another mouse model that was transgenic for CD150 was created, mice expressing the complete human gene, under the control of endogenous promoter (Welstead et al. 2005). The pattern of CD150 expression was similar to what has been described in humans: activated B and T lymphocytes and activated DCs. In mice infected intranasally by recombinant wild-type MV, a transient infection of nasal lymph nodes was observed, although they did not show clinical signs of

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disease. To improve the efficiency of MV infection, these mice were crossed on a STAT-1-deficient background and the resulting mice showed higher susceptibility to the infection and produced more viral particles. After i.n. and i.p. inoculation, infection in the thymus, spleen, and lymph nodes was detected, with increased lymph node and spleen size. Abnormally large numbers of mature neutrophils and natural killer cells caused the splenomegaly. Therefore, although STAT-1 was not required for the DC development in CD11c-CD150 mice, in this model ablation of STAT-1 significantly increased susceptibility to MV infection.

Knockin CD150 Mice In order to generate transgenic mice regulating the expression of human CD150 in the same way as murine CD150, and therefore to closely approach the immunophysiological conditions of the host, a mouse model bearing a chimeric CD150 molecule was generated (Ohno et al. 2007). As the V domain is necessary and sufficient for MV binding to CD150 (Ono et al. 2001) (Fig. 6.1), a knockin approach was used to replace the murine V domain by the human V domain, using homologous recombination in embryonic stem cells. Generated knockin mice express chimeric CD150 molecule with an expected distribution and normal signaling function. Although cells from this transgenic model could be infected in vitro with wild-type MV, these mice had to be crossed on an IFNAR-KO background to obtain a productive MV infection in vivo. In knockin-CD150 × IFNAR-KO mice, MV propagation was detected in different lymphoid organs following i.n. and i.p. wild-type MV injection. Transgenic splenocytes showed suppression of proliferative responses to concanavalin A. Thus MV infection of these mice reproduces lymphotropism and immunosuppression as in MV human infection and should serve as a useful small-animal model to study MV pathogenesis in conditions when production of interferon type I is not critical.

HMGCR-CD150 Mice To further increase the susceptibility of mice to wild-type MV infection, another CD150 transgenic model was generated using HMGCR promoter (Sellin et al. 2006). These mice ubiquitously express CD150 and present the only model reported so far where suckling mice are highly susceptible to the natural route of MV infection. Following intranasal MV inoculation, mice develop a severe neurologic syndrome, characterized by lethargy, seizures, ataxia, weight loss, and death within 3 weeks, associated with MV propagation to different organs. The highest level of replication was observed in the brain, where virus infects neurons and oligodendrocytes, but not astrocytes, although an intensive astrogliosis is observed

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Fig. 6.2 a–c Cellular targets of MV infection in the brain of HMGCR-CD150 mice. Suckling mice were infected intranasally with wild-type MV strain G954 and brains were dissected and analyzed immunohistochemically 10 days later by confocal microscopy using anti-MV nucleoprotein antibody (red, a–c) and neuron-specific anti-NF antibody (green, a); astrocyte-specific anti-GFAP antibody (green, b) and anti-GC oligodendrocytic antibody (c), as described previously (Sellin et al. 2006). Co-localization (yellow) is observed only in a and c, demonstrating that neurons and oligodendrocytes but not astrocytes are infected by MV

(Fig. 6.2). Moreover, this model was shown to be useful to test anti-measles preventive and therapeutic approaches, as a soluble recombinant CD150 molecule successfully protected HMGCR-CD150 mice from MV infection. This transgenic model, by allowing a simple readout of the efficacy of an antiviral treatment, provides a unique experimental tool to evaluate innovative anti-measles approaches. This mouse line was then used to study the mechanism of attenuation of MV by repeated cellular passages (Druelle et al. 2008). Indeed, virulence of parental and attenuated MV strains could be distinguished in this transgenic model. Moreover, HMGCR-CD150 mice were crossed on an IFNAR-KO background, where attenuated virus did not show any higher virulence. This model demonstrated that attenuation of this particular MV strain was independent of type I IFN and therefore opens new perspectives to compare the level of neurovirulence of different MV strains and further dissect MV-induced neurological disease.

CD46 × CD150 Mice Finally, to approach murine models as closely as possible to humans, a double transgenic mice was generated, expressing both known human MV receptors, CD46 and CD150 (Shingai et al. 2005). These mice present the same pattern of expression of CD46 and CD150 as humans: CD46 expression is ubiquitous and CD150 is inducible on T lymphocytes and can be upregulated on B cells and mature DCs. However, expression of both receptors on murine cells turned out to be insufficient to render them more susceptible to infection. This was not completely surprising because intracellular factors were shown to limit MV replication in murine cells (Evlashev et al. 2001). Again, to obtain higher susceptibility to infection, these double transgenic mice were crossed on an IFNAR-KO background. Consequently, following

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i.p. MV inoculation, infected DCs were detectable in lymph nodes. This transgenic model therefore helped to demonstrate that mature DCs are the main vectors of viral spread in vivo, and because of the similarity in the expression of both MV receptors compared to humans, it may give new insights into MV pathogenesis.

Conclusions There is a frequent feeling among scientists that an animal model must exactly mimic the human disease studied. However, this goal has only rarely been achieved. Many different transgenic mouse lines have been generated to study MV pathogenesis and we must admit that none of them reproduces all the aspects of MV infection in humans. Indeed, it seems clear now that if receptor expression is necessary for viral entry, additional obstacles during other steps of the viral cycle prevent expected viral pathogenesis in receptor humanized mice. In most cases, an immunodeficient background was necessary to obtain the susceptibility for MV infection in vivo. However, despite imperfections, these different transgenic models have provided remarkable insights into diverse aspects of MV pathogenesis in a relatively short period of time. Real progress has been accomplished particularly with CD150 transgenic models, since these lines are susceptible to wild-type MV strains and some of them allow MV to spread via the natural route of infection, the airways. These models are now available for the molecular dissection of the measles immunopathology, testing antiviral agents and new vaccines. In addition, since CD46 serves as a receptor for the other human viruses, these transgenic models have been recently used for adenovirus infection in vivo (DiPaolo et al. 2006) and therefore open new avenues to study other viral infections. Finally, several studies have demonstrated the existence of other way(s) of entry into human cells, independently of CD46 and CD150 (Andres et al. 2003; Hashimoto et al. 2002; Kouomou and Wild 2002; Takeuchi et al. 2003). However, the nature of this third receptor remains elusive for the moment. It is certain that its identification in the future will inspire a generation of new transgenic models and help the race towards the development of improved and more perfect models of MV pathogenesis to continue. Acknowledgements Studies cited from our laboratory were supported by institutional grants from INSERM and Fondation pour la Recherche Médicale (FRM); CIS was supported by FRM in 2006–2007.

References Andres O, Obojes K, Kim KS, ter Meulen V, Schneider-Schaulies J (2003) CD46- and CD150-independent endothelial cell infection with wild-type measles viruses. J Gen Virol 84:1189–1197 Blixenkrone-Moller M, Bernard A, Bencsik A, Sixt N, Diamond LE, Logan JS, Wild TF (1998) Role of CD46 in measles virus infection in CD46 transgenic mice. Virology 249:238–248

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Rivailler P, Trescol-Biemont MC, Gimenez C, Rabourdin-Combe C, Horvat B (1998) Enhanced MHC class II-restricted presentation of measles virus (MV) hemagglutinin in transgenic mice expressing human MV receptor CD46. Eur J Immunol 28:1301–1314 Roscic-Mrkic B, Schwendener RA, Odermatt B, Zuniga A, Pavlovic J, Billeter MA, Cattaneo R (2001) Roles of macrophages in measles virus infection of genetically modified mice. J Virol 75:3343–3351 Santoro F, Kennedy PE, Locatelli G, Malnati MS, Berger EA, Lusso P (1999) CD46 is a cellular receptor for human herpesvirus 6. Cell 99:817–827 Sellin CI, Davoust N, Guillaume V, Baas D, Belin MF, Buckland R, Wild TF, Horvat B (2006) High pathogenicity of wild-type measles virus infection in CD150 (SLAM) transgenic mice. J Virol 80:6420–6429 Shingai M, Inoue N, Okuno T, Okabe M, Akazawa T, Miyamoto Y, Ayata M, Honda K, KuritaTaniguchi M, Matsumoto M et al (2005) Wild-type measles virus infection in human CD46/ CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection. J Immunol 175:3252–3261 Slifka MK, Homann D, Tishon A, Pagarigan R, Oldstone MB (2003) Measles virus infection results in suppression of both innate and adaptive immune responses to secondary bacterial infection. J Clin Invest 111:805–810 Takeuchi K, Miyajima N, Nagata N, Takeda M, Tashiro M (2003) Wild-type measles virus induces large syncytium formation in primary human small airway epithelial cells by a SLAM(CD150)independent mechanism. Virus Res 94:11–16 Tatsuo H, Ono N, Tanaka K, Yanagi Y (2000) SLAM (CDw150) is a cellular receptor for measles virus. Nature 406:893–897 Thorley BR, Milland J, Christiansen D, Lanteri MB, McInnes B, Moeller I, Rivailler P, Horvat B, Rabourdin-Combe C, Gerlier D et al (1997) Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection. Eur J Immunol 27:726–734 Tishon A, Lewicki H, Andaya A, McGavern D, Martin L, Oldstone MB (2006) CD4 T cell control primary measles virus infection of the CNS: regulation is dependent on combined activity with either CD8 T cells or with B cells: CD4 CD8 or B cells alone are ineffective. Virology 347:234–245 Welstead GG, Iorio C, Draker R, Bayani J, Squire J, Vongpunsawad S, Cattaneo R, Richardson CD (2005) Measles virus replication in lymphatic cells and organs of CD150 (SLAM) transgenic mice. Proc Natl Acad Sci U S A 102:16415–16420 WHO (2002) Scaling up the response to infectious diseases. www.who.int/infectious-diseasereport/2002/introduction.html. Cited 26 May 2008 Yannoutsos N, Ijzermans JN, Harkes C, Bonthuis F, Zhou CY, White D, Marquet RL, Grosveld F (1996) A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection. Genes Cells 1:409–419

Chapter 7

Molecular Epidemiology of Measles Virus P.A. Rota( ), D.A. Featherstone, and W. J. Bellini

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Laboratory Surveillance for Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Background and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Utility of Molecular Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Standard Methods for Molecular Epidemiology of Measles . . . . . . . . . . . . . . . . . . . . . . Data Reporting and Surveillance Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Virus Isolation and Clinical Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New Molecular Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Vaccines and Antigenic Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Global Distribution of Measles Genotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genotypes in Areas with Endemic Measles or Frequent Outbreaks . . . . . . . . . . . . . . . . Measles Elimination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Reintroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mapping Transmission Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Genetic characterization of wild-type measles viruses provides a means to study the transmission pathways of the virus and is an essential component of laboratory-based surveillance. Laboratory-based surveillance for measles and rubella, including genetic characterization of wild-type viruses, is performed throughout the world by the WHO Measles and Rubella Laboratory Network, which serves 166 countries in all WHO regions. In particular, the genetic data can help confirm the sources of virus or suggest a source for unknown-source cases as well as to establish links, or lack thereof, between various cases and outbreaks. Virologic surveillance has helped to document the interruption of transmission of endemic measles in some regions. Thus, molecular characterization of measles viruses has provided a valuable tool for measuring the effectiveness of measles P.A. Rota Measles, Mumps, Rubella and Herpesvirus Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA, e-mail: [email protected]

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control programs, and virologic surveillance needs to be expanded in all areas of the world and conducted during all phases of measles control.

Introduction The widespread use of live attenuated vaccines for measles has dramatically reduced the worldwide incidence of measles. The disease has been eliminated in the Western hemisphere (Region of the Americas) since 2000 and the European, Eastern Mediterranean, and Western Pacific Regions of the World Health Organization (WHO) have set elimination goals for the near future (Anonymous 2005d). Despite these successes, measles remains endemic in many developing countries. Measles remains a major cause of childhood morbidity and mortality, accounting for an estimated 345,000 deaths in 2005, 87% of which were in the African and South East Asian Regions of WHO (Wolfson et al. 2007). Subsequently the African and Southeast Asian Regions have implemented a strategy of measles mortality reduction. Even counties that have measles vaccination programs experience outbreaks because of the accumulation of susceptible individuals and the constant threat of viral importations from endemic areas. Maintaining measles elimination requires achieving and maintaining very high levels of population immunity and good laboratory-based surveillance to rapidly detect and control periodic outbreaks.

Laboratory Surveillance for Measles When the incidence of measles is low, surveillance based on clinical presentation of cases has low sensitivity and specificity. Therefore, an essential component of any measles control program is laboratory-based surveillance to provide confirmation of cases and genetic characterization of circulating wild-type viruses. Routine laboratory confirmation of suspected cases is based on detection of measles-specific IgM in a single blood sample taken as soon as possible after rash onset. In some cases, molecular techniques such as RT-PCR to detect viral RNA are used to complement serologic testing. Another important aspect of laboratory surveillance for measles, and the subject of this chapter, is the genetic characterization of circulating wild-type viruses to support molecular epidemiologic studies (World Health Organization 2005a, Anonymous 2005b; Rota and Bellini 2003). Laboratory-based surveillance for measles and rubella is performed throughout the world by the WHO Measles and Rubella Laboratory Network (LabNet). This network provides for standardized testing and reporting with labs serving 166 countries in all WHO regions. A system for monitoring indicators of laboratory performance, including laboratory accreditation, and proficiency testing has been implemented in all regions (World Health Organization 2005a, Anonymous 2005b). The LabNet also supports genetic characterization of currently circulating strains of measles viruses and LabNet has been responsible for standardization of the nomenclature and

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laboratory procedures that are used to describe the genetic characteristics of wild-type measles and rubella viruses (World Health Organization 1998, 2001a, 2001b, 2003, 2005b, 2005c, 2006, 2007a). This standardization has allowed sharing of virologic surveillance data between laboratories and permitted efficient communication of this data throughout the measles control programs (World Health Organization 2007b).

Background and Methods Measles virus is an RNA virus in the genus Morbillivirus within the family Paramyxoviridae. Although other members of the genus infect various animal species, measles only infects humans and non-human primates. The negative-sense, single-stranded RNA genome is contained within a helical nucleocapsid in the virion. The genome consists of 15,894 nucleotides, which code for the six structural proteins, the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and large protein (L), and two nonstructural proteins, C and V (Griffin 2001). Although measles is a monotypic virus, genetic and antigenic variation has been detected in wild-type viruses (Rota et al. 1992; Tamin et al. 1994; Taylor et al. 1991). The nucleotide sequences of the L, M, and F genes (Bankamp et al. 1999; Rota et al. 1994b) are much less variable than the sequences of the N, P, and H genes, which have 7%–10% variability (Bankamp et al. 2008; Rima et al. 1997; Rota et al. 1992). The N and H gene sequences are most commonly used for genetic characterization of wild-type viruses. In particular, one of the most variable parts of the measles genome is the 450-nucleotide region, which codes for the COOHterminal 150 amino acids the N protein, where nucleotide variability can approach 12% between wild-type viruses (Xu et al. 1998).

Utility of Molecular Epidemiology The combination of molecular epidemiologic techniques and standard case classification and reporting provides a very sensitive means to describe the transmission pathways of measles. In particular, sequence data can help confirm the sources of virus or suggest a source for unknown-source cases as well as to establish links, or lack thereof, between various cases and outbreaks. Virologic surveillance is especially beneficial when it is possible to observe the change in viral genotypes over time in a particular country or region because this information, when analyzed in conjunction with standard epidemiologic data, has helped to document the interruption of transmission of endemic measles. Thus, molecular characterization of measles viruses has provided a valuable tool for measuring the effectiveness of measles control programs (Mulders et al. 2001; Riddell et al. 2005; Rota et al. 1996, 2002; Rota and Bellini 2003). Virologic surveillance can also help to classify suspected cases as vaccine reactions. A small proportion of measles vaccine recipients experience rash and fever 10–14 days following vaccination (Griffin 2001). During outbreaks, measles vaccine

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is administered to help control the outbreak, and in these situations, vaccine reactions may be mistakenly classified as measles cases. Since serologic methods cannot distinguish between a vaccine-induced antibody response and antibodies derived from natural disease, molecular characterization of viral isolates provides a method to confirm whether vaccine or wild-type measles virus caused the rash and fever. Molecular information is also useful for analyzing unusual or severe cases of measles infection, suspected adverse events following vaccination, and severe sequelae of measles infection such as subacute sclerosing panencephalitis (SSPE) (Bellini et al. 2005). SSPE will be discussed in more detail in Sect. 2.7.

Standard Methods for Molecular Epidemiology of Measles Before 1998, there was no uniform nomenclature or analysis protocol to describe the genetic characteristics of wild-type measles viruses. In 1998, the WHO made recommendations for a standard nomenclature for naming strains, describing genotypes, and conducting sequence analysis so that genetic data would be directly comparable between laboratories. These recommendations have been updated periodically since 1998 (World Health Organization 1998, 2001a, 2001b, 2003, 2005b, 2005c, 2006, 2007a). WHO recommends that the 450 nucleotides coding for the COOH-terminal 150 amino acids of N are the minimum amount of sequence data required for genotyping a measles virus isolate or clinical specimen. Complete H gene sequences should be obtained from representative strains or if a new genotype is suspected. Phylogenetic analysis of the H gene sequences provides additional support for the genotype assignment while monitoring amino acid substitutions that could affect antigenicity. For molecular epidemiologic purposes, the genotype designations are considered the operational taxonomic unit, while related genotypes are grouped by clades. WHO currently recognizes eight clades designated A, B, C, D, E, F, G, and H. Within these clades, there are 23 recognized genotypes, designated A, B1, B2, B3, C1, C2, D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, E, F, G1, G2, G3, H1, and H2. Some clades contain only one genotype and, in such cases, the genotype designation is the same as the clade name. Other clades contain multiple genotypes and are designated by using the clade letter (in uppercase) and genotype number. Several of the genotypes—B1, E, F, G1, D1—appear to be extinct or inactive since representatives of these genotypes have not been isolated for at least 15 years. However, the sequences of the inactive genotypes are maintained in the set of WHO reference sequences for completeness (World Health Organization 2005b). With the exception of genotype F which is based only on sequences derived from a case of SSPE, all of the genotypes have an assigned reference strain (Table 7.1, Fig. 7.1) chosen to represent the earliest isolation of virus from each genotype. Sequences from recent viral isolates are then compared to the set of WHO reference sequences, which are available from GenBank (World Health Organization 2005b) and the WHO Strain Banks, to determine the genotype. WHO has established guidelines based on both molecular biologic and epidemiologic criteria for the designation of new genotypes (World Health Organization 2001b, 2003, 2005b).

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Table 7.1 Reference strains to be used for genetic analysis of wild-type measles viruses: 2008 Genotype

Statusa

Reference strains (MVi)b

H gene accessionc

N gene accession

A B1

Active Inactive

U03669 AF079552

U01987 U01998

B2

Active

AF079551

U01994

B3

Active

C1 C2

Active Active

L46752 AJ239133 AY047365 M81898 Z80808

L46753 AJ232203 AY043459 M89921 X84872

D1 D2 D3

Inactive Active Active

Z80805 AF085198 M81895

D01005 U64582 U01977

D4 D5

Active Active

D6 D7

Active Active

D8 D9 D10 E

Active Active Active Inactive

AF079554 L46757 AF009575 L46749 AF247202 AY043461 U29285 AY127853 AY923213 Z80797

U01976 L46758 AF079555 L46750 AF243450 AY037020 AF280803 AF481485 AY923185 X84879

F

Inactive

Z80830

X84865

G1 G2 G3 H1 H2

Inactive Active Active Active Active

Edmonston-wt.USA/54 Yaounde.CAE/12.83 ‘‘Y-14” Libreville.GAB/84 “R-96” New York.USA/94 Ibadan.NIE/97/1 Tokyo.JPN/84/K Maryland.USA/77 “JM” Erlangen.DEU/90 “WTF ” Bristol.UNK/74 (MVP) Johannesburg.SOA/88/1 Illinois.USA/89/1 “Chicago-1” Montreal.CAN/89 Palau.BLA/93 Bangkok.THA/93/1 New Jersey.USA/94/1 Victoria.AUS/16.85 Illinois.USA/50.99 Manchester.UNK/30.94 Victoria.AUS/12.99 Kampala.UGA/51.00/1 Goettingen.DEU/71 “Braxator ” MVs/Madrid.SPA/94 SSPE Berkeley.USA/83 Amsterdam.NET/49.97 Gresik.INO/17.02 Hunan.CHN/93/7 Beijing.CHN/94/1

AF079553 AF171231 AY184218 AF045201 AF045203

U01974 AF171232 AY184217 AF045212 AF045217

a

Active genotypes that have been isolated within the past 15 years WHO name; other names that have been used in the literature appear in quotation marks c Sequences available at GenBank (http://www.ncbi.nlm.nih.gov) or from WHO strain banks Reproduced from World Health Organization 2005b b

Data Reporting and Surveillance Guidelines Through the efforts of LabNet, virologic surveillance has expanded on a global scale. Information on circulating genotypes has been reported from almost every country with endemic or widespread measles. Genbank contains over 1500 partial

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D4 D4 D6 D5 C2 B3

B3 B3

D6 D6 D4

D4 B3 B2 D10 D4 B3 B3 B3 B3 B2

H1 H1

D4 D4

D5

D8 D8

D5 H2

H1 D9

D3 D3

D9, G2 G3

D4

D4 B2 D2

Fig. 7.1 Global distribution of measles genotypes: 1995–2008. Figure shows geographical distribution of measles genotypes for regions that have not yet eliminated measles transmission. Figure is adapted from the Weekly Epidemiologic record of WHO (2006). Figure is based on surveillance conducted between 1995 and 2008. The region of the Americas and Australia are not shown because these countries have eliminated measles and have detected multiple genotypes from imported cases. Because countries in the European Region have a complex pattern, only the genotypes associated with major outbreaks in 2005–2008 are indicated on the map

N gene sequences with over 80% of these sequences reported since 2000 (Rota and Tian, unpublished observations). WHO maintains a database of genotype information that is reported from national and regional laboratories within LabNet. Recently, it has become apparent that comparison of the sequence information is as important as having the genotype designation. Many genotypes contain multiple co-circulating lineages and assignment of a viral sequence to one of these lineages allows for more accurate mapping of transmission pathways. For this reason, rapid exchange of both genotype and sequence information on a global scale is critical. Databases that allow rapid exchange of sequence information are being developed by the Global Specialized Laboratories within LabNet (World Health Organization 2005a). Surveillance guidelines recommend that countries involved in measles elimination collect appropriate specimens for virus isolation from every chain of transmission, while countries involved in outbreak-control and mortality reduction obtain representative specimens from measles outbreaks (World Health Organization 2007b). It is important to conduct virologic surveillance before acce-lerated control measures are initiated so that it will be possible to study the pattern of genotypes present both before and after vaccination campaigns. The best samples for virologic surveillance are throat or nasopharyngeal swabs or urine sediments since they can be used for virus isolation as well as direct RT-

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PCR. Peripheral blood mononuclear cells are a good source of virus, but this sample requires specialized laboratory techniques and biosafety procedures that are not available throughout the laboratory network. RNA from measles virus can be detected in both oral fluid samples and dried blood spots on filter paper and these samples have been used to expand virologic surveillance when the collection and transportation of the standard samples is not logistically feasible. It is noteworthy that the United Kingdom has used oral fluid for routine measles surveillance for the last decade and greatly expanded their molecular surveillance capabilities. In all cases, it is imperative to obtain the sample for virologic surveillance as soon as possible after onset of rash to maximize chances of isolating virus or detecting RNA (World Health Organization 2007b).

Virus Isolation and Clinical Samples The Vero/hSLAM cell line is now recommended for routine isolation of measles in the WHO laboratory network. These cells are Vero cells that have been transfected with a plasmid encoding the gene for the human SLAM (signaling lymphocyteactivation molecule) protein (Ono et al. 2001). SLAM has been shown to be a receptor for both wild type and laboratory-adapted strains of measles. The sensitivity of Vero/hSLAM cells for isolation of measles virus is equivalent to that of B95a cells (Kobune et al. 1990) and measles infection of Vero/hSLAM results in the characteristic CPE, syncytium formation. The advantage of the Vero/hSLAM cells compared to B95a cells is that they are not persistently infected with Epstein-Barr virus and therefore are not considered hazardous material. This provides a significant safety advantage for laboratory workers and greatly facilitates international shipments. Vero/hSLAM cells can also be used to isolate rubella viruses from clinical samples with a sensitivity that is similar to that of standard Vero cells. Though virus isolation is encouraged, many laboratories use reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify measles RNA directly from clinical specimens. The use of real-time RT-PCR assays has greatly improved sensitivity (Hubschen et al. 2008; Hummel et al. 2006).

New Molecular Techniques Though viral genotypes are assigned based on analysis of sequence data, other techniques such as restriction fragment length polymorphism and heteroduplex mobility assays have been proposed (Kreis et al. 1997; Kremer et al. 2007; Mulders et al. 2003). However, it is doubtful that these methods would provide the level of sensitivity required to accurately map transmission pathways. Array-based techniques have been used for genotyping, but these techniques are more costly and technically challenging than standard sequencing assays (Neverov et al. 2006). A potential advantage to the array-based techniques would be the ability to rapidly obtain more sequence information from selected viral isolates or samples.

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Measles Vaccines and Antigenic Variation Sequence analyses have shown that all of the measles vaccine strains are representatives of genotype A (Parks et al. 2001a, 2001b; Rota et al. 1994a). This includes both vaccines derived from the original Edmonston isolate of 1954 (e.g., Moraten, Schwarz, Edmonston-Zagreb, AIK-C) as well as vaccines derived from other wildtype viruses isolated during the 1950s and 1960s in China and Japan (e.g., Shanghai-191, Chanchun-47, CAM-70). While this suggests that genotype A viruses may have had a wide distribution in the pre-vaccine era, it is also possible that genotype A viruses were more frequently detected because they were easier to isolate in the cell culture systems available at the time. There are few samples or viruses from the pre-vaccine era that are available for genetic analysis. However, one study detected viruses with sequences in genotypes C2 and E in samples obtained during the pre-vaccine era in Denmark (Christensen et al. 2002). Genotype A viruses have been isolated from a few sporadic measles cases in the last 10 years (Wairagkar et al. 2002), but there have been no reports that this genotype has been associated with any large outbreaks. Though it is possible that wildtype genotype A viruses are still circulating, there is a strong likelihood that the more recently detected genotype A viruses are vaccine viruses or laboratory contaminants. Efforts are underway to attempt to identify a set of genetic markers to distinguish wild-type, genotype A viruses from vaccine viruses. The detection of genetic variation within wild-type measles viruses has led to the suggestion that these viruses have antigenic characteristics that allow them to circulate more efficiently in the presence of vaccine-induced immunity. Although antigenic differences between measles viruses from the various genotypes have been detected by using monoclonal antibodies and polyvalent antiserum (Giraudon et al. 1988; Santibanez et al. 2002, 2005; Tamin et al. 1994), contemporary wild-type viruses are neutralized by polyclonal antiserum to the vaccine virus (Santibanez et al. 2005; Xu et al. 1998). More importantly, measles vaccination programs, when properly administered, have been exceptionally successful in all parts of the world irrespective of the endemic genotype of wild-type virus. Studies are in progress to explore the potential for biologic differences between measles viruses from different lineages. The publication of the structure of the H protein, the major target of neutralizing antibodies, has allowed fine mapping of the antigenic sites on the molecule (Colf et al. 2007; Hashiguchi et al. 2007). The structural model shows that one of the regions that is accessible to antibody recognition includes the SLAM binding site and conservation of these key residues may account for the monotypic nature of the virus (Hashiguchi et al. 2007). Analysis of the amino acid sequences of the measles H gene show weak, if any, evidence for selective pressure (Woelk et al. 2001).

SSPE SSPE, also called Dawson’s encephalitis, is a persistent measles infection of the central nervous system. SSPE is a progressive, invariably fatal, encephalopathy

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characterized by personality changes, mental deterioration, involuntary movements, muscular rigidity, and death. SSPE usually begins 4–10 years after the patient has recovered from naturally acquired measles. Successful isolation of measles virus from brain and lymphoid tissues of SSPE patients (Horta-Barbosa et al. 1969, 1971) clearly established measles virus as the etiologic agent of the disease. However, the introduction of the live-attenuated measles vaccine raised concerns that the vaccine virus might either cause, or in some manner influence the frequency of SSPE, and led to formation of SSPE registries in a number of countries. Epidemiologic studies demonstrated a dramatic decrease in uncomplicated measles as well as the frequency of SSPE (Halsey et al. 1980). Molecular characterization of measles virus nucleic acid sequences derived from brain biopsy or autopsy has identified wild-type measles sequences with few exceptions, and not those of the vaccine strains (see review by Campbell et al. 2007). Few genotype A virus sequences have been identified (Cattaneo et al. 1989), and it is interesting that those that have been identified were from the first few SSPE isolates ever obtained. Ironically, it was suggested that Halle, the measles isolate believed to provide direct evidence for defining measles involvement in SSPE, was likely a vaccine virus laboratory contaminant. Halle virus grew very well in cell culture, a characteristic unlike any other subsequent SSPE isolate (Rima et al. 1995a). Because genotype A wild-type viruses were widely circulating in the pre-vaccine era, it is difficult to say with certainty that Halle or any other genotype A virus was not the source of infection resulting in SSPE. Measles virus genotypes found in association with SSPE clinical specimens reflect the sequences of those viruses that circulated in the geographic region where the patients acquired natural infection. For example, the measles resurgence occurring from 1989–1992 in the United States was dominated by measles virus belonging to genotype D3. Subsequent characterization of measles sequences associated with SSPE cases over the following 5–12 years identified only genotype D3 sequences. No vaccine sequences were identified from tissues of SSPE patients, regardless of the fact that literally millions of doses of measles containing vaccine were administered over the resurgence period (Bellini et al. 2005). This and other recent studies have clearly demonstrated that measles vaccine virus is not involved in the genesis of SSPE and that the use of measles vaccine not only is beneficial in preventing acute measles, but has all but eliminated SSPE from the United States (Bellini et al. 2005; Jin et al. 2002).

Global Distribution of Measles Genotypes Virologic surveillance is now well established in all WHO regions. Though surveillance in some areas is still not adequate, a global picture has emerged (World Health Organization 2006). In general, three patterns of measles genotype distribution have been described. In countries that still have endemic transmission of measles, the majority of cases are caused by several endemic genotypes that are distributed geographically (Fig. 7.2). In these cases, multiple co-circulating lineages within the

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countries to provide baseline data (World Health Organization 2006). In countries with endemic measles, there is a geographic distribution of genotypes. Some countries have more than one endemic genotype and frequently multiple, co-circulating lineages of virus are present within a genotype, indicative of multiple chains of transmission. Although the WHO African Region has made substantial progress with reducing measles mortality since 1999 (Wolfson et al. 2007), many African countries have endemic measles and several genotypes have been detected. Clade B viruses are endemic in the central and western parts of sub-Saharan Africa, and analysis of a large number of measles isolates from Nigeria, Ghana, the Gambia, Cameroon, and Sudan has supported the division of clade B into three genotypes: B1, B2, and B3 (World Health Organization 2006; El Mubarak et al. 2002; Hanses et al. 1999; Outlaw et al. 1997; Truong et al. 2001). Genotype B3 has been divided into two clusters. Genotype B3, cluster 1 viruses have been isolated from Cameroon, Ghana, and Nigeria, and as far east as Sudan, Kenya, and Tanzania, suggesting that clade B viruses are widely distributed throughout Africa (El Mubarak et al. 2002). The circulation of genotype B3 cluster 2 viruses appears to be more limited to western Africa (Kouomou et al. 2002). Genotype B2 was considered inactive since, until recently, no representative viruses had been isolated since 1984. However, genotype B2 viruses were detected in southeastern Africa in 2002–2003 primarily in association with cases and importation from Angola and in the Central African Republic (Gouandjika-Vasilache et al. 2006; Smit et al. 2005). This reemergence of what was thought to be an inactive genotype was likely the result of surveillance gaps in some countries in central Africa. Genotypes D2 and D4 have been the most frequently detected genotypes in the southern part and eastern parts of the African continent (Kreis et al. 1997; Mbugua et al. 2003; Nigatu et al. 2001), though more recent outbreaks in Kenya, Somalia, and Tanzania have been caused by genotype B3 viruses (Rota et al. 2006). A new genotype, D10, was initially detected in Uganda in 2000 and has been detected in mostly central African countries (World Health Organization 2006; Muwonge et al. 2005). The northern African countries of Tunisia, Libya, and Algeria have detected genotype B3 presumably imported from sub-Saharan countries. Morocco has also reported continued circulation of genotype C2, once one of the prevalent genotypes in Western Europe (Alla et al. 2002, 2006; Waku-Kouomou et al. 2006). Genotype D4, the most prevalent genotype detected in the Middle East and the Arabian Peninsula (Djebbi et al. 2005), has been also reported in Egypt (2006). Measles is endemic on the Indian subcontinent. Genotype D4 and D8 viruses have been isolated in India and Nepal, and genotype D4 was detected in Pakistan (Anonymous 2001b; Truong et al. 2001; Wairagkar et al. 2002). Genotype D4 and D8 viruses have also been detected in measles cases imported into the United States from both Pakistan and India (Rota et al. 1998, 2002). Recently, genotype D7 was detected in India (Vaidya et al. 2008). This genotype had been one of the most frequently detected genotypes in Europe in the early part of the decade. The recent detection in India suggests that India may have been the original source of genotype D7 in Europe, but expanded virologic surveillance in India is needed to establish the extent of circulation of genotype D7. Extensive virologic surveillance in Japan demonstrated a succession of genotypes since surveillance activities began there in the early 1990s. Genotypes D3 and

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D5 had been co-circulating in Japan for most of the 1990s (Katayama et al. 1997; Kubo et al. 2003; Sakata et al. 1993; Takahashi et al. 2000; Yamaguchi 1997) and genotype D5 viruses had been associated with many measles cases imported from Japan (Rota et al. 1998, 2002). More recently, Japan has experienced large outbreaks associated with genotypes D9 and H1 (Kubo et al. 2002; Nakayama et al. 2003; Zhou et al. 2003). In 2007, genotype D5 was apparently reintroduced in Japan and was associated with large outbreaks (Morita et al. 2007). Elsewhere in Asia, sequence analysis of wild-type measles viruses isolated in throughout the People’s Republic of China show widespread distribution of a single genotype of virus, H1, since 1993 (Xu et al. 1998; Zhang et al. 2007). Sequence analysis of over 300 samples obtained throughout the country has established that genotype H1 is clearly the endemic genotype in China. Viruses that were indistinguishable from the Chinese genotype H1 viruses were isolated during the outbreak of measles in Korea during 2000–2001 (Na et al. 2001, 2003) and were associated with imported cases in a number of countries. However, endemic circulation of geno type H1 appears to be restricted to China. Wild-type measles viruses in Vietnam are also classified as clade H, but they are sufficiently different from the Chinese viruses to be designated as a separate genotype, H2 (Liffick et al. 2001). It is interesting to note that wild-type measles viruses isolated in Thailand and Cambodia are in genotype D5 (Horm et al. 2003; Rota et al. 1998) and more closely related to viruses circulating in Japan than to viruses circulating in other parts of Asia. Until 1997, the only measles viruses representing clade G had been isolated in 1983, and this clade was considered to be inactive. In 1997, a virus belonging to clade G was isolated from an Indonesian child who was being treated at a Dutch hospital. The H and N sequences of this virus were sufficiently different from the reference strain for it to be considered a new genotype (G2) within clade G (de Swart et al. 2000). Viruses belonging to genotype G2 have recently been isolated in Indonesia and Malaysia (Rota et al. 2000). In addition, viruses from genotypes G2 and G3 have been isolated in Indonesia and East Timor. RT-PCR and sequence analyses of clinical specimens obtained from measles cases imported into Australia confirmed that G3 was present in East Timor. Genotypes G2, G3, and D9 appear to be the endemic genotypes in Indonesia, East Timor, and possibly Malaysia (Chibo et al. 2002). In some cases, the circulation of a genotype in a particular country has not been verified by virologic surveillance in the source country but was inferred based upon a consistent pattern of importations. For example, although genotype D3 viruses have never been isolated in the Philippines, there have been several instances of genotype D3 being detected in measles cases imported into the United States from the Philippines (Rota et al. 1996, 1998, 2002). In each of these cases, standard case investigation confirmed that the individuals were traveling in the Philippines during the incubation period. Because of relatively low vaccination coverage rates in some countries and the constant importation of measles from endemic regions in Asia and Africa, European countries have frequent measles outbreaks. Europe has also experienced rapid change in circulating genotypes. In the early part of this decade, genotypes C2, D6, and D7 were most frequently detected (Hanses et al. 2000; Korukluoglu et al. 2005; Rima

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et al. 1995b; Sakata et al. 1993; Santibanez et al. 1999, 2002). Genotype D6 may be endemic in Turkey (Korukluoglu et al. 2005; Korukluoglu and Zarakolu 2006). In 2005–2006, there were major measles epidemics associated with genotypes B3, D6, and D4, and these genotypes were associated with measles cases exported from Europe to other parts of the world (Korukluoglu and Zarakolu 2006; Kremer et al. 2008). Genotype D6 was associated with a larger outbreak in the Ukraine, which fueled spread cases and small outbreaks throughout Europe, while genotype D4 was detected in a large outbreak in Romania. The decreased diversity of the genotype D6 viruses along with the rapid disappearance of the previously circulating C2 and D7 viruses suggest that vaccination programs had successfully interrupted several chains of transmission. However, measles virus was reintroduced by importation and spread via highly mobile, unvaccinated communities (Kremer et al. 2008).

Measles Elimination Endemic transmission has been eliminated in many areas of the world, including the countries in the Western hemisphere. Both virologic and epidemiologic data collected in the United States between 1989 and 2000 indicated that interruption of transmission of the genotype D3 viruses that were associated with the measles resurgence of the early 1990s was achieved in 1993 and subsequently maintained (Rota et al. 1996, 1998). Analysis of viruses isolated from measles cases and outbreaks in the United Stated between 1994 and 2007 failed to detect ongoing transmission of an endemic genotype. Rather, the diversity of genotypes detected in the last 15 years is indicative of multiple, imported sources of virus (Rota et al. 1998, 2002). Likewise, the diversity of genotypes detected in Australia and Canada, and the United Kingdom is similar to that observed in the United States, suggesting frequent importation of measles and lack of endemic circulation of virus (Chibo et al. 2000, 2003; Jin et al. 1997; Ramsay et al. 2003; Rota et al. 1998, 2002). However, gaps in the vaccination program may have allowed for re-establishment of endemic transmission of measles in the United Kingdom (Asaria and MacMahon 2006). Though virologic surveillance has improved recently in South America, there is no record of the endemic genotypes that circulated before PAHO launched its very successful measles elimination efforts in the early 1990s. However, molecular epidemiologic studies have demonstrated interruption of circulation of genotype D6 viruses that were responsible for the large measles outbreak in Sao Paulo in 1997 and subsequent outbreaks in Rio de Janeiro, Argentina, Chile, Bolivia, Haiti, and the Dominican Republic (Barrero et al. 2000; Baumeister et al. 2000; Canepa et al. 2000; Hersh et al. 2000; Oliveira et al. 2002; Siqueira et al. 2001). The record low number of cases and the identification of genotypes other than D6 in association with measles cases imported into South and Central America are consistent with regional elimination (Hersh et al. 2000). During 2002, indigenous transmission in the Americas was limited to a large outbreak (>2000 cases) that started in Venezuela and spread to Colombia. Viruses isolated in Venezuela were found to be members of what was a previously

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unknown genotype, D9 (Anonymous 2002). At the time, genotype D9 was considered to be a previously undetected endemic genotype in the Americas and its discovery cast doubts on the assertion that measles had been eliminated from the region. However, shortly after the outbreaks in Venezuela and Colombia, genotype D9 viruses were found to be circulating in Java, Indonesia and were associated with measles cases imported into Australia (Chibo et al. 2003). Since genotype D9 was found to be circulating in a country with endemic measles, the more likely scenario was that genotype D9 viruses were imported into Venezuela from an unidentified index case (Anonymous 2003).

Measles Reintroduction Laboratory studies have estimated that the mutation rate of measles virus is similar to those of other RNA viruses (Schrag et al. 1999). However, the high level of genetic stability of measles field isolates was also noted by sequencing viruses from the same genotype that had been isolated several years apart (Rima et al. 1997). In addition, molecular epidemiologic studies of the measles resurgences in Brazil in 1997 (Oliveira et al. 2002) and the United States in 1989–1991 (Rota et al. 1996) suggest that there is very little sequence variation in the N and H genes within a single chain of measles transmission. Nucleotide sequences from the N genes of viruses isolated during the large outbreak in Sao Paulo, Brazil, in 1997 were nearly identical to the sequences obtained from viruses that had spread to other states in Brazil as well as other South American countries during 1997 and 1998 (Oliveira et al. 2002). The genotype D3 viruses that were isolated during the resurgence of measles in the United States during 1989–1991 showed very little sequence variability in both the H and N genes, suggesting that one strain of virus had seeded the entire country (Rota et al. 1996). Even in areas with recent endemic transmission of virus, there appears to be very little sequence variation present in viruses isolated from the same chain of transmission. For example, there was very little sequence heterogeneity in viruses obtained during outbreaks that occurred after a mass vaccination campaign in Burkina Faso, suggesting that a single introduction of virus was responsible for the outbreaks (Mulders et al. 2003). This is in contrast to the pattern observed in measles-endemic areas, which shows much more sequence variation within a genotype because the epidemiologic conditions favor maintenance of multiple chains of transmission. Therefore, measles vaccination programs can reduce the number of co-circulating chains of transmission and eventually interrupt measles transmission. However, viruses are continually being introduced from external sources, and as the number of susceptible individuals increases, sustained transmission of the newly introduced viral genotype is possible. The result of introduction into regions where vaccination programs have failed to maintain a high level of population immunity are apparent rapid changes in the endemic genotype as observed in European countries and/or circulation of viruses with very limited genetic diversity (Kremer et al. 2008; Mulders et al. 2003; Rima et al. 1995b; Santibanez et al. 2002).

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Mapping Transmission Pathways Molecular epidemiologic studies of measles virus have taught us several valuable lessons about transmission of the virus. One of these lessons is that if large measles outbreaks are occurring anywhere in the world, the viruses are soon detected throughout the world. The lineage of genotype B3 viruses that were associated with a large outbreak in Kenya in 2005 were soon detected throughout Europe, the United States, Canada, and Mexico (Fig. 7.3) (Rota et al. 2006). Likewise, there were a number of internationally spread cases and some small outbreaks that could be linked to the large outbreaks in Romania and Ukraine in 2006 (Kremer et al. 2008). Another lesson is that measles transmission can occur anywhere and that molecular techniques are often the only method for identifying the source of an outbreak or case. Exposures can occur in airports or other areas frequented by international travelers such as amusement parks. In 2005, sequence information was used to link cases that occurred in the Netherlands to an exposure in an airport in the United States (Rota et al. 2006), while in 2007 sequence data were used to link cases that occurred in Texas and Michigan to an imported case at an international youth sporting event in Pennsylvania (Anonymous 2008a). In 2008, measles cases were imported into the United States primarily from large outbreaks associated with genotypes D5 and D4 in Europe (Anonymous 2008b). Of course, there are limitations to the ability to map transmission pathways. Molecular studies can confirm independent sources of infection if different genotypes or clearly distinct lineages are detected. However, if viruses from the same lineage are detected in nonlinked cases in a particular country, the molecular data alone may not be able to differentiate between continuous circulation of virus and multiple introductions from the same source.

Challenges Genetic characterization of wild-type measles viruses provides a means to study the transmission pathways of the virus and is an essential component of laboratorybased surveillance. Virologic surveillance needs to be expanded in all areas of the world and conducted during all phases of measles control. The greatest challenge to expanding virologic surveillance for measles is to collect and transport specimens in a timely and efficient manner. The importance of obtaining samples for viral detection at first contact with the suspected case cannot be understated. In addition, local healthcare workers must have the proper supplies and training needed to obtain, process, and ship samples. This is particularly challenging in developing countries with inadequate infrastructure. The targeted introduction of new sampling techniques such as collection of dried blood on filter paper or oral fluid will help to expand virologic surveillance into remote areas (World Health Organization 2008). The WHO Laboratory Network is expanding the capacity of the national and regional laboratories for virus isolation and viral detection. The Vero/hSLAM cell line has been distributed through the network.

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Fig. 7.3 a, b Transmission of measles genotype B3 in 2005 (adapted from Rota et al. 2006). In A, the dendrogram shows the relationships among the measles reference strains representing the 23 known measles genotypes. B shows the phylogenetic analysis of nucleoprotein genes (450 nucleotides) of measles viruses from cases in the United States, Mexico, the Netherlands, Canada, Germany, and Kenya during 2005–2006. The unrooted tree includes sequences from cases in Ivory Coast in 2004 and Benin and Nigeria in 2005 as well as selected B3 sequences available from GenBank for comparison. *Collected from the Dagoretti area of Nairobi; the other Nairobi sequences were from cases in the Eastleigh area. The insert to the right highlights the transmission pathways. Note that no epidemiologic link was established with western Africa (dotted line). A separate importation of genotype B3 with a link to Spain occurred in Venezuela

Another significant challenge is the development of protocols for rapid exchange of sequence information. Timely reporting and dissemination of the genotype information will enhance our ability to track transmission of measles and to evaluate the success of vaccination programs. The planned development of databases and web sites will permit near real-time exchange of genetic information and benefit both laboratorians and public health officials.

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Siqueira MM, Castro-Silva R, Cruz C, Oliveira IC, Cunha GM, Mello M, Rota PA, Bellini WJ, Friedrich F (2001) Genomic characterization of wild-type measles viruses that circulated in different states in Brazil during the 1997 measles epidemic. J Med Virol 63:299–304 Smit SB, Hardie D, Tiemessen CT (2005) Measles virus genotype B2 is not inactive: evidence of continued circulation in Africa. J Med Virol 77:550–557 Takahashi M, Nakayama T, Kashiwagi Y, Takami T, Sonoda S, Yamanaka T, Ochiai H, Ihara T, Tajima T (2000) Single genotype of measles virus is dominant whereas several genotypes of mumps virus are co-circulating. J Med Virol 62:278–285 Tamin A, Rota PA, Wang ZD, Heath JL, Anderson LJ, Bellini WJ (1994) Antigenic analysis of current wild type and vaccine strains of measles virus. J Infect Dis 170:795–801 Taylor MJ, Godfrey E, Baczko K, ter Meulen V, Wild TF, Rima BK (1991) Identification of several different lineages of measles virus. J Gen Virol 72:83–88 Truong AT, Mulders MN, Gautam DC, Ammerlaan W, de Swart RL, King CC, Osterhaus AD, Muller CP (2001) Genetic analysis of Asian measles virus strains – new endemic genotype in Nepal. Virus Res 76:71–78 Vaidya SR, Wairagkar NS, Raja D, Khedekar DD, Gunasekaran P, Shankar S, Mahadevan A, Ramamurty N (2008) First detection of measles genotype D7 from India. Virus Genes 36:31–34 Wairagkar N, Rota PA, Liffick S, Shaikh N, Padbidri VS, Bellini WJ (2002) Characterization of measles sequences from Pune India. J Med Virol 68:611–614 Waku-Kouomou D, Alla A, Blanquier B, Jeantet D, Caidi H, Rguig A, Freymuth F, Wild FT (2006) Genotyping measles virus by real-time amplification refractory mutation system PCR represents a rapid approach for measles outbreak investigations. J Clin Microbiol 44:487–494 Woelk CH, Jin L, Holmes EC, Brown DW (2001) Immune and artificial selection in the haemagglutinin (H) glycoprotein of measles virus. J Gen Virol 82:2463–2474 Wolfson LJ, Strebel PM, Gacic-Dobo M, Hoekstra EJ, McFarland JW, Hersh BS (2007) Has the 2005 measles mortality reduction goal been achieved? A natural history modelling study. Lancet 369:191–200 World Health Organization (1998) Standardization of the nomenclature for describing the genetic characteristics of wild-type measles viruses. Wkly Epidemiol Rec 73:265–272 World Health Organization (2001a) Nomenclature for describing the genetic characteristics of wild-type measles viruses (update) Part I. Wkly Epidemiol Rec 76(32):242–247 World Health Organization (2001b) Nomenclature for describing the genetic characteristics of wild-type measles viruses (update) Wkly Epidemiol Rec 76(33):249–251 World Health Organization (2003) Update of the nomenclature for describing the genetic characteristics of wild-type measles viruses: new genotypes and reference strains. Wkly Epidemiol Rec 78(27):229–232 World Health Organization (2005a) Global Measles and Rubella Laboratory Network – update. Wkly Epidemiol Rec 80(44):384–388 World Health Organization (2005b) New genotype of measles virus and update on global distribution of measles genotypes. Wkly Epidemiol Rec 80(40):347–351 World Health Organization (2005c) Standardization of the nomenclature for genetic characteristics of wild-type rubella viruses. Wkly Epidemiol Rec 80(14):126–132 World Health Organization (2006) Global distribution of measles and rubella genotypes – update. Wkly Epidemiol Rec 81(51/52):474–479 World Health Organization (2007a) Update of standard nomenclature for wild-type rubella viruses 2007. Wkly Epidemiol Rec 82(24):216–222 World Health Organization (2007b) Manual for the diagnosis of measles and rubella infection, 2nd edn. World Health Organization, Geneva World Health Organization (2008) Measles and rubella laboratory network: 2007 meeting on the use of alternative sampling techniques for surveillance. Wkly Epdemiol Rec 83:225–232 Xu W, Tamin A, Rota JS, Zhang L, Bellini WJ, Rota PA (1998) New genetic group of measles virus isolated in the People’s Republic of China. Virus Res 54:147–156

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Chapter 8

Human Immunology of Measles Virus Infection D. Naniche

Contents Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology of Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Natural History of Clinical Measles Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immune Response to Natural Measles Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of Immunity Involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Humoral Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunogenetic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Longevity and Measles Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Immunity in Infants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Immunity in Immunocompromised Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Measles is a highly contagious disease, which was responsible for high infant mortality before the advent of an effective vaccine in 1963. In immunocompetent individuals, measles virus (MV) infection triggers an effective immune response that starts with innate responses and then leads to successful adaptive immunity, including cell-mediated immunity and humoral immunity. The virus is cleared and lifelong protection is acquired. However, changing epidemiology of measles due to vaccination as well as severe immunodeficiency has created new pockets of individuals vulnerable to measles. This chapter reviews the knowledge on effective measles-specific immune responses induced by natural infection and vaccination and explores problems arising in specific cases of immunodeficiency, infant immunity, and ineffective vaccination against measles.

D. Naniche Barcelona Center for International Health Research (CRESIB), Hospital Clinic, Institut d’Investigacions Biomedicas August Pi i Sunyer (IDIBAPS), C/Rossello 132, 4 08036, Barcelona, Spain, e-mail: [email protected].

D.E. Grippin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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Abbreviations MV IL IFN TNF TLR Th1 Th2 CTL PBMC HIV HAART

Measles virus Interleukin Interferon Tumor necrosis factor Toll-like receptors T-helper type 1 T-helper type 2 Cytotoxic T cell Peripheral blood mononuclear cells Human immunodeficiency virus Highly active antiretroviral therapy

Introduction Before the development of an effective vaccine in 1963, measles was responsible for a high level of mortality throughout the world. Essentially a rite of passage during childhood, those who survived measles infection acquired a lifelong immunity to measles in the absence of re-exposure to the virus. The landmark study of measles in the Faroe Island population during the 1846 epidemic established the basic principles of measles epidemiology, transmissibility, incubation period, and lifelong immunity. Measles entered an isolated island population that had not had an measles epidemic in 65 years. The attack rate was nearly 100% for susceptible individuals, but all persons over 65 years of age were protected by immunity from the previous outbreak (Panum 1938). Since this crucial observation regarding measles immunology, great advances have been made in the understanding of immune responses to natural measles infection and to the licensed measles vaccine; however, many fundamental questions still remain unanswered.

Epidemiology of Measles The advent of an effective attenuated measles vaccine in the 1960s and worldwide generalization of vaccination through the expanded program of immunization (EPI) in the following decades led to a dramatic decrease in measles-related mortality. In the early 1960s, over 135 million cases of measles and over 6 million measlesrelated deaths were estimated to have occurred yearly (Clements and Hussey 2004). Through intensive international efforts scaled up in the late 1990s to diversify immunization strategies with a goal of reducing the mortality due to measles, the annual number of measles deaths was reduced by 60% between 1999 and 2005 from an estimated 873,000 (634,000–1,140,000) to 345,000 (247,000–458,000) (Centers

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for Disease Control 2006; Wolfson et al. 2007). Despite high vaccine coverage levels, a small pool of susceptible individuals is sufficient for measles to occur in the form of sporadic outbreaks spaced further and further apart in time as vaccination coverage is increased. Thus occasional clusters of cases in industrialized countries and outbreaks in endemic countries occur. This is mainly due to the clusters of susceptible individuals remaining as a result of heterogeneous worldwide vaccine coverage, a 2%–5% vaccine failure, and increased international travel. Measles case fatality ratios (CFRs) vary depending on the age of infection, the nutritional status, and access to healthcare. Measles CFR was historically as high as 30% and currently may be as low as 1 in 1000 in industrialized countries, 5%– 10% in endemic areas in sub-Saharan Africa, and may reach 25% in refugee camps or other situations of overcrowding (Aaby 1988; Aaby and Clements 1989; Cutts et al. 1991; Moss 2007). Prior to mass vaccination, major epidemics occurred approximately every 2–3 years, and the highest occurrence of the disease was in children, with the highest risk of complications and death in children younger than 5 years of age and in adults over 20 years of age (Perry and Halsey 2004). In the United States, it was estimated that more than 90% of individuals had contracted measles by the age of 15 (Langmuir 1962). Vaccination dramatically changed the epidemiology of measles by shifting the peak age of infection to older children. Vaccination also shifted the burden of disease out of the age group with the highest case-fatality (infancy) and thus contributed to reducing measles-associated mortality (Moss 2007). However, this changing epidemiology created new pockets of vulnerability to measles in partially protected or unprotected infants below immunization age, and in the young adult populations with waning vaccine-induced immunity (Anders et al. 1996).

Natural History of Clinical Measles Infection Measles virus is a highly contagious disease transmitted from person to person by respiratory droplets or airborne spray to mucous membranes in the upper respiratory tract. Measles usually presents clinically with prodromal symptoms of fever, cough, and conjunctivitis occurring 10–12 days after exposure and lasting 2–4 days prior to the appearance of a characteristic rash and high fever (Griffin 2007). The virus initially spreads to the regional lymphoid tissue, where the virus undergoes extensive replication and establishes a primary viremia allowing spread of the virus to other lymphoid tissue including spleen and liver. A second phase of viremia occurs approximately 5–7 days after exposure accompanied by characteristic lymphopenia and dissemination of virus to multiple organs and epithelium. The rash marks the onset of the immune response to measles, coinciding with the appearance of measles-specific humoral and cellular immunity. The uncomplicated clinical course of measles accompanied by an effective immune response leads to decreased symptoms and clearance of detectable virus by 7–10 days after initiation of the rash. However,

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the transient immunosuppression caused by measles can lead to severe complications including otitis media, laryngotracheobronchitis, pneumonia, diarrhea, febrile seizures, and encephalitis (reviewed in Perry and Halsey 2004). In a vaccinated population, children under 5 years of age, adults over 20 years, as well as malnourished and immunocompromised individuals are at greater risk of serious complications (reviewed in Perry and Halsey 2004). The high complication rate and mortality historically associated with measles is in large part due to opportunistic infections that arise during measles-induced immunosuppression. Measles can also cause a rare fatal degenerative persistent central nervous system disease called subacute sclerosing panencephalitis (SSPE). SSPE occurs 5–15 years after measles infection in 1 per 100,000 cases of measles and results in motor disabilities, coma, and death (Jabour et al. 1969; Termeulen et al. 1983).

Immune Response to Natural Measles Infection Types of Immunity Involved In immunocompetent individuals, measles (MV) infection elicits a strong measlesspecific immune response that clears infection. Concomitantly, MV is responsible for a generalized immunosuppression that dampens immune responses to other pathogens. Much research has been dedicated to the immunological paradox by which MV-specific immune activation co-exists with nonspecific immune suppression. The mechanisms of MV-induced immunosuppression are discussed in chapter 12 of this publication and the present chapter further discusses MV-specific immune responses. The first immune response to be activated by many viral infections, including MV, is the innate immune response. Specific receptors including the Toll-like receptors (TLRs) of the innate immune system detect the presence of viruses and trigger production of proinflammatory cytokines, interferons, and TNF. However, many viruses have developed strategies to subvert these innate responses and pursue their replication. Measles infection triggers the activation of TLRs, type I interferons, cytokines, and RNA-binding proteins (Berghall et al. 2006; Bieback et al. 2002). Wild-type measles has been shown to activate TLR2, which leads to the activation of TLR responsive genes in macrophages (IL-1, IL-6, IL-12p40), thus contributing to immune activation and to the start of the adaptive immune response (Bieback et al. 2002). However, wild-type measles has been shown to induce very low levels of type I IFN and possesses mechanisms to suppress type I IFN production (Naniche et al. 2000; Shingai et al. 2007) and signaling (Fontana et al. 2008; Palosaari et al. 2003; Shaffer et al. 2003; Takeuchi et al. 2003). In parallel to activation of TLRs, MV can suppress cytokine production due to activation of TLRs such as TLR4activated IL-12 production (Hahm et al. 2007). IL-12 is thought to be a cytokine that participates in linking the innate and adaptive immune responses and its

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production can be inhibited by MV both in vitro and in vivo (Atabani et al. 2001; Karp et al. 1996; Polack et al. 2002). This reflects the generalized immunosuppression induced by MV, which impairs responses to other pathogens (discussed in chapter 12). The immune correlates of protection against measles have been defined but the relative contributions of nonspecific innate immunity and specific cellular and humoral immunity are not completely understood. The humoral arm of the immune response has been the prime focus for determining vaccine efficacy and assessing protection against measles. However, several experiments of nature and studies in monkey models have demonstrated that cell immunity is crucial for recovery from measles infection (Markowitz et al. 1988; Mitus et al. 1962; Permar et al. 2003). Children with agammaglobulinemia have a normal course of measles infection and clear the virus with no increased complications, whereas children with cellular immune deficiencies, such as leukemia, are unable to clear measles virus infection and develop progressive illness and death from measles (Burnet 1968; Good and Zak 1956; Markowitz et al. 1988). Furthermore, in CD8-lymphocyte-depleted rhesus macaques, infection with measles virus leads to more extensive viremia, disease, and death (Permar et al. 2003, 2004).

Humoral Immunity Measles-specific antibody is first detectable at the onset of the rash and titers rise rapidly thereafter (Bech 1959). Detailed kinetic studies have shown that 77% of individuals develop measles-specific IgM within 72 h after rash onset. By 11 days, 100% have detectable IgM and over 90% maintain IgM for 28 days (Helfand et al. 1997, 1999). IgG responses are generated shortly thereafter, peak at 3–4 weeks, and are lifelong (Stokes 1961). Antibody is induced to most MV proteins but appears first and most abundantly to the nucleocapsid (N) protein (Norrby and Gollmar 1972) and then to the hemagglutinin (H) and fusion (F) proteins and very little to the matrix (M) protein (Norrby et al. 1981; Stephenson and ter Meulen 1979). Measles is a monotypic virus and thus is considered to have one serotype and be subdivided into various genotypes. Thus a polyclonal measles-specific serum can neutralize all strains of measles (Bellini et al. 1994; Bellini and Rota 1998). The majority of neutralizing antibodies are specific for the H protein, although there are F-specific antibodies that neutralize MV (Black 1989; Malvoisin and Wild 1990). Measles-specific antibody can be either neutralizing or non-neutralizing. The gold standard for measuring protective neutralizing antibodies to measles is by the plaque reduction neutralizing (PRN) assay, although other ELISA and hemagglutination-inhibition assays are also used (Griffin 2007; Ratnam et al. 1995). Based on studies in outbreaks in partially vaccinated populations, a specific neutralizing antibody titer has been assigned by WHO at 200 mIU/ml as a necessary level conferring protection to measles (Chen et al. 1990; Samb et al. 1995; World Health

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Organization 1993). Quantitative aspects of humoral immunity are well characterized and there is growing information that the quality of the antibody response, including factors such as IgG subclass, complement-fixing capacity, and antibody avidity, is also important for protection. Isotype selection arises from recombination of IgG heavy chain exons resulting in antibodies containing Fc regions with different effector functions, including capacity for complement activation and opsonization (Paul 2003). IgG1 and IgG3 have been found to be the predominating antibodies elicited during acute measles, similar to other respiratory viral infections. During the convalescent phase of measles and long-term humoral memory, IgG1 and then IgG4 appear to remain relatively high, whereas IgG3 decreases (El Mubarak et al. 2004). MV neutralization activity is primarily mediated by Hspecific IgG1 and IgG3 isotypes with little contribution from IgG2 and IgG4 (Audet et al. 2006). Complement-activating capacity is primarily mediated by IgG4 (Paul 1989). Another parameter of the qualitative antibody response is an antibody’s avidity for antigen, which stems from somatic hypermutation of the variable region of the IgG (responsible for epitope recognition), leading to the process of affinity maturation, whereby antibody-producing cells secrete antibodies with progressively higher affinities for their antigen (Paul 2003). There is increasing evidence that high antibody avidity is correlated with protection or better prognosis for several viral infections and vaccine responses (Bouche et al. 2002; Griffin 2007). Measles-specific IgG undergo affinity maturation after infection, which appears to increase throughout the first 3 months after infection (El Mubarak et al. 2004). It has been shown that improper measles vaccine design can lead to short-lived inadequate antibody responses. This was the case of the formalin-inactivated measles vaccine in use in the United States between 1963 and 1968 and withdrawn because of poor protection and atypical measles syndrome upon exposure to natural measles infection (Fulginiti et al. 1967). It was demonstrated that this formalin-inactivated vaccine induced production of non-neutralizing antibodies with low-affinity and high complement-fixing capacity. This process resulted in immune complex-mediated disease leading to atypical measles pathology upon natural measles infection. This example thus highlights the importance of the quality of the antibody response in conferring protection to re-infection (Polack et al. 2003). MV-specific neutralizing antibodies correlate with protection at the time of virus exposure and play a key role in preventing measles infection following natural infection, vaccination, or passive transfer of antibodies (Albrecht et al. 1977; Black 1989; Chen et al. 1990; Halsey et al. 1985; Panum 1938). However, the role of antibodies in clearance of MV once the infection is initiated remains unclear. Studies in non-human primates have suggested that humoral immunity plays a minimal role in clearance of replicating MV (Permar et al. 2003, 2004). However, others suggest that antibodies may contribute and/or accelerate the clearance of measles infection mainly driven by the cellular immune responses (Forthal et al. 1994; Fujinami and Oldstone 1979, 1980).

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Cellular Immunity Early work has shown evidence of detectable T cell activity during the measles rash when plasma levels of IFN-gamma and soluble IL-2 receptor increase (Griffin et al. 1990). The cellular immune response has been shown to bloom at the onset of the rash as plasma levels of soluble CD8 and beta-2 microglobulin increase (Griffin et al. 1989, 1990, 1992) and measles-specific cytotoxic T cells are detected (Jaye et al. 1998; Nanan et al. 1995; Nanan et al. 2000; Vanbinnendijk et al. 1990). Acute measles has been described to generate a T-helper type 1 (Th1) response profile, including IFN-gamma and IL-2, whereas the convalescent phase of MV infection appears to be skewed towards T-cell helper type 2 (Th2) responses, including the type 2 cytokines IL-4 and IL-5 (Griffin and Ward 1993; Griffin et al. 1989, 1990). In patients with measles infection, from both industrialized and developing countries, type 2 cytokine skewing has been shown by abundant production of IL-4, presence of IL-10, and little IFN-γ after in vitro stimulation with T cell mitogens (Griffin and Ward 1993; Moss et al. 2002; Ward et al. 1991). Furthermore, MV has been shown to suppress mitogen-induced IL-12 production after in vitro infection and during measles in humans and in rhesus macaques (Atabani et al. 2001; Karp et al. 1996; Polack et al. 2002). Great advances in cellular immunology have shown that T cell immune responses are complex and involve various phases, including the effector phase, effector memory phase, and central memory phase. Effector cell types are also more heterogeneous than initially thought and encompass capacities for cytotoxicity, cytokineproduction, suppression, proliferation, and differential homing (Reiner et al. 2007). Studies have confirmed that after in vitro restimulation with measles antigens, IFNγ-producing T cells can be detected during acute measles and measles vaccination, confirming the existence of T cells with a Th1 profile during the acute response (Gans et al. 1999; Ovsyannikova et al. 2003). At later phases, months to years after natural infection or vaccination, restimulation of peripheral blood mononuclear cells (PBMCs) with MV antigens detects measles-specific T cells that can produce IFN-γ or IL-4 (Dhiman et al. 2005a; Howe et al. 2005; Nanan et al. 2000; Ovsyannikova et al. 2003). One study detected a predominance of IFN-γ-producing cells with a small percentage of individuals who exclusively produced IL-4 after MV stimulation (Dhiman et al. 2005b). Early studies specifically assessed Th1/Th2 polarization during and after measles infection or vaccination with relation to immunosuppression of responses to other non-MV antigens and nonspecific stimulation rather than as a study of the polarization of MV-specific responses. Measlesinduced Th2 polarization may thus be a manifestation of measles-induced immunosuppression occurring later in the course of infection after an effective MVspecific response has been initiated. Thus Th2 skewing may not necessarily affect an uncomplicated immune response to natural measles infection or vaccination. The kinetics of Th1/Th2 polarization during and after measles infection or vaccination

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may depend on the stage of the T cell response (acute, memory), on methods of antigen restimulation used for detection in vitro, and/or on the type of memory cell detected (effector or central memory). At a given time postinfection or vaccination, antigen-specific effector and memory T lymphocytes are present at many differentiation stages ranging from the uncommitted Th0 cells to pre-Th2/pre-Th1 and committed cells (Lanzavecchia and Sallusto 2005). Both CD8 and CD4 cells specific for various measles proteins and specific epitopes have been described in murine and non-human primate models (reviewed in van Els and Nanan 2002). All main structural MV proteins are recognized by T cells in the context of either MHC class I or class II molecules. A number of these epitopes have been confirmed in humans during natural infection or vaccination, including epitopes in the H, M, and C proteins (Jaye et al. 2003; Ota et al. 2007; van Els and Nanan 2002). Since HLA-A2 is an abundant class I histocompatibility antigen, many epitopes have been characterized for this human class I molecule (Jaye et al. 2003; Ota et al. 2007). There is no gold standard for measuring cellular immunity to measles and the diverse techniques used include ex vivo cytotoxicity assays that detect ongoing immune responses, as well as in vitro restimulation with MV antigens followed by cytotoxicity assays with ELISPOT or flow cytometry to detect memory cytokine-producing CD8 and/or CD4 T cells. The variation of techniques has introduced a degree of difficulty in comparing studies and in drawing conclusions regarding the correlates of protective immunity to measles infection both during acute infection and for long-term memory. Furthermore, it is not clear whether natural measles-induced T cell responses differ from vaccineinduced T cell responses in terms of the quantity, quality, and longevity of the T cell response.

Immunogenetic Factors Genetic factors play a role in the immune response and disease progression in various viral infections such as hepatitis B, West Nile, EBV, herpes, and HIV, as well as in vaccine responses (Helminen et al. 2001; Hurme et al. 2003; Kaslow et al. 2005; Thio et al. 2003; Yakub et al. 2005). Genetic polymorphisms that could potentially have an impact on immune response to measles infection and/or to vaccination are present in many steps of the immune response, including T cell recognition, cell signaling, cytokine production, antibody isotype switching, and many others. Several studies have assessed polymorphisms in HLA genes, cytokine, cytokine receptor genes, and MV receptor genes (SLAM and CD46) and their potential association with parameters of cellular and humoral measles immune response after measles vaccination. Associations have been identified between HLA class I and class II alleles and levels of measles-specific IFN-γ and IL-4 production (Ovsyannikova et al. 2005a; Ovsyannikova et al. 2005b). Other associations have been identified between genetic polymorphisms in IL-2, IL-10, and IL-12b receptor genes and levels of

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measles-specific antibody production and lymphoproliferation (Dhiman et al. 2007a; Ovsyannikova et al. 2006). Furthermore, polymorphisms in measles SLAM and CD46 have been found to be associated with lower measles-specific antibody responses when measured at a median of 4.8 years after a second dose of measles vaccine (Dhiman et al. 2007b). These studies have been conducted in children years after having received one dose or two doses of measles vaccine and living in areas with very little to no natural boosting from outbreaks of measles. It is not known whether these polymorphisms only play a role in immune memory or are also applicable to the initial immune response upon vaccination. It is likely, although unknown, that these polymorphisms and others can be extrapolated to the immune response after natural measles infection. Genetic polymorphisms could thus impact the efficiency, breadth, and the quality of the measles-specific immune response, which could have consequences on viral replication, transmission, and disease severity. Recently, gene expression array studies during acute measles virus infection have been conducted and have identified genes potentially involved in immunological changes both in PBMCs and in dendritic cells (Zilliox et al. 2006, 2007). Many genes were observed to be up- or downregulated during measles infection and the 1st month of follow-up. Genes included inflammatory cytokines and their receptors, chemokines and their receptors, cell surface adhesion, signaling, and transcription regulation. These studies open interesting areas of research into the immunogenetics involved in the ability to control acute measles virus infection, including in immunosuppressed patients.

Longevity and Measles Vaccination As shown by the Faroe Island example, measles immunity after natural infection is lifelong in the absence of natural boosting. However, it appears that measlesvaccine induced protective immunity after vaccination with a single dose may not be lifelong and it is yet unknown whether two doses of the vaccine can elicit lifelong immunity. Several studies have evaluated the duration of measles-specific T cell immunity after vaccination. The cytotoxic T lymphocytes (CTLs) elicited immediately after vaccination are indistinguishable from those induced after natural measles in terms of lysis, restimulation capacity, and recognition of measles proteins (Jaye et al. 1998). Short-term prospective studies have analyzed T cell responses up to 8 months after vaccination and showed that PBMCs from infants vaccinated at 6, 9, and 12 months of age were able to proliferate and secrete cytokines (IL-2 and IFN-γ) in response to in vitro measles stimulation assessed at 3 months postvaccination (Gans et al. 1999). T cell proliferative capacity has also been detected in subjects up to 15 years after vaccination (Bautista-Lopez et al. 2000; Toyoda et al. 1999). A study conducted in the United States suggested that CTL activity might be weaker in vaccinated individuals 16 years postvaccination as compared to those having had natural measles over 23 years earlier (Wu et al.

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1993). Another study showed that both measles-specific CD4 and CD8 T cells could be detected up to 34 years after vaccination and up to 47 years after natural measles infection. It was also suggested that vaccine-induced measles-specific CD4 cells may wane over time (Naniche et al. 2004). There are a greater number of studies assessing the waning of measles-specific antibody after vaccination than the waning of cell immunity including studies assessing measles-specific antibody titer years after a single dose. Studies have documented both stable and waning titers of vaccine-induced measles-specific antibody and models have been established to estimate the decay rate of MV-vaccine-induced antibody titer (Bautista-Lopez et al. 2001; Dai et al. 1991; Lee et al. 2001; Lee and Nokes 2001; Mossong et al. 2000; Weibel et al. 1980; Whittle et al. 1999). Two studies estimated the half-life of anti-MV antibody in two different geographic regions: 12 years in Canada and 5.5 years in Taiwan (Lee and Nokes 2001; Mossong et al. 2000). It is likely that the longevity of vaccine-induced humoral immunity may differ according to genetic, demographic, and environmental factors as well as to the immunocompetence status of individuals.

Measles-Specific Immunity in Infants Infant immunity to measles is a special situation whereby immunity depends on the presence and transfer of maternal antibody to the infant. Levels of transplacentally acquired measles-specific antibody depend on the amount of maternal antibody transferred and the rate of antibody decay in the infant. This results in a variable window of vulnerability between the waning of the protection conferred by maternal antibodies and generation of infant antibodies after exposure to measles or measles vaccination. The recommendation for vaccination at 12– 15 months of age was made at a time when mothers had measles-specific antibody induced by natural infection, which they transferred to their infants transplacentally. Vaccination at 12–15 months ensured that no passively transferred maternal antibody remained in the infant that could hamper vaccine efficacy. In contrast, after four decades of extensive measles vaccination, currently most infants are born to mothers who have vaccine-induced immunity, thus transferring less maternal antibody to their infants. Studies in highly vaccinated populations have shown higher susceptibility of infants to measles and a window of vulnerability with little to no protective measles antibodies as early as 4–6 months (Dabis et al. 1989; de Francisco et al. 1998; Markowitz et al. 1996; Nicoara et al. 1999; Papania et al. 1999; Tapia et al. 2005). Furthermore, in developing countries, other factors such as poor nutrition, high parity, low birth weight, HIV infection, and anemia can further contribute to lower measles antibody titers in infants (de Moraes-Pinto et al. 1998; Farquhar et al. 2005; Okoko et al. 2001a, b; Owens et al. 2006; Scott et al. 2005, 2007; Wesumperuma et al. 1999).

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In many developing countries with high exposure to measles, vaccination is recommended at 9 months of age (World Health Organization 2006), which may not be completely effective. There has been intense evaluation of the possibility of vaccinating at the youngest possible age, including trials at 6 months. However, at 6 months, there appears to be an intrinsic deficiency in generating a proper humoral response to measles (Gans et al. 2001, 2003, 2004). Compared to infants at 9 and 12 months, 6-month-old infants with no residual maternal antibody develop lower protective antibody responses after vaccination and lower IFN-γ production after MV stimulation (Gans et al. 1998). Further work has shown that it is not necessarily the quantitative production of IgG that is affected by age but rather affinity maturation and avidity of measles-vaccine-specific antibody that is lower in 6-month-old infants independent of the presence of maternal antibody (Nair et al. 2007). This is likely to be due to an impairment of the somatic hypermutation mechanism in human B cells early in life, which develops during an infant’s 1st year (Ridings et al. 1998). Overall, until vaccination at 9 or 12 months, infants initially depend on maternal antibody for protection from measles prior to the development of a long-lasting response to measles infection or vaccination. Many research efforts are aimed at the development of vaccines that could give an effective response when given at an earlier age than the current measles vaccine.

Measles Immunity in Immunocompromised Hosts In individuals with impaired cellular immunity (HIV infection, congenital immunodeficiency, leukemia, etc.), measles infection can lead to giant cell pneumonia, measles inclusion body encephalitis, and death (Markowitz et al. 1988; Mustafa et al. 1993; Rand et al. 1976; Siegel et al. 1977). Many regions of the developing world where measles remains endemic are also regions burdened with a high prevalence of human immunodeficiency virus (HIV) infection. The impact of the HIV epidemic on the immune response to natural measles infection and to vaccination is the subject of intense study, as it is unknown whether the HIV epidemic will impede measles elimination efforts. Natural measles infection in HIV-infected children leads to a prolonged phase of illness and MV shedding in PBMCs, nasopharyngeal swabs, and in urine as compared to HIV-negative children (Permar et al. 2001). This suggests that the impaired CD4 and CD8 T cell response present in HIV-infected children results in slower clearance of MV, which could thus increase in the measles transmissibility window. Extremely immunosuppressed children may not be able to clear the virus and can die from measles-induced giant-cell pneumonia (Krasinski and Borkowsky 1989; Markowitz et al. 1988; Nadel et al. 1991; Palumbo et al. 1992). Furthermore, clinical manifestations of measles in HIV-infected individuals have been described to be altered (Moss et al. 1999). Since the cellular immune response to measles is responsible for the hallmark rash, HIV-infected individuals with more marked immunosuppression may become ill with measles without developing the rash (Centers for

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Disease Control 1988; Markowitz et al. 1988; Moss et al. 1999). The rate of complications and measles-related mortality is also significantly higher in HIV-positive children as compared to HIV-negative children. Initial studies assessed measles mortality with relation to infant HIV-antibody positivity. Further studies assessing HIV infection by virological methods demonstrated that HIV-negative children born to HIV-positive mothers also have higher mortality after measles infection as compared to children born to HIV-negative women (Moss et al. 2008). Measles vaccination of HIV-infected infants is hampered by impaired immune responses and it has been observed that response rates to measles vaccination range from 25% to 72% as measured by protective measles-specific antibodies (Arpadi et al. 1996; Brena et al. 1993; Krasinski and Borkowsky 1989; Palumbo et al. 1992; Rudy et al. 1994), whereas 95% of healthy children mount protective immunity to the measles vaccine (Krugman 1977). Measles-specific antibody levels elicited by vaccination also decay more rapidly in HIV-infected children (al-Attar et al. 1995) than in HIV-negative infants. A severe dysfunction of long-term humoral memory caused by a progressive memory B cell loss during chronic HIV infection may be involved in measles-specific antibody decay (Titanji et al. 2006). However, several studies have shown effective responses and antibody maintenance after measles revaccination in children undergoing successful highly active antiretroviral therapy (HAART) (Aurpibul et al. 2007; Berkelhamer et al. 2001). Prior to measles immunization, infants are protected from measles by maternally transferred antibody. Levels of MV-protective antibodies are lower in infants born to HIV-positive women as compared to infants born to HIV-negative women, thus increasing the window of vulnerability to natural measles infection prior to vaccination age. Lower levels of MV-specific antibodies in infants born of HIVpositive mothers are likely to be due to both lower maternal antibody levels and/or impaired placental transfer (de Moraes-Pinto et al. 1996, 1998; Scott et al. 2005, 2007). Overall the HIV epidemic poses obstacles to measles elimination by (1) increasing transmissibility of natural measles infection, (2) modifying clinical manifestations, thus disrupting case-finding efforts, (3) increasing vulnerability of infants born to HIV-positive mothers, and (4) reducing efficacy of the measles vaccine in HIV-positive individuals. However, models have suggested that the impact of the HIV pandemic on population immunity to measles should not introduce an insurmountable barrier to measles control (Helfand et al. 2005; Scott et al. 2008). This is partially due to the high mortality rate of these children, which counteracts the increased vaccine failure, shorter duration of maternal antibody protection, and longer duration of MV infectiousness among HIV-infected children. However, survival is largely determined by HAART and one published model suggests that HAART treatment could lead to an increase in the population susceptible to measles if vaccine-induced protection in HIV-1infected children wanes to an equal extent in HIV-infected children irrespective of HAART (Scott et al. 2008). A combination of HAART and revaccination against measles may improve the situation. More studies on measles vaccine- induced immunity in HIV-infected infants under HAART are necessary, as are studies assessing revaccination after HAART response.

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Conclusions The immune response to natural measles infection is a complex cascade of innate, cellular, and humoral immune responses leading to lifelong immunity (Fig. 8.1). The humoral arm of the immune response has been the prime focus for determining vaccine efficacy and assessing protection against measles. However, it has clearly been demonstrated that cellular immunity is essential for effective recovery from measles infection. The quality as well as the intensity of the response to MV is crucial such that the T cell effector functions elicited, antibody IgG isotypes, and avidity play a role in determining whether the MV-specific response will be protective. Individuals with cellular immunodeficiencies have impaired immune responses to measles infection and are at increased risk of severe complications and death due to measles infection. Immunodeficiencies such as HIV infection also impair responses to measles vaccination. As a result of four decades of extensive measles vaccination, currently most infants are born to mothers who have vaccine-induced immunity rather than immunity induced by natural measles infection. It has been shown that these mothers transfer less maternal antibody to their infants, thus leaving young infants susceptible to measles in areas where measles outbreaks still occur. Two doses of measles

Measles infection

Innate immunity

DC mφ

TLR activation IFN type I/IL-12 production Humoral

CMI

YY YY CD4-Th1(early): IFN-g, IL-2 CD8-CTL CD4-Th2(late): IL-4,IL-5,IL-10

Short-term EM recovery

Maintenance by TCD4

Long-term CM

Neutralizing IgG1 IgG3 Affinity maturation Serologic memory IgG1 IgG4 >200mIU /ml

protection from re-infection

Fig. 8.1 Schema of the components of an effective anti-measles immune response. Immunodeficiency, improper vaccine design, vaccination, and severe measles-induced immunosuppression can affect various aspects of the immune cascade. CMI cell-mediated immunity, EM effector memory, CM central memory

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vaccine is currently thought to induce long-lasting protection against measles infection and studies are currently underway to develop a vaccine that would be immunogenic for infants younger than 9 months of age.

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Hurme M, Haanpaa M, Nurmikko T, Wang XY, Virta M, Pessi T, Kilpinen S, Hulkkonen J, Helminen M (2003) IL-10 gene polymorphism and herpesvirus infections. J Med Virol 70 [Suppl 1]:S48–S50 Jabbour JT, Carcia HJ, Lemmi H, Ragland J, Duenas DA, Sever JL (1969) Subacute sclerosing panencephalitis. A multidisciplinary study of eight cases. JAMA 207:2248–2254 Jaye A, Magnusen AF, Sadiq AD, Corrah T, Whittle HC (1998) Ex vivo analysis of cytotoxic T lymphocytes to measles antigens during infection and after vaccination in Gambian children. J Clin Invest 102:1969–1977 Jaye A, Herberts CA, Jallow S, Atabani S, Klein MR, Hoogerhout P, Kidd M, van Els CA, Whittle HC (2003) Vigorous but short-term gamma interferon T-cell responses against a dominant HLA-A*02-restricted measles virus epitope in patients with measles. J Virol 77:5014–5016 Karp CL, Wysocka M, Wahl LM, Ahearn JM, Cuomo PJ, Sherry B, Trinchieri G, Griffin DE (1996) Mechanism of suppression of cell-mediated immunity by measles virus. Science 273:228–231 Kaslow RA, Dorak T, Tang JJ (2005) Influence of host genetic variation on susceptibility to HIV type 1 infection. J Infect Dis 191 [Suppl 1]:S68–S77 Krasinski K, Borkowsky W (1989) Measles and measles immunity in children infected with human immunodeficiency virus. JAMA 261:2512–2516 Krugman S (1977) Present status of measles and rubella immunization in the United States: a medical progress report. J Pediatr 90:1–12 Langmuir AD (1962) Medical importance of measles. Am J Dis Child 103:224–226 Lanzavecchia A, Sallusto F (2005) Understanding the generation and function of memory T cell subsets. Curr Opin Immunol 17:326–332 Lee MS, Nokes DJ (2001) Predicting and comparing long-term measles antibody profiles of different immunization policies. Bull World Health Organ 79:615–624 Lee MS, Chien LJ, Yueh YY, Lu CF (2001) Measles seroepidemiology and decay rate of vaccineinduced measles IgG titers in Taiwan, 1995–1997. Vaccine 19:4644–4651 Malvoisin E, Wild F (1990) Contribution of measles virus fusion protein in protective immunity: anti-F monoclonal antibodies neutralize virus infectivity and protect mice against challenge. J Virol 64:5160–5162 Markowitz LE, Chandler FW, Roldan EO, Saldana M, Roach KC, Hutchins SS, Preblud SR, Mitchell CD, Scott GB (1988) Fatal measles pneumonia without rash in a child with AIDS. J Infect Dis 158:480–483 Markowitz LE, Albrecht P, Rhodes P, Demonteverde R, Swint E, Maes EF, Powell C, Patriarca PA (1996) Changing levels of measles antibody titers in women and children in the United States: impact on response to vaccination. Kaiser Permanente Measles Vaccine Trial Team. Pediatrics 97:53–58 Mitus A, Holloway A, Evans AE, Enders JF (1962) Attenuated measles vaccine in children with acute leukemia. Am J Dis Child 103:413–418 Moss WJ (2007) Measles still has a devastating impact in unvaccinated populations. PLoS Med 4: e24 Moss WJ, Cutts F, Griffin DE (1999) Implications of the human immunodeficiency virus epidemic for control and eradication of measles. Clin Infect Dis 29:106–112 Moss WJ, Ryon JJ, Monze M, Griffin DE (2002) Differential regulation of interleukin (IL)-4, IL-5, and IL-10 during measles in Zambian children. J Infect Dis 186:879–887 Moss WJ, Fisher C, Scott S, Monze M, Ryon JJ, Quinn TC, Griffin DE, Cutts FT (2008) HIV type 1 infection is a risk factor for mortality in hospitalized Zambian children with measles. Clin Infect Dis 46:523–527 Mossong J, O’Callaghan CJ, Ratnam S (2000) Modelling antibody response to measles vaccine and subsequent waning of immunity in a low exposure population. Vaccine 19:523–529 Mustafa MM, Weitman SD, Winick NJ, Bellini WJ, Timmons CF, Siegel JD (1993) Subacute measles encephalitis in the young immunocompromised host: report of two cases diagnosed by polymerase chain reaction and treated with ribavirin and review of the literature. Clin Infect Dis 16:654–660

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Palumbo P, Hoyt L, Demasio K, Oleske J, Connor E (1992) Population-based study of measles and measles immunization in human immunodeficiency virus-infected children. Pediatr Infect Dis J 11:1008–1014 Panum P (1938) Med Classics 3:829–886 Papania M, Baughman AL, Lee S, Cheek JE, Atkinson W, Redd SC, Spitalny K, Finelli L, Markowitz L (1999) Increased susceptibility to measles in infants in the United States. Pediatrics 104:e59 Paul WE (1989) In: Paul WE (ed) Peripheral T lymphocytes. Fundamental immunology. Raven Press, New York, pp 398–399 Paul WE (2003) Fundamental immunology. Lippincott Raven, Philadelphia Permar SR, Moss WJ, Ryon JJ, Monze M, Cutts F, Quinn TC, Griffin DE (2001) Prolonged measles virus shedding in human immunodeficiency virus-infected children, detected by reverse transcriptase-polymerase chain reaction. J Infect Dis 183:532–538 Permar SR, Klumpp SA, Mansfield KG, Kim WK, Gorgone DA, Lifton MA, Williams KC, Schmitz JE, Reimann KA, Axthelm MK, Polack FP, Griffin DE, Letvin NL (2003) Role of CD8(+) lymphocytes in control and clearance of measles virus infection of rhesus monkeys. J Virol 77:4396–4400 Permar SR, Klumpp SA, Mansfield KG, Carville AA, Gorgone DA, Lifton MA, Schmitz JE, Reimann KA, Polack FP, Griffin DE, Letvin NL (2004) Limited contribution of humoral immunity to the clearance of measles viremia in rhesus monkeys. J Infect Dis 190:998–1005 Perry RT, Halsey NA (2004) The clinical significance of measles: a review. J Infect Dis 189 [Suppl 1]: S4–S16 Polack FP, Hoffman SJ, Moss WJ, Griffin DE (2002) Altered synthesis of interleukin-12 and type 1 and type 2 cytokinesin rhesus macaques during measles and atypical measles. J Infect Dis 185:13–19 Polack FP, Hoffman SJ, Crujeiras G, Griffin DE (2003) A role for nonprotective complement-fixing antibodies with low avidity for measles virus in atypical measles. Nat Med 9:1209–1213 Rand KH, Emmons RW, Merigan TC (1976) Measles in adults. An unforeseen consequence of immunization? JAMA 236:1028–1031 Ratnam S, Gadag V, West R, Burris J, Oates E, Stead F, Bouilianne N (1995) Comparison of commercial enzyme immunoassay kits with plaque reduction neutralization test for detection of measles virus antibody. J Clin Microbiol 33:811–815 Reiner SL, Sallusto F, Lanzavecchia A (2007) Division of labor with a workforce of one: challenges in specifying effector and memory T cell fate. Science 317:622–625 Ridings J, Dinan L, Williams R, Roberton D, Zola H (1998) Somatic mutation of immunoglobulin V(H)6 genes in human infants. Clin Exp Immunol 114:33–39 Rudy BJ, Rutstein RM, Pinto-Martin J (1994) Responses to measles immunization in children infected with human immunodeficiency virus. J Pediatr 125:72–74 Samb B, Aaby P, Whittle HC, Seck AM, Rahman S, Bennett J, Markowitz L, Simondon F (1995) Serologic status and measles attack rates among vaccinated and unvaccinated children in rural Senegal. Pediatr Infect Dis 14:203–209 Scott S, Cumberland P, Shulman CE, Cousens S, Cohen BJ, Brown DW, Bulmer JN, Dorman EK, Kawuondo K, Marsh K, Cutts F (2005) Neonatal measles immunity in rural Kenya: the influence of HIV and placental malaria infections on placental transfer of antibodies and levels of antibody in maternal and cord serum samples. J Infect Dis 191:1854–1860 Scott S, Moss WJ, Cousens S, Beeler JA, Audet SA, Mugala N, Quinn TC, Griffin DE, Cutts FT (2007) The influence of HIV-1 exposure and infection on levels of passively acquired antibodies to measles virus in Zambian infants. Clin Infect Dis 45:1417–1424 Scott S, Mossong J, Moss WJ, Cutts FT, Cousens S (2008) Predicted impact of the HIV-1 epidemic on measles in developing countries: results from a dynamic age-structured model. Int J Epidemiol 30:356–367

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Shaffer JA, Bellini WJ, Rota PA (2003) The C protein of measles virus inhibits the type I interferon response. Virology 315:389–397 Shingai M, Ebihara T, Begum NA, Kato A, Honma T, Matsumoto K, Saito H, Ogura H, Matsumoto M, Seya T (2007) Differential type I IFN-inducing abilities of wild-type versus vaccine strains of measles virus. J Immunol 179:6123–6133 Siegel MM, Walter TK, Ablin AR (1977) Measles pneumonia in childhood leukemia. Pediatrics 60:38–40 Stephenson JR, ter Meulen V (1979) Antigenic relationships between measles and canine distemper viruses: comparison of immune response in animals and humans to individual virus-specific polypeptides. Proc Natl Acad Sci U S A 76:6601–6605 Stokes J, Reilly CM, Bunyak EB, Hilleman MR (1961) Immunologic studies of measles. Am J Hyg 74:293–303 Takeuchi K, Kadota SI, Takeda M, Miyajima N, Nagata K (2003) Measles virus V protein blocks interferon (IFN)-alpha/beta but not IFN-gamma signaling by inhibiting STAT1 and STAT2 phosphorylation. FEBS Lett 545:177–182 Tapia MD, Sow SO, Medina-Moreno S, Lim Y, Pasetti MF, Kotloff K, Levine MM (2005) A serosurvey to identify the window of vulnerability to wild-type measles among infants in rural Mali. Am J Trop Med Hyg 73:26–31 Termeulen V, Stephenson JR, Kreth HW (1983) Subacute sclerosing panencephalitis. In: FraenkelConrat H, Wagner RR (eds) Comprehensive virology. Plenum, New York, pp 105–159 Thio CL, Thomas DL, Karacki P, Gao X, Marti D, Kaslow RA, Goedert JJ, Hilgartner M, Strathdee SA, Duggal P, O’Brien SJ, Astemborski J, Carrington M (2003) Comprehensive analysis of class I and class II HLA antigens and chronic hepatitis B virus infection. J Virol 77:12083–12087 Titanji K, De Milito A, Cagigi A, Thorstensson R, Grutzmeier S, Atlas A, Hejdeman B, Kroon FP, Lopalco L, Nilsson A, Chiodi F (2006) Loss of memory B cells impairs maintenance of long-term serologic memory during HIV-1 infection. Blood 108:1580–1587 Toyoda M, Ihara T, Nakano T, Ito M, Kamiya H (1999) Expression of interleukin-2 receptor alpha and CD45RO antigen on T lymphocytes cultured with rubella virus antigen, compared with humoral immunity in rubella vaccinees. Vaccine 17:2051–2058 van Els CA, Nanan R (2002) T cell responses in acute measles. Viral Immunol 15:435–450 Van Binnendijk RS, Poelen MCM, Kuijpers KC, Osterhaus ADME, Uytdehaag FGCM (1990) The predominance of CD8+ T cells after infection with measles virus suggests a role for CD8+ class I MHC-restricted cytotoxic T lymphocytes (CTL) in recovery from measles. Clonal analyses of human CD8+ class I MHC-restricted CTL. J Immunol 144:2394–2399 Ward BJ, Johnson RT, Vaisberg A, Jauregui E, Griffin DE (1991) Cytokine production in vitro and the lymphoproliferative defect of natural measles virus infection. Clin Immunol Immunopathol 61:236–248 Weibel RE, Buynak EB, McLean AA, Roehm RR, Hilleman MR (1980) Persistence of antibody in human subjects for 7 to 10 years following administration of combined live attenuated measles, mumps, and rubella virus vaccines. Proc Soc Exp Biol Med 165:260–263 Wesumperuma HL, Perera AJ, Pharoah PO, Hart CA (1999) The influence of prematurity and low birthweight on transplacental antibody transfer in Sri Lanka. Ann Trop Med Parasitol 93:169–177 Whittle HC, Aaby P, Samb B, Cisse B, Kanteh F, Soumare M, Jensen H, Bennett J, Simondon F (1999) Poor serologic responses five to seven years after immunization with high and standard titer measles vaccines. Pediatr Infect Dis J 18:53–57 Wolfson LJ, Strebel PM, Gacic-Dobo M, Hoekstra EJ, McFarland JW, Hersh BS (2007) Has the 2005 measles mortality reduction goal been achieved? A natural history modelling study. Lancet 369:191–200 World Health Organization (1993) Immunological Basis for Immunization Module 7: Measles. WHO/EPI/GEN/98.17, World Health Organization, Geneva World Health Organization (2006) WHO AFRO Measles SIA Field Guide. World Health Organization, Geneva

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Wu VH, McFarland H, Mayo K, Hanger L, Griffin DE, Dhib-Jalbut S (1993) Measles virusspecific cellular immunity in patients with vaccine failure. J Clin Microbiol 31:118–122 Yakub I, Lillibridge KM, Moran A, Gonzalez OY, Belmont J, Gibbs RA, Tweardy DJ (2005) Single nucleotide polymorphisms in genes for 2'–5'-oligoadenylate synthetase and RNase L inpatients hospitalized with West Nile virus infection. J Infect Dis 192:1741–1748 Zilliox MJ, Parmigiani G, Griffin DE (2006) Gene expression patterns in dendritic cells infected with measles virus compared with other pathogens. Proc Natl Acad Sci U S A 103:3363– 3368 Zilliox MJ, Moss WJ, Griffin DE (2007) Gene expression changes in peripheral blood mononuclear cells during measles virus infection. Clin Vaccine Immunol 14:918–923

Chapter 9

Measles Control and the Prospect of Eradication W.J. Moss

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Goals and Strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mortality Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regional Elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Progress in Measles Mortality Reduction and Elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . Global Goals and Progress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regional Progress in Mortality Reduction and Elimination . . . . . . . . . . . . . . . . . . . . . . . . Feasibility of Measles Eradication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological Feasibility of Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical Feasibility of Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Logistical Feasibility of Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Challenges to Global Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Challenges to Achieving High Levels of Measles Vaccine Coverage . . . . . . . . . . . . . . . . . Challenges to Achieving High Levels of Population Immunity . . . . . . . . . . . . . . . . . . . . . Challenges to Sustained Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prospects for Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Remarkable progress has been made in reducing measles incidence and mortality as a consequence of implementing the measles mortality reduction strategy of the World Health Organization (WHO) and United Nations Children’s Fund (UNICEF). The revised global measles mortality reduction goal set forth in the WHO-UNICEF Global Immunization Vision and Strategy for 2006–2015 is to reduce measles deaths by 90% by 2010 compared to the estimated 757,000 deaths in 2000. The possibility of measles eradication has been discussed for almost 40 years, and measles meets many of the criteria for eradication. Global measles eradication will face a number of challenges to achieving and sustaining high levels W.J. Moss Department of Epidemiology and the W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore MD, USA, e-mail: [email protected] D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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of vaccine coverage and population immunity, including population growth and demographic changes, conflict and political instability, and public perceptions of vaccine safety. To achieve the measles mortality reduction goal, continued progress needs to be made in delivering measles vaccines to the world’s children.

Introduction Measles virus (MV) has caused millions of deaths since its emergence as a zoonosis thousands of years ago. Prior to the introduction of measles vaccine, more than 130 million cases and 7–8 million deaths due to measles were estimated to have occurred annually, and almost everyone was infected during childhood. Measles mortality declined in the first half of the twentieth century in developed countries as a consequence of improvements in living conditions, better nutritional status, and the availability of antibiotics for secondary bacterial infections. The introduction of measles vaccines beginning in the 1960s led to substantial reductions in measles incidence, morbidity, and mortality in both developed and developing countries. Measles vaccines were not available for many of the world’s children, however, until the World Health Organization (WHO) launched the Expanded Programme on Immunization (EPI) in 1974, which provided vaccines against six target diseases including measles. Global vaccine coverage against EPI targeted diseases increased from less than 5% at the start of the program to almost 80% by 1990. More recently, remarkable progress has been made in reducing measles incidence and mortality as a consequence of implementing the measles mortality reduction strategy of the WHO and United Nations Children’s Fund (UNICEF). This strategy focuses on 47 priority countries and includes: (1) achieving and maintaining more than 90% coverage with the first dose of measles vaccine in every district by the age of 12 months; (2) ensuring that all children receive a second opportunity for measles vaccination; (3) surveillance for measles cases and serological confirmation; and (4) provision of appropriate case management (WHO/UNICEF 2001). Support for these efforts comes from the Measles Initiative, a partnership started in 2001 and led by the American Red Cross, the United Nations Foundation, UNICEF, the United States Centers for Disease Control and Prevention, and the WHO.

Goals and Strategies The key to measles control is achieving and sustaining high levels of measles vaccine coverage (Fig. 9.1). Different goals for measles control have been established, necessitating different vaccination strategies. Three broad goals can be defined: mortality reduction, regional elimination, and global eradication.

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Mortality Reduction

Number of cases

Official coverage

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1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006

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Mortality reduction, the least demanding of the three goals, calls for a reduction in measles mortality from a predetermined level through reductions in incidence and case fatality. Although reducing case fatality through appropriate case management is an important component, measles mortality reduction is achieved largely through a reduction in incidence. To reduce incidence, measles vaccine is administered as a single dose through routine immunization services (Fig. 9.2), with the optimal age

WHO / UNICEF estimated coverage

Fig. 9.1 Global annual reported measles incidence and measles vaccine coverage, 1986–2006. From World Health Organization 2007a

Fig. 9.2 Immunization coverage with measles containing vaccines in infants, 2006. From World Health Organization 2008

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of immunization determined by the average age of infection and the rate of decline of maternal antibodies. In areas where measles is endemic, and the average age of infection is low, measles vaccine is routinely administered at 9 months of age in accordance with the EPI schedule (WHO 2004). In countries where MV transmission has been significantly reduced, the age at first vaccination is often increased to 12–15 months, resulting in a higher proportion of children who develop protective immunity. If vaccination coverage is sufficiently high, substantial reductions in incidence and mortality occur, the period between epidemics lengthens, and the age distribution shifts toward older children and young adults, further contributing to a reduction in measles case fatality. Mortality reduction can also be achieved by proper case management, including the administration of vitamin A to persons with measles (D’Souza and D’Souza 2002) and prompt antibiotic treatment of secondary bacterial pneumonia (Duke and Mgone 2003). Provision of vitamin A through polio and measles vaccination campaigns further contributes to the reduction in measles mortality (WHO 2005).

Regional Elimination Measles elimination is the interruption of MV transmission within a defined geographic area, such as country, continent, or WHO region. Small outbreaks of primary and secondary cases may occur following importation from outside the region, but sustained transmission does not occur. Because of the high infectivity of MV and the fact that not all persons develop protective immunity following vaccination, a single dose of measles vaccine does not achieve a sufficient level of population immunity to eliminate measles. A second opportunity for measles immunization is necessary to provide protective immunity to children who fail to respond to the first dose, as well as to immunize those children who were not previously vaccinated. Two strategies to administer the second dose of measles vaccine have been used. In countries with sufficient health infrastructure, and where children routinely receive well-child care beyond the 1st year of life, the second dose of measles vaccine is administered through routine immunization services, typically prior to the start of school (4–6 years of age). High coverage levels can be ensured by school entry requirements (CDC 2007). A second approach, first developed by the Pan American Health Organization (PAHO) for South and Central America (PAHO 1999) and modeled after polio eradication strategies, involves mass immunization campaigns (called supplementary immunization activities or SIA) to deliver the second dose of measles vaccine. This strategy was successful in eliminating measles in South and Central America (de Quadros et al. 1996, 2004) and has resulted in a marked reduction in measles incidence and mortality in much of sub-Saharan Africa (Otten et al. 2005; WHO 2006b). The PAHO strategy consists of four subprograms: catch-up, keep-up, follow-up, and mop-up. The catch-up activity consists of a one-time, mass immunization campaign that targets all children within a broad age range regardless of whether they

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have previously had wild-type MV infection or measles vaccination. The goal is to rapidly achieve a high level of population immunity and interrupt MV transmission. These campaigns are conducted over a short period of time, usually several weeks, and during a low transmission season. Under the PAHO strategy, children 9 months to 14 years of age were targeted for vaccination, a substantial proportion of the total population in many countries. The appropriate target age range depends upon the age distribution of measles cases. In regions where measles is endemic, the majority of older children are likely to be immune. Nevertheless, seroprevalence studies usually are not conducted prior to catch-up campaigns, and this broad age range first adopted by PAHO has been widely used in sub-Saharan Africa and Asia. These campaigns require significant financial investments and the commitment of large numbers of personnel; extensive logistical planning to transport and store vaccines, maintain cold chains, and dispose of syringes and needles; and community mobilization to ensure participation. If successful, SIA are cost effective (Dayan et al. 2004; Uzicanin et al. 2004) and can abruptly interrupt MV transmission, with dramatic declines in incidence and mortality. Keep-up refers to the need to maintain greater than 90% coverage with the first dose of measles vaccine through routine immunization services. Follow-up refers to periodic mass campaigns to prevent the accumulation of susceptible children. Followup campaigns typically target children 9 months to 4 years of age, a narrower age group than targeted in catch-up campaigns. Follow-up campaigns should be conducted when the estimated number of susceptible children reaches the size of one birth cohort, generally every 3–5 years after the catch-up campaign. These campaigns need to be conducted indefinitely because of the potential risk of MV importation, or until the health infrastructure is sufficiently developed so that the second opportunity can be provided through routine immunization services. Mop-up campaigns target difficult-to-reach children in areas of measles outbreaks or low vaccine coverage. Difficult to reach children include those living on the street or in areas of conflict. Measles elimination also requires active surveillance for measles cases to assist outbreak response (Grais et al. 2008) and to monitor progress in mortality reduction and elimination. Blood samples should be collected from all suspected measles cases for confirmation by detection of IgM antibodies to MV. Measles outbreaks require serological investigation of the first five to ten cases (WHO/UNICEF 2001), with other cases linked epidemiologically. Less invasive oral fluid or dried blood spot samples also may be used, and urine or nasopharyngeal specimens should be obtained for virus isolation and genetic characterization (Bellini and Helfand 2003). To support these activities, the WHO established the Global Measles Laboratory network in 2000 and the Measles and Rubella Laboratory Network (LabNet) in 2003, comprising over 700 laboratories in more than 160 countries (CDC 2005).

Eradication The Dahlem Conference on Disease Eradication (1997) defined eradication as the permanent reduction to zero of the global incidence of infection caused by a specific

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pathogen as a result of deliberate efforts, with the consequence that interventions would no longer be necessary (Dowdle and Hopkins 1998). The pathogen need not be extinct. Smallpox virus, for example, exists in government laboratories in the United States and Russia, although the disease is eradicated and vaccination activities have ceased (Stone 2002). Eradication is the most ambitious goal, requiring sustained high levels of financial investment, political commitment, and public cooperation. Presumably, measles eradication would be achieved using strategies similar to those described for elimination but applied and coordinated globally.

Progress in Measles Mortality Reduction and Elimination Global Goals and Progress In 2003, the World Health Assembly endorsed a resolution to reduce the number of deaths attributed to measles by 50% by the end of 2005 compared with 1999 estimates. This target was met. Global measles mortality in 2005 was estimated to be 345,000 deaths (uncertainty bounds 247,000 and 458,000 deaths), a 60% decrease from 1999 (Wolfson et al. 2007). Further reductions in global measles mortality were achieved in 2006, with an estimated 242,000 deaths (uncertainty bounds 173,000 and 325,000 deaths) (Fig. 9.3) (WHO 2007b). These estimates are not based on active surveillance for measles deaths but instead are derived from models of measles incidence and mortality, using estimated measles vaccine coverage and case fatality rates. The revised global measles mortality reduction goal set forth in the WHOUNICEF Global Immunization Vision and Strategy (GIVS) for 2006–2015 is to reduce measles deaths by 90% by 2010 compared to the estimated 757,000 deaths in 2000 (WHO/UNCF 2005). The financial resources required to achieve the

1 000 000 800 000 600 000 400 000 200 000 2000

2001

2002

2003

2004

2005

2006

Fig. 9.3 Estimated global measles deaths, 2000–2006. Note the estimated number of global deaths is similar to the reported incidence shown in Fig. 1 because of the different methods used to derive the estimates. From World Health Organization 2007a

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measles control goals for 2001–2005 was estimated to be US $984 million, with approximately two-thirds for the operational costs of conducting SIA and one-third for the purchase of vaccines and injection equipment (WHO/UNICEF 2001). The cost of scaling up childhood immunization services to reach the WHO-UNICEF GIVS goal of reducing mortality due to all vaccine-preventable diseases by twothirds by 2015 was recently estimated to be US $35 billion (range US $ 13–40 billion) for the 72 poorest countries of the world, including US $8.7 billion for the purchase of vaccines (Wolfson et al. 2008).

Regional Progress in Mortality Reduction and Elimination Four WHO regions set measles elimination goals: Americas (2000), Europe (2010), Eastern Mediterranean (2010), and Western Pacific (2012). Measles elimination was achieved in the United States in 2000 and in the Americas by November 2002. The two regions where most measles deaths occur (Table 9.1), Africa and SouthEast Asia, have measles mortality reduction goals, although many countries in these regions have implemented the WHO-UNICEF strategy of providing a second opportunity for measles vaccination through SIA, resulting in dramatic declines in

Table 9.1 Estimated number of deaths from measles and coverage of first-dose measles vaccine through routine immunization services, by WHO region, 2000 and 2006 WHO region

2000 % Measles deaths Coverage (uncertainty with 1st bounds) dose of measles vaccine

2006 % Coverage with 1st dose of measles vaccine

Measles deaths (uncertainty bounds)

Africa

56

396,000 73 (290–514,000)

36,000 (26–49,000)

Americas

92

% Decrease in measles deaths 2000–2006

91

<1000

93

<1000

–

Eastern 73 Mediterranean

96,000 (71–124,000)

83

23,000 (16–34,000)

76

Europe

91

<1000

94

<1000

–

South-East Asia

60

240,000 65 (173–316,000)

178,000 26 (128–234,000)

Western Pacific

86

25,000 (17–35,000)

5,000 (3–7,000)

Total

72

757,000 80 (551–990,000)

Adapted from World Health Organization 2007b

93

81

242,000 68 (173–325,000)

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incidence and mortality. The largest decrease in measles mortality was in Africa, where measles mortality decreased 91% from 2000 to 2006, accounting for 70% of the global reduction in measles mortality (WHO 2007b). Progress in measles mortality reduction in the South-East Asia region was not as marked because several populous countries have not conducted SIA and routine immunization coverage remains suboptimal (WHO 2007b). Low vaccination coverage and susceptibility among adults hinder measles elimination in parts of Europe and Australia (Andrews et al. 2008). Conflicts and political instability in several countries of the Eastern Mediterranean region, including Afghanistan, Iraq, Lebanon, Somalia, and Sudan, have impeded measles control, but a catch-up SIA was conducted in parts of Pakistan in 2007 (CDC 2008c). Although measles was declared eliminated from the United States in 2000, recent outbreaks highlight the challenges in sustaining elimination. First, despite very high levels of measles vaccine coverage and population immunity overall (McQuillan et al. 2007), clustering of susceptible persons can lead to outbreaks. In 2005, a 17-year-old girl with measles returned to Indiana from Romania and attended a church gathering of over 500 people (Parker et al. 2006). Despite 98% measles vaccination coverage in the state of Indiana (among sixth-graders), 34 cases of measles resulted, of whom 94% were unvaccinated. Second, the widespread circulation of MV and ease of global travel allows for MV importation. Perhaps surprisingly, these importations frequently arise from countries with ample resources for measles control. A multistate outbreak of measles in 2007 occurred at an international sporting event after importation from Japan (CDC 2008a), the country responsible for the most imported cases of measles into the US over the past several years (Takahashi and Saito 2008). Thousands of measles cases occur annually in Japan as a consequence of low vaccination coverage resulting from the relaxing of vaccination requirements after an outbreak of aseptic meningitis from the Urabe mumps vaccine strain (Gomi and Takahashi 2004). Third, as measles control efforts are increasingly successful in reducing disease incidence, public perceptions of the risk of measles diminish and are replaced by concerns of possible adverse events associated with measles vaccine. An outbreak of measles in San Diego in 2008, imported from Switzerland, resulted in 11 additional cases in unvaccinated children and two generations of secondary cases (CDC 2008b). Among the nine cases older than 1 year of age, eight were unvaccinated because of personal exemption beliefs.

Feasibility of Measles Eradication The possibility of measles eradication has been discussed for almost 40 years (Sencer et al. 1967), beginning in the late 1960s when smallpox eradication was nearing completion and the long-term protective immunity induced by measles vaccine became evident. Three criteria are deemed important for disease eradication: (1) humans must be critical to transmission; (2) sensitive and specific diagnostic tools must exist; and (3) an effective intervention must be available

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(Dowdle and Hopkins 1998). Interruption of transmission in large geographical areas for prolonged periods further supports the feasibility of eradication. Measles is thought by many experts to meet these criteria (de Quadros 2004; Orenstein et al. 2000).

Biological Feasibility of Measles Eradication MV meets many of the biological criteria for disease eradication. MV has no nonhuman reservoir, infection can be readily diagnosed after onset of a rash, and MV has not mutated to significantly alter immunogenic epitopes (Moss and Griffin 2006). Although MV displays sufficient genetic variation to conduct molecular epidemiologic analyses (WHO 2006a), the epitopes against which protective antibodies develop have remained stable, likely because of functional constraints on the amino acid sequence and tertiary structure of the MV surface proteins (Frank and Bush 2007). Where MV differs from smallpox and polio viruses is that it is more highly infectious, necessitating higher levels of population immunity to interrupt transmission. Outbreaks can occur in populations in which less than 10% of individuals are susceptible. The contagiousness of MV is best expressed by the basic reproductive number Ro, which represents the mean number of secondary cases that would arise if an infectious agent were introduced into a completely susceptible population. Ro is a function not only of the infectious agent, but also of the host population. The estimated Ro for MV is 12–18, in contrast to 5–7 for smallpox and polio viruses and 2–3 for SARS-coronavirus. The high infectivity of MV implies that a high level of population immunity (approximately 92%–94%) is required to interrupt MV transmission (Gay 2004).

Technical Feasibility of Measles Eradication Measles vaccines are safe and effective and have interrupted MV transmission in large geographic areas, providing the critical tool for measles eradication. Despite progress in measles mortality reduction and elimination, there are several limitations of the licensed vaccines that may make measles eradication more challenging. First, attenuated measles vaccines are inactivated by heat and a cold chain must be maintained to support measles immunization activities. Second, in contrast to oral polio vaccines, measles vaccines must be injected subcutaneously or intramuscularly, necessitating trained healthcare workers, needles, syringes, and proper disposal of hazardous waste. Third, both maternally acquired antibodies and immunological immaturity reduce the protective efficacy of measles vaccination in early infancy, hindering effective immunization of young infants (Gans et al. 1998). Fourth, the attenuated measles vaccine has the potential to cause serious adverse events, such as lung or brain infection, in

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severely immunocompromised persons (Angel et al. 1998; Monafo et al. 1994). Lastly, a second opportunity for measles vaccination must be provided to achieve sufficient levels of population immunity to interrupt MV transmission. The duration of immunity following measles vaccination is more variable and shorter than following wild-type MV infection, but persists for decades even in countries where measles is no longer endemic and immunological boosting from wild-type MV infection does not occur (Amanna et al. 2007; Dine et al. 2004). Although antibody levels induced by vaccination may decline over time and become undetectable, immunological memory persists and most vaccinated persons produce a MV-specific immune response without clinical symptoms following exposure to wild-type MV.

Logistical Feasibility of Measles Eradication The elimination of measles in large areas, such as the Americas, suggests that measles eradication is feasible with current vaccination strategies (CDC 1997; de Quadros 2004; Meissner et al. 2004). Perhaps the major logistical challenge to measles eradication will be sustaining the financial resources, political will, and public confidence to implement widespread and coordinated measles vaccination and surveillance activities.

Challenges to Global Measles Eradication Global measles eradication will face a number of challenges to achieving and sustaining high levels of vaccine coverage and population immunity. Serious discussion of measles eradication is not likely to take place before polio eradication is achieved. Garnering the necessary political and public support for measles eradication will be extremely difficult should polio eradication efforts fail.

Challenges to Achieving High Levels of Measles Vaccine Coverage Sustainability of Current Measles Control and Elimination Strategies To eradicate measles, high levels of population immunity need to be sustained through coverage with two doses of measles vaccine. Because the first dose of measles vaccine is administered through routine immunization services, strengthening the primary healthcare system will further this goal. However, the long-term sustainability of mass vaccination campaigns is unclear as these activities make

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additional demands on the resources and staff of the primary healthcare system and require continued public support in the face of decreasing disease burden and perception of public health importance. The polio eradication campaign in Nigeria, for example, was perceived negatively to trigger a massive outbreak response for just three confirmed cases of polio in Adamawa State in 2005, whereas hundreds of deaths due to measles did not result in a comparable response (Schimmer and Ihekweazu 2006). Countries dependent upon SIA to deliver the second dose of measles vaccine will need to strengthen their primary health care systems to provide this dose beyond the 1st year of life. Ensuring the necessary supply of measles vaccine as eradication efforts progress will be critical (Costa et al. 2003), requiring close collaboration with vaccine manufacturers and the understanding that vaccination may decrease or cease should eradication be achieved.

Public Perceptions of Vaccine Safety and Public Health Loss of public confidence in vaccines can significantly impair control efforts, as demonstrated by the poliovirus outbreaks in northern Nigeria, which subsequently spread across several continents (Katz 2006). Numerous measles outbreaks have occurred in communities opposed to vaccination on religious or philosophical grounds (CDC 2000) or because of unfounded fears of serious adverse events (Feikin et al. 2000). Garnering the political will and public support for measles eradication is likely to be difficult in countries where the burden of disease due to measles is not recognized and unfounded fears of serious vaccine adverse events are common. Much public attention has focused on a purported association between measlesmumps-rubella (MMR) vaccine and autism following publication of a report in 1998 hypothesizing that MMR vaccine may cause a syndrome of autism and intestinal inflammation (Wakefield et al. 1998). The events that followed, and the public concern over the safety of MMR vaccine, led to diminished vaccine coverage in the United Kingdom and provide important lessons in the misinterpretation of epidemiologic evidence and the communication of scientific results to the public (Offit and Coffin 2003). As a consequence, measles outbreaks became more frequent and larger in size (Jansen et al. 2003). Several epidemiological studies and comprehensive reviews of the evidence rejected a causal relationship between MMR vaccination and autism (DeStefano and Thompson 2004; Madsen et al. 2002).

Conflict and Political Instability Maintaining high levels of measles vaccine coverage in areas of conflict and political instability is challenging (Senessie et al. 2007), and devastating measles outbreaks occur frequently in refugee populations and internally displaced populations (Connolly et al. 2004). Measles case fatality rates in such settings have been as high as 20%–30% (Salama et al. 2001). Progress in global control has made outbreaks of

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measles less likely in some regions, although outbreaks continue to occur in refugee and internally displaced populations with low levels of immunity (Kamugisha et al. 2003). Polio vaccination campaigns have been successfully conducted during scheduled cease-fires in regions of conflict (“days of tranquility”) (Tangermann et al. 2000) and similar results should be achievable for measles vaccination campaigns, although more highly skilled healthcare workers are needed to administer parenteral measles vaccine than oral poliovirus vaccine.

Population Growth and Demographic Changes The world’s population is predicted to increase 2.5 billion from 2007 to 2050, from the current 6.7 billion to 9.2 billion (UN 2007). This increase is equivalent to the total number of people in the world in 1950. Population growth will be greatest in the less developed regions of the world, increasing from 5.4 billion in 2007 to 7.9 billion in 2050 (UN 2007), where the proportion of people younger than 15 years of age is greatest. Specifically, the population of the 50 least developed countries will likely more than double, increasing from 0.8 billion in 2007 to 1.7 billion in 2050 (UN 2007). This increase in global population, particularly in less developed countries, will require additional resources, personnel, and vaccine supplies just to maintain current levels of vaccine coverage. Most of the predicted population growth will take place in urban areas of less developed countries (Cohen 2003). Currently, more than half of the world’s population lives in urban areas, and one in three urban residents live in slums, with a much higher proportion in sub-Saharan Africa and Asia (Dye 2008; UNPF 2007). Measles elimination may be particularly difficult in impoverished areas of large cities in Africa and Asia where several factors converge to facilitate MV transmission, including the high population density and difficulties in achieving high vaccination coverage. A critical question regarding measles eradication is whether the epidemiological conditions are sufficiently different in the large, densely populated cities of Africa and Asia than in the Americas to hinder measles eradication efforts.

Challenges to Achieving High Levels of Population Immunity Impact of the HIV-1 Pandemic In regions of high HIV prevalence and crowding, such as urban centers in subSaharan Africa, HIV-infected children may play a role in the sustaining MV transmission (Moss et al. 1999). Children with defective cell-mediated immunity can develop measles without the characteristic rash (Moss et al. 1999), hampering diagnosis, and HIV-infected children have prolonged shedding of MV RNA (Permer et al. 2001), potentially increasing the period of infectivity. Children born to HIVinfected mothers have lower levels of passively acquired maternal antibodies,

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increasing susceptibility to measles at an earlier age than children born to uninfected mothers (Moss et al. 2002; Scott et al. 2007), and protective antibody levels following vaccination wane within 2–3 years in many HIV-infected children not receiving antiretroviral therapy (Moss et al. 2007), creating a potential pool of susceptible children (Tejiokem et al. 2007). Thus, population immunity could be reduced in regions of high HIV-1 prevalence despite high levels of measles vaccine coverage. Counteracting the increased susceptibility of HIV-infected children is their high mortality rate, particularly in sub-Saharan Africa, such that these children do not live long enough to build up a sizeable pool of susceptible children (Helfand et al. 2005). Successful control of measles in the countries of southern Africa suggests that the HIV-1 epidemic is not a major barrier to measles control (Biellik et al. 2002; Otten et al. 2005). This may change with increased access to antiretroviral therapy, which may prolong survival without enhancing protective immunity in the absence of revaccination. Using a dynamic, age-structured mathematical model, the prevalence of measles increased after introduction of antiretroviral therapy into a hypothetical population of children with a high prevalence of HIV-1 infection (Scott et al. 2008).

Challenges to Sustained Measles Eradication Potential Use of Measles Virus as an Agent of Bioterrorism The high infectivity of MV is a characteristic suitable to a bioterrorist agent, but high levels of measles vaccination coverage throughout the world would protect many persons from the deliberate release of MV. Genetic engineering of a MV strain that was not neutralized by antibodies induced by the current measles vaccines would likely have reduced infectivity, as suggested by the fact that wild-type MV has not mutated to alter neutralizing epitopes. Whether the threat from bioterrorism precludes stopping measles vaccination after eradication is unclear but, at the least, a single-dose rather than a two-dose measles vaccination strategy could be adopted (Meissner et al. 2004).

Prospects for Measles Eradication The measles eradication end-game is likely to be different than that for smallpox or polio viruses (Gounder 1998; Morgan 2004). In contrast to polioviruses, prolonged shedding of potentially virulent vaccine viruses and environmental viral reservoirs will not be challenges to measles eradication. Although MV can be carried by persons during the incubation period, transmission from mobile, asymptomatic carriers is not as common as with poliovirus. However, higher levels of population immunity are necessary to interrupt MV transmission, more

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highly skilled healthcare workers are required to administer measles vaccines, and containment through case detection and ring vaccination will be more difficult for MV than smallpox virus because of infectivity before rash onset. New tools, such as aerosol administration of measles vaccines (Low et al. 2008), will facilitate mass vaccination campaigns, allowing less highly trained workers to administer vaccine and diminishing the medical waste disposal problems. Critics of eradication programs claim they can divert resources from primary healthcare and are imposed on countries or communities from outside. Enormous resources and efforts may be required to eradicate the few remaining measles cases, and the economic and social costs of eradication need to be carefully considered (Cutts and Steinglass 1998). Despite enormous progress, measles remains a leading vaccine-preventable cause of childhood mortality worldwide, and continues to cause outbreaks in communities with low vaccination coverage rates in industrialized nations. To achieve the measles mortality reduction goal, continued progress needs to be made in delivering measles vaccines to the world’s children.

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Madsen KM, Hviid A, Vestergaard M, Schendel D, Wohlfahrt J, Thorsen P, Olsen J, Melbye M (2002) A population-based study of measles, mumps, and rubella vaccination and autism. N Engl J Med 347:1477–1482 McQuillan GM, Kruszon-Moran D, Hyde TB, Forghani B, Bellini W, Dayan GH (2007) Seroprevalence of measles antibody in the US population, 1999–2004. J Infect Dis 196:1459–1464 Meissner HC, Strebel PM, Orenstein WA (2004) Measles vaccines and the potential for worldwide eradication of measles. Pediatrics 114:1065–1069 Monafo WJ, Haslam DB, Roberts RL, Zaki SR, Bellini WJ, Coffin CM (1994) Disseminated measles infection after vaccination in a child with a congenital immunodeficiency. J Pediatr 124:273–276 Morgan OW (2004) Following in the footsteps of smallpox: can we achieve the global eradication of measles? BMC Int Health Hum Rights 4:1 Moss WJ, Griffin DE (2006) Global measles elimination. Nat Rev Microbiol 4:900–908 Moss WJ, Cutts F, Griffin DE (1999) Implications of the human immunodeficiency virus epidemic for control and eradication of measles. Clin Infect Dis 29:106–112 Moss WJ, Monze M, Ryon JJ, Quinn TC, Griffin DE, Cutts F (2002) Prospective study of measles in hospitalized human immunodeficiency virus (HIV)-infected and HIV-uninfected children in Zambia. Clin Infect Dis 35:189–196 Moss WJ, Scott S, Mugala N, Ndhlovu Z, Beeler JA, Audet SA, Ngala M, Mwangala S, NkongaMwangilwa, Ryon JJ, Monze M, Kasolo F, Quinn TC, Cousens S, Griffin DE, Cutts FT (2007) Immunogenicity of standard-titer measles vaccine in HIV-1-infected and uninfected Zambian children: an observational study. J Infect Dis 196:347–355 Offit PA, Coffin SE (2003) Communicating science to the public: MMR vaccine and autism. Vaccine 22:1–6 Orenstein WA, Strebel PM, Papania M, Sutter RW, Bellini WJ, Cochi SL (2000) Measles eradication: is it in our future? Am J Public Health 90:1521–1525 Otten M, Kezaala R, Fall A, Masresha B, Martin R, Cairns L, Eggers R, Biellik R, Grabowsky M, Strebel P, Okwo-Bele JM, Nshimirimana D (2005) Public-health impact of accelerated measles control in the WHO African Region 2000–03. Lancet 366:832–839 Pan American Health Organization (1999) Measles eradication. Field guide. Washington, DC, Pan American Health Organization Parker AA, Staggs W, Dayan GH, Ortega-Sanchez IR, Rota PA, Lowe L, Boardman P, Teclaw R, Graves C, LeBaron CW (2006) Implications of a 2005 measles outbreak in Indiana for sustained elimination of measles in the United States. N Engl J Med 355:447–455 Permar SR, Moss WJ, Ryon JJ, Monze M, Cutts F, Quinn TC, Griffin DE (2001) Prolonged measles virus shedding in human immunodeficiency virus-infected children, detected by reverse transcriptase-polymerase chain reaction. J Infect Dis 183:532–538 Salama P, Assefa F, Talley L, Spiegel P, van Der V, Gotway CA (2001) Malnutrition, measles, mortality, and the humanitarian response during a famine in Ethiopia. JAMA 286:563–571 Schimmer B, Ihekweazu C (2006) Polio eradication and measles immunisation in Nigeria. Lancet Infect Dis 6:63–65 Scott S, Moss WJ, Cousens S, Beeler JA, Audet SA, Mugala N, Quinn TC, Griffin DE, Cutts FT (2007) The influence of HIV-1 exposure and infection on levels of passively acquired antibodies to measles virus in Zambian infants. Clin Infect Dis 45:1417–1424 Scott S, Mossong J, Moss WJ, Cutts FT, Cousens S (2008) Predicted impact of the HIV-1 epidemic on measles in developing countries: results from a dynamic age-structured model. Int J Epidemiol 37:356–367 Sencer DJ, Dull HB, Langmuir AD (1967) Epidemiologic basis for eradication of measles in 1967. Public Health Rep 82:253–256 Senessie C, Gage GN, von Elm E (2007) Delays in childhood immunization in a conflict area: a study from Sierra Leone during civil war. Confl Health 1:14

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Chapter 10

Measles: Old Vaccines, New Vaccines D.E. Griffin( ) and C.-H. Pan

Contents Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current Understanding of Protective Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inactivated Vaccines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Atypical Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Live Attenuated Virus Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determinants of Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of New Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of Vaccines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Isolation of measles virus in tissue culture by Enders and colleagues in the 1960s led to the development of the first measles vaccines. An inactivated vaccine provided only short-term protection and induced poor T cell responses and antibody that did not undergo affinity maturation. The response to this vaccine primed for atypical measles, a more severe form of measles, and was withdrawn. A live attenuated virus vaccine has been highly successful in protection from measles and in elimination of endemic measles virus transmission with the use of two doses. This vaccine is administered by injection between 9 and 15 months of age. Measles control would be facilitated if infants could be immunized at a younger age, if the vaccine were thermostable, and if delivery did not require a needle and syringe. To these ends, new vaccines are under development using macaques as an animal model and various combinations of the H, F, and N viral proteins. Promising studies have been reported using DNA vaccines, subunit vaccines, and virus-vectored vaccines. D.E. Griffin Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe St. Rm E5132 Baltimore, MD 21205, USA, e-mail: [email protected]

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Abbreviations FIMV HI HIV LAV MV

Formalin-inactivated measles vaccine Hemagglutination inhibition Human immunodeficiency virus Live attenuated virus vaccine Measles virus

Introduction Measles is a childhood rash disease associated with substantial morbidity and mortality and was an early target for development of a vaccine. The first attempts at vaccination by the Scottish physician Francis Home in 1749 were based on the principles of variolation. He inoculated individuals with blood taken from measles patients during the early stages of the rash with the reasoning that introduction of disease through the skin would lessen the effects on the lung (Home 1759). However, morbillization was in general unsuccessful and tended to result in transmission of measles (Hektoen 1905). Subsequent approaches between 1920 and 1940 designed to inactivate or attenuate the virus by culture in chick embryos also met with limited success (Maris et al. 1949). In 1954, the isolation of measles virus (MV) in tissue culture from the blood of a child with measles by Enders and Peebles opened the way for vaccine development (Enders and Peeble 1954). The Edmonston strain of MV was successfully propagated in human and monkey cells and led to the simultaneous development of both inactivated and live attenuated virus vaccines. Inactivated virus vaccines were developed using formalin or tween-ether for viral inactivation, but proved unsuccessful (Rauh and Schmidt 1965; Warren and Gallian 1962). A live attenuated virus vaccine (LAV), based on adaptation of MV to growth in chick cells (Enders et al. 1962), provided long-term protection from measles, and derivatives of this vaccine are used worldwide today (Peradze and Smorodintsev 1983). Prior to the widespread use of measles vaccine, measles was estimated to result in 5–8 million deaths each year. The decline in mortality from measles in developed countries can be attributed in part to improved nutrition and medical care, but mostly to effective delivery of LAV. However, in resource-poor countries, measles remains the second most common cause of vaccine-preventable deaths in children (CDC 2006). This is due in part to difficulty with delivery of two doses of vaccine to a high proportion of the population and to the inability to effectively immunize infants under the age of 9 months, leaving a window of vulnerability to infection (Moss and Griffin 2006). The adverse outcomes associated with certain vaccines have hampered new approaches to measles immunization, but have also increased interest in an understanding of the determinants of vaccine-induced protective immunity (Lambert et al. 2005). On the one hand, the early formalin-inactivated measles vaccine was not only poorly protective, but it also primed for a more severe disease known as atypical

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measles (Nader et al. 1968; Rauh and Schmidt 1965). Further, LAV was associated with increased mortality in girls when given in a high dose to young infants in countries with high rates of childhood mortality (Garenne et al. 1991; Holt et al. 1993). The biologic bases for these problems with prior MV vaccines are incompletely understood. Definition of protective immunity to measles could enlighten not only the development of new measles vaccines, but also the development of other viral vaccines.

Current Understanding of Protective Immunity Antibody Antibody can protect from MV infection and may contribute to recovery from infection (Albrecht et al. 1997; Endo et al. 2001). Antibody to MV is sufficient for protection because infants are protected by maternal antibody (Albrecht et al. 1997) and passive transfer of immune serum can modify or interfere with measles vaccination and can partially protect children from measles after exposure (Reilly et al. 1961). In infants, the level of maternal antibody correlates with failure of response to vaccination (Albrecht et al. 1997). In outbreaks, antibody levels correlate with protection from disease with a plaque reduction neutralizing titer (PRNT) of 120 mIU/ml generally considered the level needed to prevent disease (Chen et al. 1990). Furthermore, in vaccine studies the best correlate of protection from infection is the level of neutralizing antibody (Polack et al. 2000). Most neutralizing antibodies are directed to the H glycoprotein. Major conformational epitopes have been localized to regions between amino acids 368 and 396 and in the SLAM-binding region (Erlt et al. 2003; Liebert et al. 1994). Most of these epitopes are predicted to be a part of exposed surfaces on top of the molecule (Erlt et al. 2003; Langedijk et al. 1997). Although the H gene sequence is variable (Rota et al. 1992), MV is relatively stable in the regions required for receptor binding (Hashiguchi et al. 2007) and the current vaccines developed in the 1960s continue to provide protection from all wild-type strains of MV. Antibody to the highly conserved F protein also contributes to virus neutralization, probably by preventing fusion of the virus membrane with the cell membrane at the time of virus entry (Malvoisin and Wild 1990; Polack et al. 2000). Antibodies to other viral proteins, particularly the nucleocapsid (N) protein, are induced after infection with wild-type or vaccine strains of MV, but their role in protection is unclear (Graves et al. 1984).

Cellular Immunity The cellular immune response is also important for development of protective immunity. CD4+ T cells are essential for development of an antibody response that is mature with respect to avidity, isotype, and durability. Formation of germinal

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centers, a major site for class switch recombination and somatic hypermutation of immunoglobulin genes and for development of long-lived plasma cells is T celldependent (Fazilleau et al. 2007). CD8+ T cells are important for the early control of virus replication (Permar et al. 2003) and are also likely to contribute to protective immunity. T cell epitopes are present in many MV proteins and are particularly abundant in the H protein (Oh et al. 2006; Ota et al. 2007).

Inactivated Vaccines Development Inactivated MV vaccines were developed using formalin or tween-ether and the Edmonston B strain of MV (Warren and Gallian 1962). The alum-precipitated formalin-inactivated measles vaccine (FIMV) developed in the United States was given in a three-dose regimen and licensed in 1963 (Carter et al. 1962; Warren and Gallian 1962). Recipients of the inactivated vaccine developed moderate levels of neutralizing and hemagglutination inhibiting (HI) antibodies, but low levels of complement fixing (CF) antibodies (Carter et al. 1962; Feldman et al. 1962; Norrby et al. 1975). The vaccine was protective when exposure to measles occurred within several months after immunization (Feldman et al. 1962; Fulginiti and Kempe 1963; Measles Vaccines Committee 1968). However, antibody titers declined rapidly, and recipients again became susceptible to measles (Fulginiti and Kempe 1963; Rauh and Schmidt 1965). When infected, these previously FIMV-vaccinated individuals had a tendency to develop a more severe disease, atypical measles (Nader et al. 1968; Rauh and Schmidt 1965).

Atypical Measles Atypical measles was characterized by a higher and more prolonged fever, unusual skin lesions and severe pneumonitis compared to measles in unvaccinated persons (Fulginiti et al. 1967; Nader et al. 1968). The rash was often accompanied by evidence of hemorrhage or vesiculation and began on the extremities rather than the trunk. The pneumonitis included distinct nodular parenchymal lesions and hilar adenopathy (Gremillion and Crawford 1981; Young et al. 1970). Abdominal pain, hepatic dysfunction, headache, eosinophilia, pleural effusions, and edema were also described. Atypical measles was reported up to16 years after receipt of the inactivated vaccine. Administration of LAV after two to three doses of FIMV did not eliminate subsequent susceptibility to atypical measles and was sometimes associated with severe reactions at the site of LAV inoculation (Buser 1967; Chatterji and Mankad 1977; Fulginiti et al. 1968).

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Hypotheses about the pathogenesis of atypical measles included an abnormally intense cellular immune response (Fulginiti et al. 1968), an inability of the inactivated vaccine to induce local respiratory tract immunity (Bellanti et al. 1969) and a lack of production of antibody to F, which allowed virus to spread from cell to cell despite the development of antibody to H (Merz et al. 1980; Norrby et al. 1975). Studies in rhesus macaques have shown that FIMV induces no cytotoxic T cell response and that antibody does not undergo affinity maturation and production is short-lived (Polack et al. 1999, 2003a). The low-avidity antibody induced by FIMV can neutralize in vitro infection with viruses that use CD46 as a receptor, as routinely measured by PRNT assays in Vero cells, but cannot neutralize infection with wild-type viruses that primarily use SLAM as a receptor (Polack et al. 2003a). Subsequent infection with MV induces an anamnestic antibody response, but the antibody is also of low avidity and cannot neutralize wild-type virus. This leads to formation of complexes of non-neutralizing antibody and MV, resulting in immune complex deposition, vasculitis, and pneumonitis (Plowright and Ferris 1962; Polack et al. 2003a). The nature of the defect in immune priming exhibited by FIMV has not yet been identified. These problems of short-lived immunity and predisposition to enhanced disease, in the face of simultaneous development of the more successful LAV, led to the withdrawal of this vaccine in 1967.

Live Attenuated Virus Vaccines Development The process of adaptation of MV grown in primary human and monkey kidney and amnion cells to cells of nonsusceptible hosts, such as the chick embryo, led successfully to the development of LAV strains (Enders et al. 1960; Katz et al. 1958, 1959; Milovanovic et al. 1957; Schwarz and Zirbel 1959) (see the chapter by S.L. Katz, this volume). The first attenuated live measles vaccine was developed by passage of the Edmonston strain of MV in chick embryo fibroblasts to produce the Edmonston B virus (Enders et al. 1962). Inoculation of this virus into primates produced no clinical symptoms, no detectable viremia, and no spread to the respiratory tract (Auwaerter et al. 1999), but did induce an immune response that protected the monkeys from subsequent challenge with wild-type virus (Enders et al. 1960). The Edmonston B strain of LAV protected children from measles (Krugman 1971) and was licensed in March 1963, but induced fever and rash in a large proportion of children (Katz et al. 1960). Reactions were reduced when MV antibody was given at the same time as the vaccine (Ad Hoc Advisory committee on Measles Control 1963; Milovanovic 1965; Reilly et al. 1961; Stokes et al. 1961). More extensive passage of the Edmonston B virus in chick embryo fibroblasts at reduced temperature produced the more attenuated vaccines, Schwarz and Moraten, in

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extensive use today (Rota et al. 1994; Schwarz 1962). Other Edmonston-derived vaccine strains (e.g., Zagreb, AIK-C) and attenuated strains developed independently (e.g., CAM, Leningard-16, Shanghai-191) are also successful LAVs (Hirayama 1983; Jianzhi and Zhihui 1983; Peradze and Smorodintsev 1983; Sakata et al. 1978). Few differences have been described among MV vaccine strains (all genotype A) regardless of the geographic origin of the parent virus (Rota et al. 1994). However, there may be some biologic differences. For instance, Edmonston-Zagreb is produced in human diploid cells, rather than chick embryo fibroblasts, and may be more immunogenic in young infants and when delivered by the aerosol route than other strains (Cutts et al. 1997). Lyophilized LAV is relatively stable, but the reconstituted vaccine rapidly loses infectivity. LAV is inactivated by light and heat and after reconstitution loses about half of its potency at 20°C and almost all potency at 37°C within 1 h (Melnick 1996). Therefore, a cold chain must be maintained for the vaccine prior to and after reconstitution. Attenuated strains replicate less efficiently in vivo than wild-type MV (Auwaerter et al. 1999; Van Binnendijk et al. 1994), perhaps due to altered cellular tropism (Condack et al. 2007). However, LAV induces both neutralizing antibody and cellular immune responses qualitatively similar to that induced by natural disease, although titers are lower (Krugman 1971; Ovsyannikova et al. 2003). Antibodies first appear 12–15 days after vaccination and peak at 1–3 months. In many countries, LAV is combined with other live attenuated virus vaccines such as those for mumps, rubella (MMR) and varicella (MMRV). These measlescontaining vaccines have proven safe and effective and have saved the lives of many millions of children (Bellini et al. 1994; Wolfson et al. 2007).

Determinants of Response Age The recommended age of vaccination varies from 6 to 15 months. The probability of seroconversion and the amounts of antibody induced are determined by the levels of persisting MV-specific maternal antibody and the age of the infant at the time of vaccination (Cutts et al. 1995; Gans et al. 1998, 2004; Redd et al. 2004; Reilly et al. 1961). Levels of passively acquired antibody are dependent on the mother’s level of antibody, on the transfer of antibody across the placenta and on the rate of decay in the infant (Caceres et al. 2000). The cellular immune response is induced more readily than antibody in young infants with maternal antibody (Gans et al. 2004). As measles is controlled in a region, an increasing proportion of mothers will have measles immunity induced by vaccination rather than natural infection. This will result in lower levels of passively acquired antibody in infants and the possibility of lowering the age of vaccination (Lennon and Black 1986; Maldonado et al. 1995; Markowitz et al. 1996; Pabst et al. 1992). Currently, the proportions of children that

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develop protective levels of antibody based on age of vaccination are approximately 85% at 9 months and 95% at 12 months (Cutts et al. 1995). The recommended age for measles vaccination varies regionally and is a balance between the optimum age for seroconversion and the probability of acquiring measles before that age (Cutts et al. 1995). In areas where measles remains prevalent, measles vaccination is routinely performed at 9 months, whereas in areas with little measles, vaccination is often at 12–15 months. During epidemics and for human immunodeficiency virus (HIV)infected infants in developing countries, vaccination at 6 months with a second dose at 9 or 12 months is recommended (Hutchins et al. 2001; WHO/UNICEF 1989).

Route LAV is administered subcutaneously or intramuscularly. However, there is substantial interest in alternate routes of delivery that would not require needles and syringes. Neither oral nor intranasal administration is effective (Black and Sheridan 1960; Stittelaar et al. 2002a), but respiratory delivery may be more promising. There are several ongoing efforts to develop and evaluate aerosol delivery of aqueous and dry powder forms of LAV (Cutts et al. 1997; de Swart et al. 2006, 2007). Aerosol administration of the aqueous vaccine is highly effective in boosting preexisting antibody and may hold promise for use in older children (Cutts et al. 1997; Sabin 1991; Sabin et al. 1983). Respiratory routes of vaccination have also been advocated as a means to lower the age of immunization (Sabin et al. 1983, 1985). However, the primary immune response to aerosolized measles vaccine, particularly in young infants, is lower than it is to subcutaneous administration (Low et al. 2008; Wong et al. 2004, 2006). The reasons for this are not known, but may be related to dose, efficiency of delivery, or ability of the vaccine virus to establish infection in the respiratory tract. Responses of macaques to respiratory delivery of a dry powder vaccine are lower than to aqueous vaccine for reasons that are not yet clear (de Swart et al. 2007).

Host Determinants Genetic background affects the likelihood of seroconversion and antibody titers (Dhiman et al. 2007; Ovsyannikova et al. 2004, 2006). Common childhood illnesses at the time of vaccination may also have an effect (Migasena et al. 1997). Any potential decrease in seroconversion must be balanced against the loss of the opportunity for vaccination and the consequent risk of the child acquiring measles. Similar compromises must be considered with respect to immunizing individuals infected with HIV-1 (Cutts et al. 1999). LAV is contraindicated in individuals with severe deficiencies of cellular immunity because of the possibility of progressive pulmonary or CNS infection (Bitnun et al. 1999; Mawhinney et al. 1971; Monafo et al. 1994). However, LAV has generally been well tolerated in HIV-infected

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children and adults, although the antibody responses are of lower avidity and less durable than those of HIV-uninfected children (Brena et al. 1993; McLaughlin et al. 1988; Moss et al. 2007; Rudy et al. 1994). Because of the potential severity of wildtype MV infection in HIV-infected individuals (Budka et al. 1996; CDC 1988; Moss et al. 2008), LAV is recommended for administration without respect to HIV status in most countries. However, LAV is not recommended for those with known low CD4+ T cell counts (Committee on Infectious Diseases and Committee on Pediatric AIDS 1999) because of the possibility of progressive infection (Angel et al. 1998; Goon et al. 2001).

Dose The dose of MV routinely used for immunization is between 103 and 104 plaqueforming units. Overall, the efficacy of a single dose of measles vaccine in infancy is estimated at 80%–95% (Gans et al. 2004). When 100-fold higher doses were used, seroconversion in younger infants improved and in 1990 the WHO recommended the high-titer vaccine for use in countries with significant measles transmission (WHO 1990). However, subsequent follow-up of children receiving high-titer vaccines in countries with high childhood mortality showed an increased mortality in girls over the subsequent 2–3 years and the recommendation was withdrawn (Cutts and Markowitz 1994; Holt et al. 1993; Knudsen et al. 1996). Mortality was due to a relative increase in deaths due to other infections (Aaby et al. 1996). The pathogenesis of delayed increased mortality after the high-titer vaccine is not understood, but occurred primarily in those who developed a rash after vaccination and may be related to long-term suppression of immune responses to other pathogens similar to that induced by measles (Seng et al. 1999) or to a change in the sequence of vaccine delivery (Aaby et al. 2003a, 2003b, 2006). The duration of vaccine-induced immunity is variable. In general, levels of antibody are lower after vaccination than after recovery from natural disease and MVspecific antibody and CD4+ T cells decay with time (Christenson and Bottiger 1994; Eghafona et al. 1991; Krugman 1971; Naniche et al. 2004). Secondary vaccine failure rates of 5% have been estimated at 10–15 years after vaccination, but are probably lower when vaccine is given after the age of 1 year (Anders et al. 1996; Mathias et al. 1989; Ozanne and d’Halewyn 1992). Decreasing antibody titers do not necessarily imply a complete loss of protective immunity, as a secondary immune response usually develops after re-exposure to MV, with a rapid rise in antibody and absence of clinical disease (Ozanne and d’Halewyn 1992). Because of the high infectivity of MV and the fact that not everyone develops protective immunity following vaccination, a single dose of measles vaccine does not achieve a sufficient level of population immunity to eliminate endemic MV transmission (see the chapter by P.A. Rota et al., this volume). Therefore, to achieve 95% immunity, a second dose of vaccine is necessary to immunize persons who missed or did not respond to the first dose (CDC 2000; Cutts et al. 1999; Gay 2004; Orenstein et al. 2006). The two-dose strategy has been credited with elimination of

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indigenous measles in many countries in which it has been employed (Cutts et al. 1999). Two broad strategies to administer the second dose have been used. In countries with sufficient infrastructure, the second dose can be delivered as a part of routine vaccination, typically prior to the start of school with school entry requirements enforcing the policy (Orenstein 2006). A second approach uses mass supplementary immunization campaigns to deliver the second dose of vaccine in a wide geographic area (de Quadros et al. 2004). The goal is to rapidly achieve a high level of population immunity and interrupt MV transmission. However, even highly immunized populations in countries that have eliminated endemic transmission are vulnerable to localized outbreaks associated with importation from areas where measles remains endemic (Rota et al. 2002).

Development of New Vaccines Rationale A new vaccine would be advantageous if it would allow vaccination of infants before 6 months of age. This would both close the window of susceptibility between decay of maternal antibody and vaccination and facilitate vaccine delivery by allowing measles vaccine to be given along with other WHO Expanded Program for Immunization (EPI) vaccines (El Kasmi and Muller 2001). Additional motivations for development of a new vaccine would be to eliminate the need for a cold chain, to avoid the use of needles and syringes for delivery (Mitragotri 2005), and to provide a vaccine that would be safe for immunocompromised individuals. Such a vaccine would need to induce protective immunity and overcome the obstacles of immunologic immaturity and maternal antibody present in young infants. Several animal models, including transgenic mice and cotton rats, have been used for testing potential new measles vaccines, but only non-human primates, particularly rhesus macaques, develop a disease similar to that of humans and offer the opportunity for assessing both protection from wild-type MV challenge and priming for enhanced disease (Auwaerter et al. 1999; Combredet et al. 2003; El Mubarak et al. 2007; Polack et al. 1999; Van Binnenkijk et al. 1995). A number of experimental vaccines have been developed and there is an increasing understanding of measles protective immunity. Vaccinations with individual MV proteins expressed in viral or bacterial vectors or as DNA, peptides, or proteins have been explored in mice and cotton rats (El Kasmi and Muller 2001; Wyde et al. 2000). A more limited portfolio of experimental vaccines have been studied in macaques (Polack et al. 2000; Premenko-Lanier et al. 2006;Stittelaar et al. 2000b, 2002c; Van Binnenkijk et al. 1997). The choices of MV antigens to be used in a new vaccine have focused on proteins required for induction of neutralizing antibody and CD4+ and CD8+ T cell responses. Neutralizing antibody is directed primarily against H and to a lesser

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degree against F. However, in several studies it has been observed that the level of neutralizing antibody is higher when H is used alone for vaccination rather than in combination with F (Pan et al. 2005; Pasetti et al. 2007; Polack et al. 2000). The reason for this is not clear, but may be related to the fact that H primes for a type 2 cytokine response while F primes for a type 1 response and that these responses are cross-modulated when H and F are given together (Polack et al. 2003b). H contains many human epitopes presented by HLA-class I (Oh et al. 2006; Ota et al. 2007) and induces both CD4+ and CD8+ T cell responses in macaques (Pan et al. 2005, 2008; Polack et al. 2000); N has occasionally been included to enhance the opportunity for induction of T cell responses.

Types of Vaccines DNA Delivery of viral genes into host cells for processing and antigen presentation without the need for virus infection make DNA vaccines an attractive possibility for development. In addition, DNA vaccines are thermostable, inexpensive to manufacture, do not induce antivector immunity, and induce strong T cell responses. Immunization with DNA expressing N did not protect against intracerebral challenge with rodent-adapted MV (Fooks et al. 1996), but DNA expressing H or F delivered by gene gun, intramuscularly or by various mucosal routes induced good humoral and cellular responses in mice and cotton rats (Cardoso et al. 1996; Etchart et al. 1997; Pasetti et al. 2003; Yang et al. 1996). Several DNA vaccines have been tested in juvenile cynomolgus or rhesus macaques. Transdermal delivery of two doses of plasmids encoding MV proteins elicited low serum antibody responses, but priming for more rapid antibody and cellular immune responses and a tendency for lower viremia after challenge (Stittelaar et al. 2002b). Intradermal or gene gun delivery of two doses of nonadjuvanted DNA encoding H, F, or H and F elicited cytotoxic T cell responses and sustained antibody responses of variable titer (Polack et al. 2000). Protection from challenge 2 years after the initial immunization correlated with the levels of neutralizing antibody. All monkeys having antibody levels less than 120 mIU/ml (one immunized with F, one with H, and one with H + F) developed a rash and viremia. However, the rashes were mild and there was no suggestion of atypical measles. Monkeys with intermediate levels of antibody developed viremia without a rash (partial protection) and monkeys with high levels of antibody did not develop a viremia or rash (full protection). These studies indicated that DNA vaccines could protect from measles and that DNA immunization did not predispose to atypical measles. Studies of the same or similar vaccines in infant macaques have shown induction of lower levels of antibody, particularly in the face of maternal antibody, and limited protection from challenge (Premenko-Lanier et al. 2006).

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More recent studies have focused on improved plasmid design and the use of adjuvants to enhance immunogenicity of DNA vaccines and more reliably exceed the threshold of antibody needed for complete protection. New plasmids have used different promoter and expression strategies. Adjuvants have included cytokines (Premenko-Lanier et al. 2004) and formulation of the DNA with complex carbohydrates or lipids designed to improve DNA entry into cells, to provide a depot for prolonged release or to stimulate B cell production of antibody (Kaslow 2004). Improved plasmid design has included codon optimization and use of alphavirus DNA vectors (e.g., SINCP) for a layered DNA/RNA expression of antigen (Dubensky et al. 1996). In this approach, the DNA used for immunization consists of the genes coding for the alphavirus nonstructural proteins, the subgenomic promoter, and the MV protein. When the DNA is transcribed to mRNA, the nonstructural proteins are translated, replicate the RNA, and produce abundant subgenomic mRNAs that code for the antigen. Amplification of the mRNAs results in large amounts of synthesized protein (Boorsma et al. 2003; Dubensky et al. 1996; Herweijer et al. 1995). In addition, these vaccines activate innate immune pathways to increase immunogenicity of the antigens encoded (Leitner et al. 2003). Sindbis virus-based replicon plasmid (SINCP)-based vaccines expressing H and F have been used successfully to prime responses of juvenile and infant macaques to LAV (Pasetti et al. 2007). A number of DNA adjuvants have also been studied. Adsorption of DNA onto biodegradable cationic polylactide co-glycolide (PLG) microparticles (Denis-Mize et al. 2000; O’Hagan et al. 2001) delivers the DNA vaccine to and activates antigenpresenting cells, increases DNA persistence and recruits mononuclear cells to the site (Denis-Mize et al. 2003). An improvement over naked DNA for induction of both antibody and T cell responses has been demonstrated in rodents and primates for other candidate vaccines (Mollenkipf et al. 2004; O’Hagan et al. 2001, 2004; Otten et al. 2005). PLG formulation improved the antibody responses of mice, but not the responses of macaques, to MV DNA vaccination (Pan et al. 2008). PLG/ SINCP-H given intramuscularly elicited short-lived neutralizing antibody and memory T cells in juvenile rhesus macaques that provided partial protection from challenge. The PLG/SINCP-H vaccine was less effective when given intradermally with lower antibody and T cell responses than after intramuscular inoculation. Monkeys vaccinated intradermally were not protected from rash or viremia after challenge. Monkeys vaccinated with low-dose PLG/SINCP-H intradermally had higher viremias, more severe rashes and higher eosinophil counts after challenge than monkeys that received the control vaccine, suggesting exacerbated disease (Pan et al. 2008). Another class of adjuvants that has been explored is cationic lipids. Cationic lipids are safe and well tolerated in humans and other animals (Nabel et al. 1993; Parker et al. 1995). Vaxfectin consists of an equimolar mixture of the cationic lipid GAP-DMORIE [(+)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide)] and a neutral co-lipid DPyPE (1,2-diphytanoylsn-glydero-3-phosphoethanolamine) (Hartikka et al. 2001). Immunization with

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Vaxfectin-formulated, codon-optimized DNAs expressing the MV H and F proteins elicited strong antibody and T cell responses in mice and monkeys and provided complete protection against rash and viremia after challenge with wild-type MV in both juvenile and infant rhesus macaques. Two doses of vaccine delivered either intradermally or intramuscularly to juvenile or infant rhesus macaques induced MV-specific antibody responses that were durable, neutralizing, and of high avidity, as well as MV-specific interferon (IFN)-γ-producing memory T cells.

Recombinant and Subunit Proteins and Peptides Recombinant proteins, peptides, and proteins purified from MV have been used for vaccine development. In mice, peptides from H and F have been used alone and in combination to induce neutralizing antibody (El Kasmi and Muller 2001) and H protein expressed in tobacco plants and delivered orally could boost the response to a DNA vaccine (Webster et al. 2002). Immune-stimulating complexes (iscoms) incorporating the F and H proteins into a matrix with quilaja saponins, phospholipids, and cholesterol stimulated HI and hemolysis-inhibiting antibodies in mice (Stittelaar et al. 2000a). Immunized mice could be protected from intracerebral challenge with a neurotropic strain of MV (Condack et al. 2007; Varsanyi et al. 1987). In cynomolgus macaques, iscoms induced durable MV-specific antibody in the presence and absence of passively transferred antibody and provided partial protection from challenge (Stittelaar et al. 2002c; Van Binnendijk et al. 1997). MV proteins from infected cells mixed with a proteosome adjuvant consisting of Neisseria meningitidis outer membrane proteins and Shigella flexneri 2a lipopolysaccharide stimulated neutralizing antibody in mice after intranasal administration (Chabot et al. 2005) and could boost the responses of rhesus macaques to a DNA vaccine (Pasetti et al. 2007).

Vectored by Other Viruses or Bacteria Several viruses have been used to express MV proteins and tested as experimental vaccines. The first studies were done with vaccinia virus expressing H, F, and/or N and induced immune responses in mice, rats, and macaques (El Kasmi and Muller 2001). This vaccine was not able to stimulate an antibody response in the presence of passively acquired antibody, but did provide partial protection from challenge, presumably mediated by the observed MV-specific T cell responses (Van Binnendijk et al. 1997). Subsequently, studies of the replication-defective modified vaccinia virus Ankara (MVA) expressing H and F showed that two doses of 108 pfu delivered intramuscularly and intranasally 1 month apart elicited neutralizing antibody and T cell responses in juvenile cynomolgus macaques in the presence and absence of passively transferred antibody (Sittelaar et al. 2000b). These monkeys were all protected from challenge 1 year after vaccination. This vaccine is safe in immunosuppressed macaques (Stittelaar et al. 2001).

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Alphavirus replicon particle vaccines expressing MV H induced high-titered, dose-dependent, MV-neutralizing antibody after a single vaccination in mice. Vaccination of juvenile rhesus macaques with a single dose, and infant macaques with two doses, of 108 particles induced sustained levels of high-titered MV-neutralizing antibody and IFN-γ-producing memory T cells. Most monkeys were protected from disease, but not from viremia when challenged 18 months later (Pan et al. 2005). Recombinant Bacille-Calmette-Guérin, the mycobacteria used for neonatal immunization against tuberculosis, has been engineered to express the MV N protein for immunization of infant rhesus macaques (Zhu et al. 1997). Cellular immune responses were elicited after challenge and there was reduced lung inflammation, but monkeys were not protected from systemic infection.

Summary and Future Directions The current live attenuated measles virus vaccine is safe and has been effective in eliminating endemic MV transmission in regions that have successfully delivered two doses of vaccine to a high proportion of the population. Challenges to measles control remain in many regions due to difficulty with delivery of two doses as a part of routine childhood vaccination programs. Development of a new vaccine that did not require a cold chain or delivery with needle and syringe and that could be safely and effectively used in infants under the age of 6 months could facilitate measles control in many parts of the world. Investigation of the efficacy of experimental measles vaccines has advanced our knowledge of the determinants of protective immunity and may lead to new vaccines to aid in the control of measles. Acknowledgements Work from the authors’ laboratory was supported by research grants from the National Institutes of Health (AI 23047) and the Bill and Melinda Gates Foundation.

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Polack FP, Auwaerter PG, Lee S-H, Nousari HC, Valsamakis A, Leiferman KM, Diwan A, Adams RJ, Griffin DE (1999) Production of atypical measles in rhesus macaques: evidence for disease mediated by immune complex formation and eosinophils in the presence of fusion-inhibiting antibody. Nat Med 5:629–634 Polack F, Lee S, Permar S, Manyara E, Nousari H, Jeng Y, Mustafa F, Valsamakis A, Adams R, Robinson H, Griffin D (2000) Successful DNA immunization against measles: neutralizing antibody against either the hemagglutinin or fusion glycoprotein protects rhesus macaques without evidence of atypical measles. Nat Med 6:776–781 Polack FP, Hoffman SJ, Crujeiras G, Griffin DE (2003a) A role for nonprotective complementfixing antibodies with low avidity for measles virus in atypical measles. Nat Med 9:1209–1213 Polack FP, Hoffman SJ, Moss WJ, Griffin DE (2003b) Differential effects of priming with DNA vaccines encoding the hemagglutinin and/or fusion proteins on cytokine responses after measles virus challenge. J Infect Dis 187:1794–1800 Premenko-Lanier M, Rota P, Rhodes G, Bellini W, McChesney M (2004) Prior DNA vaccination does not interfere with the live-attenuated measles vaccine. Vaccine 22:762–765 Premenko-Lanier M, Hodge G, Rota P, Tamin A, Bellini W, McChesney M (2006) Maternal antibody inhibits both cellular and humoral immunity in response to measles vaccination at birth. Virology 350:429–432 Rauh LW, Schmidt R (1965) Measles immunization with killed virus vaccine. Am J Dis Child 109:232–237 Redd SC, King GE, Heath JL, Forghani B, Bellini WJ, Markowitz LE (2004) Comparison of vaccination with measles-mumps-rubella vaccine at 9, 12, and 15 months of age. J Infect Dis 189 [Suppl 1]:S116–S122 Reilly CM, Stokes J Jr, Buynak EB, Goldner H, Hilleman MR (1961) Living attenuated measlesvirus vaccine in early infancy. Studies of the role of passive antibody in immunization. N Engl J Med 265:165–169 Rota JS, Hummel KB, Rota PA, Bellini WJ (1992) Genetic variability of the glycoprotein genes of current wild-type measles isolates. Virology 188:135–142 Rota JS, Wang ZD, Rota PA, Bellini WJ (1994) Comparison of sequences of the H, F, and N coding genes of measles virus vaccine strains. Virus Res 31:317–330 Rota PA, Liffick SL, Rota JS, Katz RS, Redd S, Papania M, Bellini WJ (2002) Molecular epidemiology of measles viruses in the United States, 1997–2001. Emerg Infect Dis 8:902–908 Rudy BJ, Rutstein RM, Pinto-Martin J (1994) Responses to measles immunization in children infected with human immunodeficiency virus. J Pediatr 125:72–74 Sabin AB (1991) Measles, killer of millions in developing countries: strategy for rapid elimination and continuing control. Eur J Epidemiol 7:1–22 Sabin AB, Arechiga AF, de Castro JF, Sever JL, Madden DL, Shekarchi I, Albrecht P (1983) Successful immunization of children with and without maternal antibody by aerosolized measles vaccine. JAMA 249:2651–2662 Sabin AB, Albrecht P, Takeda AK, Ribeiro EM, Veronesi R (1985) High effectiveness of aerosolized chick embryo fibroblast measles vaccine in seven-month-old and older infants. J Infect Dis 152:1231–1237 Sakata H, Hirayama M, Kimura M (1978) A ten-year follow-up study on measles vaccination in Japan: evaluation of the efficacy analyzed on a computer system. Japan. J Med Sci Biol 31:339–356 Schwarz AJF (1962) Preliminary tests of a highly attenuated measles vaccine. Am J Dis Child 103:216–219 Schwarz AJF, Zirbel LW (1959) Propagation of measles virus in non-primate tissue culture. I. Propagation in bovine kidney tissue culture. Proc Soc Exp Biol Med 102:711–714 Seng R, Samb B, Simondon F, Cisse B, Soumare M, Jensen H, Bennett J, Whittle H, Aaby P (1999) Increased long term mortality associated with rash after early measles vaccination in rural Senegal. Pediatr Infect Dis J 18:48–52

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Stittelaar KJ, Boes J, Kersten GF, Spiekstra A, Mulder PG, de Vries P, Roholl PJ, Dalsgaard K, van den DG, van Alphen L, Osterhaus AD (2000a) In vivo antibody response and in vitro CTL activation induced by selected measles vaccine candidates, prepared with purified Quil A components. Vaccine 18:2482–2493 Stittelaar KJ, Wyatt LS, de Swart RL, Vos HW, Groen J, van Amerongen G, Van Binnendijk RS, Rozenblatt S, Moss B, Osterhaus AD (2000b) Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies. J Virol 74:4236–4243 Stittelaar KJ, Kuiken T, de Swart RL, van Amerongen G, Vos HW, Niesters HG, van Schalkwijk P, van der KT, Wyatt LS, Moss B, Osterhaus AD (2001) Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed macaques. Vaccine 19:3700–3709 Stittelaar KJ, de Swart RL, Vos HW, van Amerongen G, Agafonov AP, Nechaeva EA, Osterhaus AD (2002a) Enteric administration of a live attenuated measles vaccine does not induce protective immunity in a macaque model. Vaccine 20:2906–2912 Stittelaar KJ, de Swart RL, Vos HW, van Amerongen G, Sixt N, Wild TF, Osterhaus AD (2002b) Priming of measles virus-specific humoral- and cellular-immune responses in macaques by DNA vaccination. Vaccine 20:2022–2026 Stittelaar KJ, Vos HW, van Amerongen G, Kersten GF, Osterhaus AD, de Swart RL (2002c) Longevity of neutralizing antibody levels in macaques vaccinated with Quil A-adjuvanted measles vaccine candidates. Vaccine 21:155–157 Stokes J Jr, Hilleman MR, Weibel RE, Buynak EB, Halenda R, Goldner H (1961) Efficacy of live, attenuated measles-virus vaccine given with human immune globulin. A preliminary report. N Engl J Med 265:507–513 Van Binnendijk RS, van der Heijden RWJ, van Amerongen G, Uytdehaag FGCM, Osterhaus ADME (1994) Viral replication and development of specific immunity in macaques after infection with different measles virus strains. J Infect Dis 170:443–448 Van Binnendijk RS, van der Heijden RWJ, Osterhaus ADME (1995) Monkeys in measles research. Curr Top Microbiol Immunol 191:135–148 Van Binnendijk RS, Poelen MCM, van Amerongen G, de Vries P, Osterhaus ADME (1997) Protective immunity in macaques vaccinated with live attenuated, recombinant, and subunit measles vaccines in the presence of passively acquired antibodies. J Infect Dis 175:524–532 Varsanyi TM, Morein B, Love A, Norrby E (1987) Protection against lethal measles virus infection in mice by immune-stimulating complexes containing the hemagglutinin or fusion protein. J Virol 61:3896–3901 Warren J, Gallian MJ (1962) Concentrated inactivated measles-virus vaccine: preparation and antigenic potency. Am J Dis Child 103:248–253 Webster DE, Cooney ML, Huang Z, Drew DR, Ramshaw IA, Dry IB, Strugnell RA, Martin JL, Wesselingh SL (2002) Successful boosting of a DNA measles immunization with an oral plant-derived measles virus vaccine. J Virol 76:7910–7912 World Health Organization (1990) Expanded programme on immunization. Global Advisory Report, Part 1. Wkly Epidemiol Rep 65:5–12 WHO/UNICEF (1989) Joint WHO/UNICEF statement on early immunization for HIV-infected children. Global programme on AIDS and expanded programme on immunization. Wkly Epidemiol Rec 7:48 Wolfson LJ, Strebel PM, Gacic-Dobo M, Hoekstra EJ, McFarland JW, Hersh BS (2007) Has the 2005 measles mortality reduction goal been achieved? A natural history modelling study. Lancet 369:191–200 Wong-Chew RM, Islas-Romero R, Garcia-Garcia ML, Beeler JA, Audet S, Santos-Preciado JI, Gans H, Lew-Yasukawa L, Maldonado YA, Arvin AM, Valdespino-Gomez JL (2004) Induction of cellular and humoral immunity after aerosol or subcutaneous administration of EdmonstonZagreb measles vaccine as a primary dose to 12-month-old children. J Infect Dis 189:254–257 Wong-Chew RM, Islas-Romero R, Garcia-Garcia ML, Beeler JA, Audet S, Santos-Preciado JI, Gans H, Lew-Yasukawa L, Maldonado YA, Arvin AM, Valdespino-Gomez JL (2006)

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Immunogenicity of aerosol measles vaccine given as the primary measles immunization to nine-month-old Mexican children. Vaccine 24:683–690 Wyde PR, Stittelaar KJ, Osterhaus AD, Guzman E, Gilbert BE (2000) Use of cotton rats for preclinical evaluation of measles vaccines. Vaccine 19:42–53 Yang K, Mustafa F, Valsamakis A, Santoro JC, Griffin DE, Robinson HL (1996) Early studies on DNA-based immunizations for measles virus. Vaccine 15:888–892 Young LW, Smith DI, Glasgow LA (1970) Pneumonia of atypical measles. Residual nodular lesions. Am J Roentgenol 110:439–448 Zhu Y, Fennelly G, Miller C, Tarara R, Saxe I, Bloom B, McChesney M (1997) Recombinant Bacille Calmette-Guérin expressing the measles virus nucleoprotein protects infant Rhesus macaques from measles virus pneumonia. J Infect Dis 176:1445–1453

Chapter 11

Measles Virus for Cancer Therapy S.J. Russell( ) and K.W. Peng

Contents Oncolytic Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why Attenuated Measles Viruses Are Attractive Oncolytic Agents . . . . . . . . . . . . . . . . . . . . Tumor Targeting Through CD46 Density Discrimination. . . . . . . . . . . . . . . . . . . . . . . . . . Safety to Population. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncolytic Activity of Wild-Type and Attenuated (Vaccine Strain) Measles Viruses . . . . . Engineering Attenuated Measles Viruses to Enhance Their Utility as Oncolytic Agents. . . . Noninvasive Monitoring of Measles Virus Spread . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arming the Virus to Combat Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Engineering Measles Virus Tropism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Translation of Recombinant Oncolytic Measles Viruses . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Choice of Animal Models for Toxicology Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacology and Toxicology Testing of MV-CEA and MV-NIS . . . . . . . . . . . . . . . . . . . Current Status of Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enhancing the Efficacy of Oncolytic Measles Viruses: Immune Evasion and Immune Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of Cell Carriers to Deliver Oncolytic Measles Viruses to Sites of Tumor Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Combination Therapy with Oncolytic Measles Viruses and Immunosuppressive Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current Status and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Measles virus offers an ideal platform from which to build a new generation of safe, effective oncolytic viruses. Occasional so-called spontaneous tumor regressions have occurred during natural measles infections, but common tumors do not express SLAM, the wild-type MV receptor, and are therefore not susceptible to the virus. Serendipitously, attenuated vaccine strains of measles virus have adapted to use CD46, a regulator of complement activation that is expressed in higher abundance on human tumor cells than on their nontransformed counterparts. S.J. Russell Mayo Clinic, Department of Molecular Medicine, 200 1st Street SW, Rochester, MN 55905, USA, e-mail: [email protected]

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For this reason, attenuated measles viruses are potent and selective oncolytic agents showing impressive antitumor activity in mouse xenograft models. The viruses can be engineered to enhance their tumor specificity, increase their antitumor potency, and facilitate noninvasive in vivo monitoring of their spread. A major impediment to the successful deployment of oncolytic measles viruses as anticancer agents is the high prevalence of preexisting anti-measles immunity, which impedes bloodstream delivery and curtails intratumoral virus spread. It is hoped that these problems can be addressed by delivering the virus inside measles-infected cell carriers and/or by concomitant administration of immunosuppressive drugs. From a safety perspective, population immunity provides an excellent defense against measles spread from patient to carers and, in 50 years of human experience, reversion of attenuated measles to a wild-type pathogenic phenotype has not been observed. Clinical trials testing oncolytic measles viruses as an experimental cancer therapy are currently underway.

Oncolytic Viruses Viruses that replicate selectively in neoplastic tissues (oncolytic viruses) hold considerable promise as novel therapeutic agents for the treatment of human malignancies and many such agents are currently under investigation, both in preclinical studies and in human clinical trials (Aghi and Martuza 2005; Hermiston 2006; Liu et al. 2007; Tai and Kasahara 2008). The existence of viruses was not recognized until the turn of the nineteenth century, but ever since that time, they have continued to attract considerable interest as possible agents of tumor destruction (Kelly and Russell 2007; Sinkovics and Horvath 2000). Clinical observations suggested that, given the right set of conditions, cancers would sometimes regress during naturally acquired virus infections (Kelly and Russell 2007; Bluming and Ziegler 1971; Zygiert 1971; Hoster et al. 1949). Clinical trials were therefore conducted in which a variety of different human and animal viruses were administered to cancer patients (Kelly and Russell 2007; Sjoùozi et al. 1988; Asada 1974; Okuno et al. 1978; Newman and Southam 1954; Southam and Moore 1952). Most often, these viruses were arrested by the host immune system and did not significantly impact tumor growth (Huebner et al. 1956). However, in a few immunosuppressed patients, the infection took and tumors regressed, although all too often, this was associated with unacceptable morbidities due to infection of normal tissues. Attempts to address the specificity problem continued throughout the 1950s and 1960s, but the results, although encouraging, were not compelling, and with the advent of anticancer chemotherapy, the concept of using replication competent viruses as anticancer agents was largely eclipsed (Kelly and Russell 2007). However, by the 1980s it was clear that even the combination of surgery, radiotherapy, and anticancer chemotherapy was failing to substantially impact cancer mortality and with the advent of modern virology accompanied by powerful reverse genetic systems, there came a resurgence of interest in oncolytic viruses (Russell and Peng 2007; Campbell and Gromeier 2005; Lichty et al. 2004). During

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the past two decades, oncolytic virotherapy has reestablished itself as a respectable field of research and there are new and numerous ongoing early-phase clinical trials testing a wide variety of oncolytic viruses representing many virus families (Liu et al. 2007; Lorence et al. 2007; Thorne et al. 2007; Forsyth et al. 2008; Yu and Fang 2007; Shen and Neumunaitis 2006; Kinoh and Inoue 2008; Nakamura and Russell 2004).

Why Attenuated Measles Viruses Are Attractive Oncolytic Agents Safety concerns arising from the use of oncolytic viruses for human cancer therapy can be divided into two areas: risk to the patient and risk to the population (Russell 1994). To minimize risk to the patient, an ideal oncolytic virus should be selective for the tumor, nonpathogenic for normal host tissues, nonpersistent, and genetically stable. To minimize risk to the population, in addition to the above characteristics, the virus should be nontransmissible and preferably derived from a virus to which the population is generally immune (Russell 1994). Attenuated measles viruses fulfill the above requirements. During the past 50 years, live attenuated measles viruses have been administered as vaccines to more than a billion people and the safety record has been outstanding (Griffin 2001; Nakamura and Russell 2004). Very occasionally, in people with severely compromised immune functions, the viral vaccine has propagated and caused disease in the recipient. However, even in this extreme circumstance, as in the case of an HIVinfected patient with virtually no CD4 lymphocytes who succumbed to measles pneumonia 9 months after vaccination (Anonymous 1996), there was no evidence that the offending virus had reverted to a pathogenic phenotype capable of spreading and causing disease in normal people.

Tumor Targeting Through CD46 Density Discrimination Wild-type pathogenic and attenuated measles viruses have different receptor tropisms (Yanagi et al. 2006). Most importantly, attenuated vaccines strains such as MV-Edm are capable of using CD46 as a cell entry receptor (Naniche et al. 1993; Dorig et al. 1993). Wild-type measles viruses do not, in general, use CD46 as a cell entry receptor, but acquire the CD46 tropism during tissue culture adaption via a mutation in the H-attachment protein coding sequence that changes the amino acid at position 481 in the H-protein, from asparagine to tyrosine (Hsu et al. 1998; Xie et al. 1999; Rota et al. 1994). Attenuated measles virus strains carrying this mutation are typically selected when wild-type measles stocks are applied to CD46positive SLAM-negative cell monolayers (for example, Vero cells) (Nielsen et al. 2001). CD46, also known as membrane cofactor protein, is ubiquitously expressed by all human cells except erythrocytes (Riley-Vargas et al. 2004). CD46 plays an

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important role in protecting autologous cells from complement attack by serving as a cofactor for Factor I-mediated inactivation of C3b and C4b, thus blocking the complement cascade at the C3 activation stage (Liszewski and Atkinson 1992). Fortuitously for those wishing to use attenuated measles as a oncolytic agent, CD46 is frequently overexpressed on human cancer cells compared with their normal nontransformed counterparts, possibly as a mechanism to protect the cancer cells from complement mediated lysis (Fishelson et al. 2003; Durrant and Spendlove 2001; Hara et al. 1995; Seya et al. 1990). Overexpression of CD46 has been documented in gastrointestinal, hepatocellular, colorectal, endometrial, cervical, ovarian, breast, renal, and lung carcinomas, also in leukemias and multiple myeloma, and has been found to limit the therapeutic potential of monoclonal antibody therapy (Seya et al. 1990; Ong et al. 2006; Bjorge et al. 1997; Varsano et al. 1998; Blok et al. 2000; Simpson et al. 1997; Murray et al. 2000; Kinugasa et al. 1999; Juhl et al. 1997; Gorter et al. 1996; Thorsteinson et al. 1998; Yamakawa et al. 1994). CD46 mediates not only the attachment and entry of attenuated measles viruses, but also drives the process of virus-induced cell-to-cell fusion between a virusinfected cell and its neighboring cells. Using engineered Chinese hamster ovary (CHO) cells expressing a range of CD46 densities, it was shown that intercellular fusion between infected and uninfected cells was minimal at low CD46 receptor density, but increased dramatically above a threshold CD46 expression level (Anderson et al. 2004). Virus entry, by contrast, increased progressively with increasing CD46 receptor density, showing no dramatic all-or-nothing threshold effects. Thus, at low CD46 receptor densities typical of normal cells, attenuated measles virus is able to infect but intercellular fusion is negligible. At higher CD46 receptor densities typical of tumor cells, infection leads to extensive intercellular fusion (the classical cytopathic effect of measles virus), culminating in a dramatically increased level of cell killing (Anderson et al. 2004). In one recent study, the levels of CD46 expression on myeloma cells was found to be much higher (49,130 per cell) than levels on corresponding normal bone marrow cells (7340 per cell) from 38 myeloma patients (Ong et al. 2006). Potent cytopathic effects of extensive intercellular fusion were observed in the myeloma cells after measles infection, but not in the normal bone marrow cells (Fig. 11.1). Also, the extent of measles virusinduced cell fusion varied from patient to patient, but correlated with CD46 expression levels on the myeloma cells, colony-forming assays demonstrated that measles was not cytotoxic to the normal bone marrow progenitor cells (Ong et al. 2006). Discrimination between cells with high and low CD46 receptor densities provides a compelling basis for the oncolytic specificity of attenuated measles viruses and establishes them as a highly promising CD46 targeted cancer therapeutic agent (Ong et al. 2006; Anderson et al. 2004). There may be additional factors contributing to the tumor specificity of attenuated measles virus besides CD46 receptor density. One possible contributing factor could be a higher intrinsic membrane fusogenicity in tumor cells, making them more likely to undergo intercellular fusion when infected by measles virus, regardless of the surface density of CD46 (Ong et al. 2006). Another factor might be that there are deficiencies in the innate antiviral responses of tumor cells that render

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C

A

MM cell B

CD46 density

1000

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10 PC

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Fig. 11.1 A–C Attenuated measles virus preferentially targets CD46, which is overexpressed on cancer cells, including multiple myeloma cells. A Cytospin of a bone marrow aspirate showing myeloma cells. B Bone marrow aspirates obtained from myeloma patients were separated into plasma cells (myeloma) and nonplasma cells (all normal hemapoietic cells in the marrow). Cells were stained with an anti-CD46 antibody and the numbers of CD46 receptors/cell were determined using BD-QuantiBrite Beads. (Ong et al. 2006). Primary myeloma cells express sevenfold higher CD46 receptors than normal nonplasma cells in the bone marrow. C Primary myeloma cells expressing high levels of CD46 receptors (left) are more susceptible to the cytopathic effects of measles induced syncytial formation compared to normal bone marrow cells isolated from the same bone marrow aspirates. (Ong et al. 2006)

them more highly susceptible to virus infection (Balachandran and Barber 2007). For example, the interferon-α/β and RNA-dependent protein kinase response pathways are often impaired in tumor cells but not in normal cells, and this is the mechanism underlying the tumor selectivity of a number of RNA viruses currently being tested for cancer therapy, such as vesicular stomatitis virus and reovirus (Campbell and Gromeier 2005; Basler and Garcia-Sastre 2002; Strong et al. 1998; Stojdl et al. 2003). These same innate viral control mechanisms may also serve, albeit to a lesser extent, to control the propagation of attenuated measles viruses.

Safety to Population The main public safety concern associated with the therapeutic use of an oncolytic virus is accidental emergence of a new viral pathogen capable of epidemic spread in the human population (Russell 1994). Serious epidemics arise when pathogenic viruses gain access to susceptible populations under conditions that favor transmission between individual members of the population. In selecting a virus from which to develop an oncolytic antitumor agent for human use, consideration should be

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given to its mutability, potential pathogenicity, potential transmissibility, and prevalence in the population. As mentioned above, even though the measles vaccine has been given to more than one billion people over the past 50 years, there has never yet been a documented reversion to wild-type measles (Griffin 2001). Moreover, in contrast to viruses such as influenza which are inherently unstable, requiring new vaccines every year, measles virus has remained very stable and has been effectively controlled by essentially the same vaccine for decades (Griffin et al. 2008). Even if one considers the worst case scenario wherein an attenuated measles virus used for cancer therapy might revert back to the wild-type pathogenic strain, the risk of virus transmission from patients to carers and then into the population is limited by the high prevalence of anti-measles immunity. At the current time, because of the successful childhood measles vaccination programs, which give lifelong protection, more than 80% of the people in the world are currently measles immune (McQuillan et al. 2007). For this reason, attenuated measles viruses raise significantly fewer safety concerns than oncolytic viruses derived from other virus families.

Oncolytic Activity of Wild-Type and Attenuated (Vaccine Strain) Measles Viruses Occasional so-called spontaneous tumor regressions of Hodgkin’s disease and Burkitt’s lymphoma have been documented after measles infections (Bluming and Ziegler 1971; Zygiert 1971; Taqui et al. 1981; Mota 1973; Ziegler 1976). Perhaps the most compelling was the case history of an 8-year-old African boy who presented to a clinic in Uganda with a 4-month history of painless right orbital swelling. A biopsy specimen of the right retroorbital tumor was histologically diagnostic of Burkitt’s lymphoma but at the time of planned initiation of therapy, he was noted to have a generalized measles rash. On the same day, the right orbital tumor was noted to be regressing and because of the presumed measles infection, he was given no chemotherapy for the Burkitt’s lymphoma. During the course of the next 2 weeks, his rash disappeared and he seroconverted to measles. At the same time, the tumor regressed completely and remained in complete remission for at least 4 months after the measles infection in the absence of antineoplastic therapy. The mechanism underlying the rapid tumor regression that was observed in this remarkable case history was never elucidated, but Burkitt’s lymphomas are known to express high levels of SLAM and are therefore susceptible to infection by wild-type measles viruses (Nagy et al. 2002; Nakamura et al. 2005). The timing of the regression, coinciding with the period during which measles virus burden and measles-induced immunosuppression are at their peak, supports the contention that the tumor cells were directly destroyed by the virus. It could be argued on the basis of case histories such as the one described above that there would be some value to treating cancer patients with a wild-type pathogenic strain of measles. However, there are strong safety and efficacy arguments against this. From a safety perspective, measles is a serious and unpleasant illness,

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which is highly transmissible to nonimmune subjects and is sometimes fatal (Griffin et al. 2008). Regarding efficacy, as discussed previously, most human malignancies lack receptors for wild-type measles viruses (Cocks et al. 1995) and therefore are not even theoretically susceptible to its possible oncolytic actions. Attenuated vaccine strains of measles virus, by contrast, have far greater appeal as possible oncolytic agents for intentional administration to human cancer patients. Most of the measles vaccine strains in current use belong to the Edmonston lineage, which comprises a number of closely interrelated laboratory-adapted substrains derived from a 1954 clinical isolate (from the throat of a child named David Edmonston) that has been passaged extensively in tissue culture, resulting in a loss of pathogenicity (Anonymous 1996; Griffin et al. 2008; Enders and Peebles 1954). The MV-Edm vaccine was initially licensed in 1963 but was found to be reactogenic, causing fever and rash in measles-naïve children (Katz 1965, 1996). The virus was passaged further in other cells, including chick embryo fibroblasts, giving rise to more highly attenuated vaccine strains, including MV-Moraten, which was licensed as Attenuvax in the United States in 1968 and the Edmonston-Zagreb strain (MV-EZ), a strain which has been used extensively in Europe (Griffin 2001). In contrast to wild-type measles viruses, all the laboratory-adapted Edmonston strains of measles virus have acquired the ability to use CD46 as a receptor to mediate virus entry and intercellular fusion (Naniche et al. 1993; Dorig et al. 1993; Nielsen et al. 2001). Preclinical studies to test the oncolytic potential of attenuated Edmonston lineage viruses were first conducted at the turn of the twentieth century using a genetically modified derivative of the Edmonston-B strain (MV-Edm tag), which was rescued from a molecular clone of the viral genome and was subsequently amplified on Vero cells (Radecke et al. 1995). The Edm-tag strain and its genetically engineered derivatives (see below in this paragraph) were found to be selectively destructive to human tumor cells in culture (Ong et al. 2006) and showed promising antitumor activity in several mouse xenograft models of different human malignancies, including lymphoma, multiple myeloma, ovarian, colorectal, breast, and liver cancer and glioma (Grote et al. 2001; Peng et al. 2001, 2002a; Phuong et al. 2003; Blechacz et al. 2006; McDonald et al. 2006; Hoffmann et al. 2006). However, clinical testing of these engineered measles strains presented considerable logistic challenges, including the need to develop new manufacturing processes, validate the clinical products, and perform extensive toxicology testing to confirm their safety. For these reasons, there was a strong impetus to take the simple path and explore the possibility of using agents that had already been approved for human use, such as the Moraten and Edmonston-Zagreb measles vaccines, as oncolytic agents for cancer therapy. For this reason, the oncolytic potential of the commercially available Moraten measles vaccine was tested in a murine intraperitoneal model of human ovarian cancer and was compared with the efficacy of a recombinant MV-Edm tagderived viral strain (Myers et al. 2005). In vitro, the Moraten strain was able to infect human ovarian cancer cells but caused intercellular fusion with greatly delayed kinetics compared to the MV-Edm tag strain. However, in vivo studies in ovarian cancer models showed that both viruses had equivalent antitumor potency causing significant prolongations in survival after intraperitoneal administration of

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a total dose of 106 infectious units (Myers et al. 2005). Unfortunately, since the Moraten vaccine is packaged and sold commercially in doses of only 103 TCID50, it was considered impractical to proceed to a clinical trial in which, it was anticipated, MV-Moraten doses in excess of 108 TCID50 (i.e., 100,000 times the vaccination dose) would be required for oncolytic efficacy. While there have been no human studies evaluating the oncolytic potential of the MV-Moraten vaccine strain, a phase I clinical study using the EdmonstonZagreb vaccine strain was conducted in Zurich in patients with cutaneus T cell lymphoma (Heinzerling et al. 2005). A total of five patients were enrolled in this study and the Edmonston-Zagreb vaccine strain of measles virus was administered directly into accessible cutaneous tumors for up to a total of four doses. Each virus injection was preceded by two subcutaneous injections of interferon-alpha (9 million units subcutaneously), at 72 h and 24 h previously. This was a dose escalation study with a minimum intratumoral dose of 100 TCID50 and a maximum dose of 1000 TCID50 of the MV-Edmonston-Zagreb virus. The treatment was very well tolerated and five of six MV-injected lesions showed clear regression. In two of the patients, distant noninjected lesions improved, but they remained unchanged in the other three of five. In all five patients, there was a slight increase in the anti-measles antibody titer after therapy. These very promising data were published in October 2005 and follow-on studies are eagerly awaited (Heinzerling et al. 2005).

Engineering Attenuated Measles Viruses to Enhance Their Utility as Oncolytic Agents A reverse genetic system for the generation of recombinant measles viruses derived from MV-Edm tag was first reported in 1996 (Radecke et al. 1995). This opened the door for the generation of recombinant measles viruses encoding additional transcription units as well as the engineering of viral structural and nonstructural protein coding sequences to modulate virus biology (Fig. 11.2). Genome engineering has therefore emerged as an important approach whereby new versions of the MV-Edm tag virus can be generated as a way to enhance its performance in cancer therapy. Efforts to date have focused on the generation of recombinant viruses whose in vivo spread can be noninvasively monitored as well as the generation of viruses that can more effectively combat host innate immune responses or that can more accurately recognize and destroy neoplastic tissues. Each of these approaches is discussed separately in Sect. 3.1–3.3.

Noninvasive Monitoring of Measles Virus Spread Pharmacokinetics describes the fate of a drug in the body, including its absorption, distribution, biotransformation, and excretion. Unfortunately, pharmacokinetic issues

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B. Imaging

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ablate CD46

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H xx xx

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display cell targeting ligands

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Fig. 11.2 Genetic engineering of attenuated measles virus for cancer therapy. A The virus can be engineered to express soluble marker proteins (e.g., MV-CEA), which are secreted into the circulation, thus enabling noninvasive monitoring of the profiles of viral gene expression over time by sampling body fluids (Peng et al. 2002). B The virus can be engineered to express the sodium iodide symporter (MV-NIS), which concentrates radioiodine in the infected cell, thus enabling noninvasive monitoring of the sites of MV infection by gamma camera, SPECT-CT, or PET-CT imaging (Dingli et al. 2004). Virotherapy can also be enhanced by a timely dose of beta-emitting I-131 to result in synergistic killing of MV-infected tumors. C Arming of the virus with genes (e.g., P gene from wild-type measles) that enable the virus to combat the innate antiviral immunity (Haralambieva et al. 2007). D Targeting virus entry, the H glycoprotein of measles virus can accommodate addition of large polypeptides (e.g., single-chain antibodies) as C-terminal extensions on the H protein. Mutations in the H protein that ablate fusion via CD46 and SLAM have been identified and incorporated in the retargeted viruses. The displayed ligand redirects binding of the virus to the new receptor to mediate virus entry and syncytial formation via the targeted receptor (Nakamura et al. 2005)

have not been adequately addressed in previous human virotherapy studies and this is proving to be a significant impediment to the intelligent clinical development of these agents. Ideally, it should be possible to noninvasively monitor the in vivo spread and elimination of an oncolytic virus in a treated cancer patient and to determine the profile of viral gene expression over time. In the absence of data on virus kinetics, it is not possible to know whether a failed response to therapy is due to lack of infection, inadequate virus spread, weakened cytopathic effect, or premature virus elimination. Noninvasive monitoring should therefore be an integral component of human studies of oncolytic measles viruses and has the potential to facilitate the tailoring of virotherapy protocols for individual patients. Two approaches have been taken to facilitate in vivo monitoring of the spread of oncolytic measles viruses (Fig. 11.2). In the first approach, additional transcription

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units coding for soluble marker peptides were inserted into the viral genome (Peng et al. 2002b). It was reasoned that ideal marker peptides should be nonimmunogenic with no biological function and a constant circulation half-life. They should also be efficiently secreted from virus-infected cells into the blood stream. The soluble extracellular domain of human carcinoembryonic antigen (CEA) and the β subunit of human chorionic gonadotrophin (βhCG) were therefore chosen for this approach. Oncolytic measles viruses expressing each of these transgenes from an additional transcription unit inserted upstream of the N gene were constructed (Peng et al. 2002b). The marker peptides did not compromise virus replication and the kinetics of virus propagation in CD46 transgenic mice could be easily followed by measuring concentrations of the virally encoded marker peptides in serum. When mice bearing human tumor xenografts were challenged with the trackable viruses, different kinetic profiles of marker gene expression could be correlated with distinct therapeutic outcomes (Peng et al. 2002b, 2006). In subsequent studies, the MV-CEA virus was shown to retain its potent oncolytic activity in preclinical models of ovarian cancer (Peng et al. 2002a; Hasegawa et al. 2006b) and subsequently in brain cancer (Phuong et al. 2003), which provided the impetus for its advancement to phase I clinical testing in patients with each of these malignancies (see later). While virally encoded soluble marker peptides do provide for noninvasive monitoring of the total burden of virus-infected cells and tissues in the body, they do not provide any anatomical information about the location of virus-infected cells (Fig. 11.2). To this end, a second recombinant measles virus was generated coding for the human thyroidal sodium iodide symporter (NIS) (Dingli et al. 2004). NIS is a membrane ion channel expressed on thyroid follicular cells, which efficiently transports iodine (required for thyroxine production) into cells against its concentration gradient (Dadachova and Carrasco 2004). Thyroidal NIS expression has been exploited for more than 50 years in clinical practice for thyroid imaging (with I-123) or ablation (with I-131) and for systemic therapy of well-differentiated thyroid malignancies (Riesco-Eizaguirre and Santisteban 2006; Mazzaferri and Kloos 2001). MV-NIS-infected cells are able to concentrate radioactive iodine from the bloodstream, enabling the status of an infection to be monitored by serial noninvasive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging using I-123 or I-124 as tracers, respectively (Dingli et al. 2005a; Carlson et al. 2006). The approach has been used for noninvasive monitoring of intratumoral virus propagation in preclinical models of multiple myeloma, pancreatic, hepatocellular, and ovarian carcinoma, and has further been used to enhance the therapeutic potency of measles virotherapy by judiciously timed administration of the β-emitting radioiodine isotope I-131 (Blechacz et al. 2006; Hasegawa et al. 2006b; Dingli et al. 2003, 2004; Carlson et al. 2006). Interestingly the recombinant measles virus in which NIS was inserted between the H and L cistrons is able to propagate as efficiently as the MV-Edm tag strain from which it was derived and is potently oncolytic even in the absence of I-131 (Dingli et al. 2004). Based on these favorable characteristics, the MV-NIS virus has been advanced to phase I clinical testing in patients with multiple myeloma (see later).

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The interaction of a replicating measles virus with tumor cells and with the host immune system represents a complex dynamical system that can be analyzed mathematically as a problem in population dynamics (Dingli et al. 2006; Wein et al. 2003). The outcome of this type of therapy depends in a complex way on the intricate interactions between the various populations involved and modeling of the kinetics of virotherapy may aid in improved understanding of treatment outcomes and in the design of improved therapeutic protocols. Mathematical models of cancer virotherapy with recombinant measles viruses and of radiovirotherapy using the combination of MV-NIS and radioactive I-131 have been developed and provide a reasonably good fit to experimental data (Dingli et al. 2006; Bajzer et al. 2008).

Arming the Virus to Combat Innate Immunity Pathogenic measles viruses are capable of combating the cellular innate immune response. They do this by means of the P/VC proteins encoded in the phospho protein (P) transcription unit (Gotoh et al. 2001). The P and V proteins are particularly implicated in measles immune evasion and have been shown to inhibit interferoninduced STAT nuclear translocation and to suppress STAT1 and STAT2 phosphorylation (Palosaari et al. 2003; Shaffer et al. 2003; Takeuchi et al. 2003; Devaux et al. 2007). Attenuated measles viruses are typically mutated in their P and V proteins and are therefore unable to efficiently suppress innate immune responses (Ohno et al. 2004). As a consequence, wild-type measles isolates induce significantly lower release of interferon upon infecting peripheral blood lymphocytes when compared to attenuated strains (Ohno et al. 2004; Waehler et al. 2007). As a general rule, tumor cells are thought to have defects in their interferon response pathways such that they are unable to mount effective innate antiviral responses (Stojdl et al. 2000). However, this is not an absolute deficiency. Oncolytic measles viruses derived from the MV-Edm tag infectious clone were shown to induce significantly higher levels of interferon in both normal and tumor cells when compared to a wild-type measles virus (Haralambieva et al. 2007). Moreover, pretreatment of tumor cells with interferon prior to measles infection did significantly compromise viral gene expression. Based on these observations, a chimeric measles virus based on the Edm-tag platform, but armed with a wild-type P gene, was generated and evaluated both in vitro and in vivo for antineoplastic activity (Haralambieva et al. 2007). As expected, the chimeric virus exhibited a reduced capacity to induce interferon in infected cells, and when intravenously administered to SCID mice bearing human myeloma xenografts, showed greater oncolytic potency. This study served to emphasize that oncolytic measles viruses can be subject to control by the innate immune defenses of human tumor cells and may therefore be more effective when engineered to rearm them with wild-type proteins that silence the innate immune response. This engineering strategy does raise the legitimate concern that rearming an attenuated measles virus with a wild-type P gene to permit more effective immune evasion may generate a more pathogenic agent and

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thereby compromise patient safety. However, measles virus virulence and pathogenicity are known to be complex and do not depend solely on P/V/C proteins (Bankamp et al. 2008). Clinical testing of oncolytic measles viruses that have been engineered to more effectively combat the innate intracellular immune response is not currently being pursued. However, depending upon the clinical outcomes (i.e., efficacy vs toxicity) of ongoing clinical trials that are using highly attenuated measles viruses, there may be a good rationale for testing such fortified viruses in the future.

Engineering Measles Virus Tropism At this time, measles is still the only virus that can be efficiently retargeted through a broad range of cellular receptors without significant reductions in entry efficiency (Nakamura and Russell 2004; Waehler et al. 2007). However, while the retargeting of measles virus entry is without question an extraordinary technical triumph, its utility has yet to be proven. The original rationale for attempting to redirect the tropisms of oncolytic measles viruses was the incorrect assumption that, because it is expressed ubiquitously, CD46 would not be tumor-selective. However, as outlined previously, the oncolytic strains of measles virus in current use do efficiently discriminate between the high density of CD46 expressed on tumor cells and the lower densities of this receptor present on nontransformed cells (Ong et al. 2006; Anderson et al. 2004; Peng et al. 2002a). As such, these viruses already discriminate and selectively destroy cancer cells by targeting CD46. For this reason, unless significant toxicities arising from collateral damage to normal tissues are encountered in ongoing clinical studies using the nontargeted viruses MV-CEA and MV-NIS in patients with ovarian cancer, glioma, and multiple myeloma, it will be difficult to justify the use of fully retargeted viruses. However, it is possible that certain tumor types will prove to have lower levels of CD46 receptor expression, in which case there may be a stronger case for using viruses with alternative, engineered receptor tropisms. In addition, for intravenously administered viruses, the ability to interact with the lumenal surface of vascular endothelial cells lining tumor blood vessels might lead to significant enhancements in virus uptake at sites of tumor growth. Initial efforts to engineer foreign polypeptides into the measles virus coat were focused on the fusion (F) protein. But even small modifications to the extreme Nterminus of this protein completely destroyed its ability to provide fusion support (F. Morling and S.J. Russell, unpublished observations). In contrast to the F protein, the Hemagglutinin (H), a type II membrane glycoprotein, was able to tolerate the insertion of large polypeptide sequences at or close to its extreme C-terminus without compromising its ability to be incorporated into virus particles and to provide fusion support functions. Building upon this observation, recombinant viral genomes coding for chimeric H-glycoproteins with a variety of different C-terminal extensions were constructed and rescued as recombinant infectious viral particles. In this way, it was possible to display a wide variety of different ligands on the viral surface,

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including growth factors (human EGF and IGF 1), single-chain antibodies (against CEA CD38, CD20, EGF receptor, and others), single-chain T cell receptors, and snake venom peptides such as Echistatin (Schneider et al. 2000; Hammond et al. 2001; Peng et al. 2003a, 2004; Bucheit et al. 2003; Hallak et al. 2005). In each case, the recombinant viruses were shown to display multiple copies of the respective polypeptides on the surface and were endowed not only with new binding specificities, but also with expanded tropisms. Thus each recombinant virus was able to bind and enter cells via its targeted receptor and thereafter to drive the process of intercellular fusion between the infected cell and neighboring receptor positive cells. Having determined that the tropisms of recombinant measles viruses could be expanded by surface display of cell binding polypeptides, the next step was to engineer the underlying H protein to ablate the natural virus tropisms for CD46 and SLAM (Vongpunsawad et al. 2004; Nakamura et al. 2004). Several different mutations known to interfere with binding to either CD46 (Xie et al. 1999; Lecouturier et al. 1996) or SLAM (Vongpunsawad et al. 2004) were therefore engineered into an H-expression construct coding for a chimeric H protein displaying a single-chain antibody against CD38. Numerous different permutations of CD46- and SLAMablating mutations were tested in a screening system, wherein the H-expression constructs were co-transfected with an F-expression plasmid into cells expressing receptors for CD46, SLAM, or CD38, respectively (T. Nakamura and Russell, unpublished observations). In this way, a construct with mutations at residues 481 (Y to A) and 533 (R to A) was found to efficiently mediate antibody-targeted cell fusion, even when the displayed domain was replaced with single-chain antibodies recognizing alternative cellular targets (EGFR and EGFRvIII) (Nakamura et al. 2004). Subsequently, these fully retargeted H-coding sequences were engineered into recombinant measles virus genomes and the corresponding viruses were recovered (Hadac et al. 2004). In each case, recombinant viruses displaying single-chain antibodies on a doubly ablated H protein displayed the expected (fully retargeted) host range properties and could be used to mediate targeted destruction of tumors expressing the appropriate cognate receptor in living mice. One technical issue associated with the rescue and propagation of fully retargeted viruses was the requirement that the targeted receptor should be expressed on the cells being used as a substrate for virus growth. This was addressed in two ways, either by generating Vero cells expressing the targeted receptors (Hadac et al. 2004) or, alternatively, by means of a pseudo-receptor system using Vero-α His cells expressing a membrane-anchored single-chain antibody that recognizes a hexahistidine peptide (H6) (Kaufmann et al. 2002). The H6 peptide was then incorporated at the extreme C-terminus of the chimeric H proteins of fully retargeted viruses, such that they could be efficiently rescued and propagated on the Vero-α His cells (Nakamura et al. 2005). This system has subsequently been used to generate fully retargeted viruses displaying single-chain antibodies with diverse receptor specificities. At the current time, the list includes viruses retargeted to CD38, EGFR, EGFRvIII, α-folate receptor, HER2/neu, CD20, CD19, CD52, prostate specific membrane antigen (PSMA), and the myeloma antigens HM1.24 and Wue-1 (S.J. Russell, unpublished observations) (Nakamura et al. 2005; Hasegawa et al. 2006a,

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2007; Ungerechts et al. 2007b; Paraskevakou et al. 2007; Allen et al. 2006). Interestingly, where tested, the new tropisms conferred on measles viruses by displayed scFvs are stably maintained during multiple serial virus passages without reversion to native receptor usage. While one conclusion from the aforementioned studies is that measles virus has a remarkably flexible and adaptable entry mechanism that can utilize multiple alternative cellular receptors, it has also become apparent that there can be significant differences in the efficiency of virus entry and intercellular fusion depending on the precise specificity and affinity of the displayed ligand. To further explore the relationship between the affinity of a displayed ligand for its targeted receptor and the behavior of the retargeted viruses, a panel of six recombinant HER2/neu retargeted measles viruses was generated displaying a panel of single-chain antibodies with identical HER2/neu receptor specificities but with dissociation constants ranging from 1.6 × 10−6 M to 1.5 × 10−11 M (Hasegawa et al. 2007). Comparisons of the infectivities and cytopathic effects of these viruses on a panel of cell lines expressing different surface densities of the HER2/neu receptor gave quite unexpected results, showing that there was a functional threshold affinity level above which infection and intercellular fusion proceeded with equal efficiency, even when affinity increased over 1000-fold above the threshold level. Below the threshold, infection was minimal. This affinity threshold was shown to correlate inversely with receptor density such that higher affinities were required to fuse cells with lower receptor densities. Thus, depending on their receptor affinities, retargeted measles viruses are able to discriminate efficiently between cells expressing different densities of a targeted receptor (Hasegawa et al. 2007). Several in vivo therapy studies have been published in which fully retargeted viruses have been administered to mice bearing human tumor xenografts by intratumoral, intraperitoneal, or intravenous routes, resulting in retardation of tumor growth and prolongation of survival. Examples include the use of measles viruses targeted to EGF receptor in ovarian cancer and glioma models, viruses targeted to the α-folate receptor in an ovarian cancer model, EGFRvIII-targeted viruses for glioma, and CD38- or CD20-targeted viruses for the treatment of two different mouse lymphoma models (Nakamura et al. 2005; Hasegawa et al. 2006a; Paraskevakou et al. 2007; Allen et al. 2006; Ungerechts et al. 2007a). In general, the retargeted viruses showed at least equivalent antitumor activity compared to the parental MV-Edm tag strain, but with reduced capacity to cause damage to the brains of CD46 transgenic mice. As discussed previously, it is still an open question whether fully retargeted measles viruses can offer therapeutic advantages over the CD46 specific viruses that are currently undergoing clinical testing. However, it is hoped that measles viruses that can interact more efficiently with markers expressed on the lumenal surfaces of the endothelial cells lining tumor blood vessels will more efficiently localize to sites of tumor growth when infused into the bloodstream. Exploratory studies using measles viruses displaying αvβ3 integrin-binding peptides (cyclic RGD and Echistatin) as extensions of their H proteins have shown that they can, at least, interact with the lumenal surface of developing neovessels in the chick chorioallantoic membrane and that they can be transcystosed across the vascular

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endothelium of these vessels into the interstitial space (K.W. Peng and S.J. Russell, unpublished observations).

Clinical Translation of Recombinant Oncolytic Measles Viruses Of the recombinant measles viruses discussed in the previous section, two (MVCEA and MV-NIS) are currently being administered to cancer patients in phase I clinical trials. MV-CEA is being infused into the peritoneal cavities of patients with advanced treatment-refractory ovarian cancer, and in a second trial, it is being administered into the tumor bed after surgical excision of high-grade tumors of the brain. MV-NIS is being administered intravenously to patients with advanced treatment-refractory multiple myeloma. Approval for these clinical protocols was, in each case, preceded by a detailed FDA review of the clinical protocol design, the manufacturing process, the purity, identity, and sterility of the manufactured product, the results of preclinical efficacy studies, and the results of comprehensive toxicology and biodistribution studies conducted in appropriate animal models.

Manufacture Based on the doses of MV-CEA and MV-NIS that were required to mediate tumor regression in mouse xenograft models of human cancer, it was projected that doses of virus in the region of 109 TCID50 would be required for clinical efficacy. A single dose of the Moraten measles vaccine contains somewhere between 103 and 104 TCID50 and existing manufacturing processes used by major measles vaccine suppliers were unsuitable for the generation of high titer viral stocks required for oncolytic applications (Meyers et al. 2005). A new process was therefore developed for the scaled manufacture and partial purification of oncolytic measles viruses using the immortal Vero cell line as substrate. Manufacture of clinical grade lots of both MV-CEA and MV-NIS was then completed in a pilot manufacturing facility at Mayo Clinic Rochester, adhering to the principles of Good Manufacturing Practices (GMP).

Choice of Animal Models for Toxicology Studies Because they are derived from attenuated laboratory adapted strains of measles virus, the host range properties of MV-CEA and MV-NIS are quite distinct from those of wild-type pathogenic measles virus strains. As mentioned previously, these viruses efficiently exploit CD46 as a receptor for binding, cell entry, and cell fusion and this receptor usage is a critical factor influencing the choice of an appropriate animal model for pharmacology and toxicology studies. As for other vaccine strains of

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measles viruses, because they are highly attenuated (i.e., nonpathogenic), there is no model in which to study their pathogenesis. The three animal models that have proven to be of considerable value for FDA-mandated preclinical studies of biodistribution and toxicity are rhesus monkeys (Old World primates), squirrel monkeys (New World primates), and interferon α/β receptor knockout CD46 transgenic mice. CD46 transgenic mouse models were originally created in the hope that they might facilitate the study of measles virus pathogenesis (Mrkic et al. 1998; Oldstone et al. 1999; Manchester and Rall 2001). However, dissemination of viruses belonging to the Edmonston lineage has only ever been observed in CD46 mice lacking the interferon α/β receptor (IFNARko xCD46-Ge) (Mrkic et al. 2000). These mice express human CD46 in a tissue distribution that mimics the pattern of CD46 expression in humans, including low to absent expression on erythrocytes (Mrkic et al. 1998; Kemper et al. 2001). After intranasal challenge with MV-tag, the animals showed local virus replication in the respiratory epithelium followed by dissemination via the lymph node system, similar to the pattern of wild-type measles virus dissemination in the human host (Mrkic et al. 1998, 2000). Infection of cells of the monocyte macrophage lineage is prominent in this model (Mrkic et al. 2000; Kemper et al. 2001; Peng et al. 2003b). An alternative and better-established model for the study of measles virus pathogenesis is the rhesus monkey, which, like other Old World non-human primates, develops a measles-like illness when challenged with wild-type measles virus (Kobune et al. 1996; van Binnendijk et al. 1994). However, rhesus monkeys do not provide, by any means, a perfect model in which to test the virulence of tissue culture adapted measles viruses that use CD46 as a receptor (Kobune et al. 1996; Auwaerter et al. 1999; Takeda et al. 1998; Kobune et al. 1990). Previous studies have shown repeatedly that rhesus monkey virulence studies of attenuated measles viruses are not predictive of their reactogenicity (i.e., the ability to cause measles-like illness) in humans (Aldous et al. 1961; Collard et al. 1961; Katz et al. 1960; Schwarz et al. 1960). Numerous attenuated measles viruses have been tested in primates during the development of new measles virus vaccines (Schwarz et al. 1960; Enders et al. 1960; Goffe and Laurence 1961) and even though they have scored completely negative in this macaque-virulence test, they have proven to be reactogenic in humans, causing a morbilliform rash, often accompanied by fever and malaise in a high percentage of recipients (Aldous et al. 1961; Collard et al. 1961; Katz et al. 1960; Schwarz et al. 1960). The key deficiency of the macaquevirulence model is that the tissue distribution of CD46 in macaque differs significantly from humans. Most importantly, CD46 is abundant on macaque red blood cells, but is absent from human red blood cells (Hsu et al. 1997). CD46 binding strains of measles virus, therefore, are agglutinated on macaque, but not human red blood cells, an interaction that most likely impedes virus dissemination in the primates. The macaque model was therefore considered to be suboptimal for toxicity studies in which recombinant measles viruses are administered into the bloodstream, but was considered relevant when studying the toxicity of intracerebral MV-CEA (see Sect. 4.3).

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Unlike Old World primates, New World primates such as the squirrel monkey (Saimiri sciureus) express a truncated CD46 molecule to which MV-Edm does not attach (Hsu et al. 1997). The red cells of New World monkeys are, therefore, not agglutinated by attenuated measles viruses, but these animals do get a measles-like illness when challenged with wild-type measles virus, making them an informative model in which to test the toxicities of oncolytic measles viruses arising from their interactions with the wild-type measles receptor SLAM. Squirrel monkeys challenged with wild-type measles virus develop a classical measles-like illness characterized by fever, coryza rash, immunosuppression, and subsequent recovery (Kobune et al. 1996).

Pharmacology and Toxicology Testing of MV-CEA and MV-NIS To support the first clinical protocol, a phase I trial of intraperitoneal administration of MV-CEA in patients with recurrent ovarian cancer, the formal biodistribution and toxicology studies were conducted in IFNARko xCD46-Ge mice (Peng et al. 2003b). The key findings were that MV-CEA administered into the peritoneal cavity efficiently infected peritoneal macrophages and these trafficked to abdominal draining lymph nodes, as well as to the marginal zones of the spleen. CEA expression peaked between days 2 and 5 and returned to baseline by day 10, due to virus elimination. There was no evidence of virus shedding in the urine or respiratory secretions, but there was some evidence for long-term persistence of viral genomes in the spleens of infected animals. Measles viruses encoding different marker genes were also employed for biodistribution studies and green fluorescent protein proved to be highly informative. Mesothelium and ovarian surface epithelium were remarkably resistant to infection but peritoneal macrophages were susceptible. Large numbers of infected macrophages could be detected in the greater omentum concentrated in milky spots. Infected macrophages were also identified outside the peritoneal cavity at diaphragmatic stomata along lymphatic vessels and in the parathymic lymph nodes. Eventually, the cells escaped into the blood stream and could be identified in the marginal zones of the white pulp of the spleen. In toxicology studies, MV-CEA was administered into the peritoneal cavity (maximum dose 107 TCID50) and the animals were closely monitored for activity level, general appearance, body weight, key hematological and biochemical parameters, and serum CEA levels (K.W. Peng et al., unpublished observations). Necropsies were conducted on days 14, 28, and 91 after virus administration and all organs were sent for histopathological analysis. The study was essentially negative, with no significant toxicities encountered at any dose level. Dose-response studies in a SKOV3.ip1 intraperitoneal ovarian cancer xenograft model revealed that the virus was equally effective over a wide range of dose levels, from 6 × 107 down to 6 × 103 TCID50. Analysis of CEA profiles in the treated mice was highly informative, illustrating the variability of virus kinetics at different dose levels. The highest doses of

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virus were associated with higher initial levels of tumor cell killing, but the final outcome of MV-CEA therapy at all dose levels was a partial equilibrium between virus and tumor, resulting in significant slowing of tumor growth and enhanced survival of the mice (Peng et al. 2006). In support of the phase I brain cancer study of intratumoral and/or resection cavity administration of MV-CEA in patients with recurrent glioblastoma multiforme, toxicology studies were conducted both in IFNARko xCD46-Ge mice and in rhesus monkeys (Allen et al. 2008). In both cases, the virus was delivered by intracerebral inoculation and animals were carefully observed for signs of neurological impairment or other organ damage. MV-Edm is known to be neuropathogenic in measles-naïve IFNARko xCD46-Ge mice and the animals were therefore preimmunized by intraperitoneal administration of MV-GFP for 1 month prior to the initiation of toxicology. The animals were closely monitored for 90 days after intracerebral inoculation with MV-CEA, but there was no evidence for neurotoxicity, neither clinically nor upon histological examination of brain sections at various time points after virus administration. For toxicity testing in monkeys, five adult measles-immune rhesus macaques received intracerebral injections of MV-CEA (105 or 106 TCID50) or vehicle control into the frontal lobe on days 1 and 5. The animals were monitored closely thereafter. Monitoring studies included clinical observation, analysis of blood samples, throat swabs, and cerebral-spinal fluid and MRI imaging of the brain. There was no evidence in this study for MV-CEA-mediated neurotoxicity. In support of the phase I clinical trial testing intravenous administration of MVNIS, with or without cyclophosphamide, in patients with multiple myeloma, preclinical pharmacology and toxicology studies were conducted in SCID mice bearing subcutaneous myeloma xenografts, nontumor-bearing IFNARko xCD46-Ge mice, and in measles-naïve squirrel monkeys (Myers et al. 2007). Dose-response studies conducted in the KAS-6/1 myeloma xenograft model demonstrated that a single intravenous dose of 4 × 106 TCID50/kg of MV-NIS was the minimum dose that reliably led to tumor regression and prolongation of survival. Toxicity studies in IFNARko xCD46-Ge mice were negative up to a single intravenous dose of 4 × 108 TCID50/kg. A single dose of cyclophosphamide given prior to virus administration did significantly impede the antiviral immune response in this model, leading to delayed virus elimination. Virus-associated toxicities were not observed in measlesnaïve squirrel monkeys, even at very high intravenous doses of 108 TCID50/kg of MV-NIS given alone or in combination with cyclophosphamide. Viral mRNA was detected in cheek swabs harvested from the squirrel monkeys on days 1, 2, 8, 15, and 22 after MV-NIS administration and copy numbers were higher at all time points in the cyclophosphamide-treated animals, with peak levels seen on day 8. Based on these studies, a safe starting dose of MV-NIS for the clinical protocol was set at 1.5 by 104 TCID50/kg (106 TCID50 per patient) increasing to a maximum of 1.5 × 107 TCID50/kg (109 TCID50 per patient) with a single-dose of 10 mg/kg cyclophosphamide administered 24 h earlier by intravenous infusion.

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Current Status of Clinical Trials Each of the three phase I clinical trials discussed above is progressing satisfactorily. The first patient to receive a recombinant measles virus had advanced ovarian cancer and received a series of six intraperitoneal infusions of MV-CEA starting in July 2004. To date, 20 patients have been treated in the ovarian cancer study at seven dose levels (103 –109 TCID50). The study is progressing satisfactorily and the results will be published in the near future. Two patients with glioblastoma multiforme have so far been enrolled in the intracerebral MV-CEA study and six patients with multiple myeloma have been enrolled for intravenous administration of MV-NIS at two dose levels (106 TCID50 and 107 TCID50). Each of these clinical studies is progressing satisfactorily, but detailed reports of the trial outcomes will not be published until the trials have been completed.

Enhancing the Efficacy of Oncolytic Measles Viruses: Immune Evasion and Immune Suppression For successful virotherapy of disseminated malignancies, oncolytic measles viruses will have to be delivered via the bloodstream. Optimal treatment outcomes will require efficient delivery via the bloodstream to sites of tumor growth, and this should be followed by efficient intratumoral spread, leading to tumor destruction. Each of these processes can be severely constrained by anti-measles immunity; delivery by humoral immunity, and spread by cell-mediated immunity (Fig. 11.3). Considerable attention has therefore been focused on the use of cells as delivery vehicles to circumvent the humoral anti-measles immune response, and on the use of immunosuppressive drugs to combat cell-mediated anti-measles immunity. These two approaches are discussed below.

Use of Cell Carriers to Deliver Oncolytic Measles Viruses to Sites of Tumor Growth Irrespective of the presence of antiviral antibodies, many intravenously administered viruses are sequestered in the microcirculations of lung, liver, and spleen, where they are phagocytosed by macrophages (Wang and Yuan 2006; Fisher 2006). It is the small percentage of intravenously administered viruses escaping this fate that infect the tumor tissue and mediate the regressions that are seen in preclinical models. However, these viruses are highly vulnerable to the neutralizing activities of antiviral antibodies, which have a dramatic titer-dependent effect on the speed of virus inactivation (Scallan et al. 2006; Klasse and Sattentau 2002). Also, antibody

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Solution No anti-MV Immunity

Delivery Spread

Tumor Eliminated!

MV

Give Immunosuppressive drugs

Delivery

Anti-MV CTLs

Spread

X Tumor Remains

Delivery

Anti-MV Antibodies

Spread

Use cells as delivery vehicle

X X Tumor Remains

Fig. 11.3 Considerations for future improvements of measles virotherapy. Ideally, intravascularly administered viruses will reach the tumor sites to result in infection of tumor cells, viral spread, and elimination of the tumors. However, anti-measles antibodies can potentially inhibit delivery of the viruses and viral spread in the infected tumor can be inhibited by cell-mediated immunity. To combat these barriers to successful therapy, virus-infected cell carriers can be exploited to act as Trojan horses to deliver virus to the tumor sites and cell-mediated immunity can be controlled by judicious use of immunosuppressive agents such as cyclophosphamide

titers increase progressively with each successive exposure to the virus (Reid et al. 2002; Hangartner et al. 2006). Because of prior measles infection or measles vaccination, approximately 90% of Americans have protective titers of anti-measles antibodies, which may limit the therapeutic efficacy of systemically administered measles viruses (McQuillan et al. 2007; Audet et al. 2006). In contrast to the general population, patients with multiple myeloma have profound suppression of their humoral immune responses and low antibody titers to measles virus, making them ideal candidates for systemic oncolytic measles virus therapy (Dingli et al. 2005b; Jacobson and Zolla-Pazner 1986). However, most patients suffering from other cancers do have healthy protective titers of anti-measles antibodies. The protective properties of anti-measles antibodies were well demonstrated in the prevaccination era when serum from convalescing measles patients was used as postexposure prophylaxis for children at risk (Zingher and Mortimer 2005; Zordman et al. 1944). Interestingly, serotherapy was only effective if administered within 6 days of measles virus exposure, indicating that it cannot prevent the cell-associated viremia and dissemination of measles virus via the bloodstream that occurs toward the end of

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the incubation period (days 10–14 after initial exposure, coincident with the prodromal symptoms and rash) (Griffin 2001). Importantly, cell-free viremia has not been recorded in natural measles infections (Griffin 2001; Osunkoya et al. 1990). The virus enters via the respiratory tract, migrates locally to lymphoid tissues and then enters the bloodstream, only inside cells, typically monocytes or lymphocytes, which transport it to distant sites where it causes the typical rash and other disease features (Griffin 2001). On the basis of the above insights into how measles virus can travel via the bloodstream, even the presence of antiviral antibodies, there is considerable interest in the use of infected cell carriers to deliver oncolytic measles viruses to sites of tumor growth (Munguia et al. 2008). Not only does this approach have the potential to avoid neutralization by antiviral antibodies, but might also prevent mislocalization of the virus in liver and spleen, as well as aiding extravasation from tumor blood vessels. In vitro studies demonstrated that in contrast to infection by naked virions, heterofusion between infected cell carriers and tumor cells was more resistant to antibody neutralization (Iankov et al. 2007; Ong et al. 2007). Moreover, systemic and intraperitoneal injection of measles-infected cells successfully transferred infection in vivo to sites of tumor growth (using xenograft models of lymphoma, hepatocellular carcinoma, and multiple myeloma) even in the presence of neutralizing antibodies (Iankov et al. 2007; Ong et al. 2007). Monocytes, endothelial progenitor cells, and T lymphocytes were also shown to have potential as measles virus carriers, but considerable optimization of the approach is required since, to date, the enhancements in efficacy that have been achieved using this approach have been very small.

Combination Therapy with Oncolytic Measles Viruses and Immunosuppressive Drugs There are many drugs with immunosuppressive properties currently being used in the clinic, either for the treatment of autoimmune disease or to suppress the rejection of transplanted organs. Their profiles of activity vary, some being more effective against a particular subset of lymphocytes, with others having a broader spectrum of activity. While there is a strong rationale for combining immunosuppressive therapy with oncolytic measles virotherapy, progress in this research endeavor has been hampered by the lack of suitable animal models in which a measles-susceptible tumor is allowed to grow in a measles-susceptible animal with an intact immune system. However, despite the lack of suitable experimental models, attempts have nevertheless been made to determine whether cyclophosphamide might be capable of suppressing anti-measles immune responses and thereby enhancing its therapeutic potency. Cyclophosphamide is known to be highly toxic to proliferating lymphocytes and can modulate both primary and anamnestic immune responses to a variety of antigenic challenges, including virus infection (Steinberg 2001). Precise

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effects vary with the dose of antigen, the dose of cyclophosphamide, and the timing of cyclophosphamide administration relative to the antigenic challenge (Hill 1975). Suppression of B cell and T cell responses is most pronounced when cyclophosphamide is administered at the same time or within 3 days following antigen challenge, but is also seen when the drug is administered 1 or 2 days before exposure to antigen, particularly when higher doses of cyclophosphamide are used (Aisenberg and Davis 1968). When a single dose of cyclophosphamide was administered to IFNARko xCD46-Ge mice a few hours prior to intraperitoneal or intravenous injection of MV-CEA or MV-NIS, respectively, elimination of the viruses was substantially delayed and the primary anti-measles antibody response was suppressed. A similar delay in virus elimination was observed when squirrel monkeys were pretreated with cyclophosphamide prior to infusion of MV-NIS, and these data provided part of the justification for the ongoing MVNIS clinical trial in which, in later cohorts, the patients will be pretreated with cyclophosphamide before they receive the virus. Additional preclinical studies were conducted in which cyclophosphamide was compared with other immunosuppressive agents and found to be substantially superior to dexamethasone, an equipotent but less toxic than whole-body irradiation (R.M. Myers et al., unpublished observations). Toward the development of an informative immunoincompetent murine model to experimentally test the role of immunosuppressive agents in measles virus therapy, a CEA-retargeted measles virus was used to treat a CEA-positive murine colon adenocarcinoma implanted in syngeneic C57BL/6 mice (Ungerechts et al. 2007a). This targeted virus was also armed with the prodrug convertase purine nucleotide phosphorylase (PNP). Systemic delivery of MV-PNP-anti CEA had no substantial oncolytic activity, but in combination with the prodrug, 6-methyl purine-2'-deoxyroboside, it was therapeutic, revealing synergistic affects between virus and prodrug. Immunosuppression with cyclophosphamide was shown to retard the appearance of measles-neutralizing antibodies and also to enhance oncolytic efficacy.

Current Status and Future Prospects Measles virotherapy has recently emerged as a safe and highly promising experimental approach to the treatment of human cancer. Clinical testing is still at an early stage and it remains possible that efficacy will be limited by preexisting anti-measles immunity. However, ongoing research emphasizing the use of engineered viruses, infected cell carriers, and supplemental therapy within immunosuppressive drugs is already offering viable strategies to enhance the potency of these agents, even in the face of preexisting anti-measles immunity. Acknowledgements Dr. Stephen Russell and Dr. Kah-Whye Peng are supported by funds from the NIH/NCI (CA100634 and CA129966 to Dr. Stephen Russell and CA118488 to Dr. KahWhye Peng). A grant from the Rapid Access to Intervention Development (RAID) Program of

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the NCI supported the manufacture of MV-NIS and toxicology studies in transgenic mice and squirrel monkeys. Efforts of numerous individuals including clinical collaborators (Drs. Evanthia Galanis, Angela Dispenzieri, Lynn C. Hartmann, David Dingli), members of the virus manufacturing facility (Drs. Mark Federspiel and Linda Gregory, Guy Griesmann, Kirsten Langfield, Julie Sauer, Sharon Stephan, Henry Walker, Troy Wegman, Cindy Whitcomb) and members of the pharmacology/toxicology group (Rae Myers, Suzanne Greiner, Mary Harvey, Pam Ryno, Nathan Jenks, Emily Mader) are gratefully acknowledged.

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Takeda M, Kato A, Kobune F, et al (1998) Measles virus attenuation associated with transcriptional impediment and a few amino acid changes in the polymerase and accessory proteins. J Virol 72:8690–8696 Takeuchi K, Kadota SI, Takeda M, et al (2003) Measles virus V protein blocks interferon (IFN)alpha/beta but not IFN-gamma signaling by inhibiting STAT1 and STAT2 phosphorylation. FEBS Lett 545:177–182 Taqi AM, Abdurrahman AB, Yakubu AM, et al (1981) Regression of Hodgkin’s disease after measles. Lancet 1:1112 Thorne SH, Hwang TH, O’Gorman WE, et al (2007) Rational strain selection and engineering creates a broad-spectrum, systemically effective oncolytic poxvirus JX-963. J Clin Invest 117:3350–3358 Thorsteinsson L, O’Dowd GM, Harrington PM, et al (1998) The complement regulatory proteins CD46 and CD59, but not CD55, are highly expressed by glandular epithelium of human breast and colorectal tumour tissues. APMIS 106:869–878 Ungerechts G, Springfield C, Frenzke ME, et al (2007a) An immunocompetent murine model for oncolysis with an armed and targeted measles virus. Mol Ther 15:1991–1997 Ungerechts G, Springfield C, Frenzke ME, et al (2007b) Lymphoma chemovirotherapy: CD20targeted and convertase-armed measles virus can synergize with fludarabine. Cancer Res 67:10939–10947 van Binnendijk RS, van der Heijden RW, van Amerongen G, et al (1994) Viral replication and development of specific immunity in macaques after infection with different measles virus strains. J Infect Dis 170:443–448 Varsano S, Rashkovsky L, Shapiro H, et al (1998) Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro, and an insight into mechanism(s) of resistance. Clin Exp Immunol 113:173–182 Vongpunsawad S, Oezgun M, Braun W, et al (2004) Selectively receptor-blind measles viruses: identification of residues necessary for SLAM- or CD46-induced fusion and their localization on a new hemagglutinin structural model. J Virol 78:302–313 Waehler R, Russell SJ, Curiel DT (2007) Engineering targeted viral vectors for gene therapy. Nat Rev Genet 8:573–587 Wang Y, Yuan F (2006) Delivery of viral vectors to tumor cells: extracellular transport, systemic distribution, and strategies for improvement. Ann Biomed Eng 34:114–127 Wein LM, Wu JT, Kirn DH (2003) Validation and analysis of a mathematical model of a replication-competent oncolytic virus for cancer treatment: implications for virus design and delivery. Cancer Res 63:1317–1324 Xie M, Tanaka K, Ono N, et al (1999) Amino acid substitutions at position 481 differently affect the ability of the measles virus hemagglutinin to induce cell fusion in monkey and marmoset cells co-expressing the fusion protein. Arch Virol 144:1689–1699 Yamakawa M, Yamada K, Tsuge T, et al (1994) Protection of thyroid cancer cells by complementregulatory factors. Cancer 73:2808–2817 Yanagi Y, Takeda M, Ohno S et al (2006) Measles virus receptors and tropism. Jpn J Infect Dis 59:1–5 Yu W, Fang H (2007) Clinical trials with oncolytic adenovirus in China. Curr Cancer Drug Targets 7:141–148 Ziegler JL (1976) Spontaneous remission in Burkitt’s lymphoma. Natl Cancer Inst Monogr 44:61–65 Zingher A, Mortimer P (2005) Convalescent whole blood, plasma and serum in the prophylaxis of measles: JAMA, 12 April, 1926; 1180–1181. Rev Med Virol 15:407–418; discussion 418–421 Zygiert Z (1971) Hodgkin’s disease: remissions after measles. Lancet 1(7699):593

Chapter 12

Measles Virus-Induced Immunosuppression S. Schneider-Schaulies( ) and J. Schneider-Schaulies

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Features Associated with MV-Induced Immunosuppression . . . . . . . . . . . . . . . . . . . Lymphopenia Mechanisms of Lymphocyte Depletion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Regulation of APC Viability and Function-Modulated Instruction and/or Active Lymphocyte Silencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dendritic Cells: Maturation Program and General Functions . . . . . . . . . . . . . . . . . . . . . . . MV-Induced Effects on Dendritic Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viral Proteins Effective at T Cell Silencing and Modulation. . . . . . . . . . . . . . . . . . . . . . . . . . The N Protein. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Immunosuppression is the major cause of infant death associated with acute measles and therefore of substantial clinical importance. Major hallmarks of this generalized modulation of immune functions are (1) lymphopenia, (2) a prolonged cytokine imbalance consistent with suppression of cellular immunity to secondary infections, and (3) silencing of peripheral blood lymphocytes, which cannot expand in response to ex vivo stimulation. Lymphopenia results from depletion, which can occur basically at any stage of lymphocyte development, and evidently, expression of the major MV receptor CD150 plays an important role in targeting these cells. Virus transfer to T cells is thought to be mediated by dendritic cells (DCs), which are considered central to the induction of T cell silencing and functional skewing. As a consequence of MV interaction, viability and functional differentiation of DCs and thereby their expression pattern of co-stimulatory molecules and soluble mediators are modulated. Moreover, MV proteins expressed by these cells actively silence T cells by interfering with signaling pathways essential for T cell activation. S. Schneider-Schaulies Institute for Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany, e-mail: [email protected]

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Introduction Measles virus (MV) is the causative agent of acute measles, a well-defined clinical entity usually contracted by children. The virus is highly contagious, yet causes acute infection only once in the lifetime of an individual, indicating that anti-viral immune responses are efficiently generated and persistent. In spite of the availability of this efficient vaccine, more than 30 million cases of acute measles are annually reported worldwide with approximately 345,000 cases of infant deaths. Although MV can and does cause severe and sometimes lethal complications, including pneumonia and encephalitis also in industrialized countries, the majority of fatal cases develop in Third World countries on the basis of the transient, pronounced general immunosuppression induced by the virus during and for several weeks after acute measles (Katz 1995). There, exposure occurs early in infancy when fading maternal antibodies prevent successful vaccination but are no longer protective against wild-type MV infection (Black et al. 1986; Lennon and Black 1986; Ryon et al. 2002). Further aggravated by malnutrition, secondary infections then often follow a severe or fatal course (Dollimore et al. 1997). Furthermore, MV-induced immunosuppression also favors reactivation and exacerbation of persistent infections in children and young adults (Tamashiro et al. 1987). More recently, immunosuppression caused by viral infection in infants where the immune system is still immature has been proposed to play an important role in the establishment of persistent CNS MV infections (Oldstone et al. 2005).

General Features Associated with MV-Induced Immunosuppression In the immunocompetent host, MV induces an efficient virus-specific immunity that controls spread and leads to clearance of MV and to a most likely lifelong immunity to clinical reinfection. An initial Th1 cell activation as marked by soluble CD4, CD8, IL-2R, and β2, microglobulin switches to a prolonged Th2dominated response (reviewed in Griffin 1995 and the chapters by P.A. Rota et al. and W.J. Moss, this volume). The contribution of components of the innate immunity in MV control in humans is less well understood. The term “anergy” was first coined by von Pirquet in 1908 to describe the loss of DTH reactions to tuberculin in MV-infected individuals. MV-induced immunosuppression is commonly characterized by (1) a marked lymphopenia, which is rapidly resolved (Ryon et al. 2002), (2) a cytokine imbalance skewed toward a prolonged Th2 response, which suppresses cellular immunity to secondary infections (Griffin and Ward 1993), and (3), the inability of peripheral blood lymphocytes (PBL) to expand in response to polyclonal or antigen-specific stimulation ex vivo (Borrow and Oldstone 1995; Schneider-Schaulies et al. 2001). The extent to which these parameters are mechanistically decisive for or represent surrogates of MV-induced immunosuppression has not been resolved because skewing to an early Th2 response

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and slightly impaired proliferative responses of PBLs ex vivo have also been related to MV vaccination which, when applied at standard doses, does not cause immunosuppression (Ward and Griffin 1993; Hussey et al. 1996). Thus, there is still a need to understand basic mechanisms underlying MV-induced immunosuppression such as targeting of specific host cells and their relative importance for immunomodulation in suitable experimental systems. These involve small models mainly involving genetically modified mice, ferrets, and cotton rats (detailed in the chapters by S. Pillet et al., S. Niewiesk, C.I. Sellin and B. Horvat, and B. Hahm, this volume) and experimental infection in non-human primates, which has continued to provide exciting insights into cell targeting in primary infection (reviewed in the chapter by R.L. de Swart, this volume). It will be extremely important to evaluate to what extent the plethora of findings obtained in vitro, mainly referred to within this chapter, are reflected or at least compatible with an in vivo infection.

Lymphopenia Mechanisms of Lymphocyte Depletion Measles is associated with lymphopenia affecting B cells, monocytes, neutrophils, as well as CD4 and CD8 T cells. No genetic or gender-specific factors are known, yet the extent of lymphopenia correlates with age and severity of the disease (Okada et al. 2000, 2001). In contrast to B cell frequencies, which can still be below control levels for up to 6 weeks, T cell numbers return to normal after 10 days and the CD4/CD8 ratio remains constant over time (Arneborn et al. 1983; Okada et al. 2001; Ryon et al. 2002). Figures published on the frequency of infected peripheral blood mononuclear cells (PBMCs) vary considerably depending on the stage of disease analyzed and the methods used, however, generally do not exceed 2% at the onset of the acute phase (Schneider-Schaulies et al. 1991; Forthal et al. 1992; Esolen et al. 1993). This correlates with findings recently obtained in experimentally infected rhesus macaques (de Swart et al. 2007; see the chapter by R.L. de Swart, this volume). Not surprisingly, expression patterns of the major MV uptake receptor, CD150, directly correlate with infected cell types. Thus, the peripheral CD150+ B cell compartment was identified as preferentially targeted, and this may account for B cell lymphopenia as observed in humans (Okada et al. 2000). Peripheral blood monocytes do not express CD150 and were found not to be infected in this model, while earlier findings in humans suggested these were a major infected peripheral cell population (Esolen et al. 1993). In addition, infection of T lymphocytes both in PBMCs and secondary lymphatic tissues was documented in macaques (de Swart et al. 2007), and this mirrors findings obtained after in vitro infection of human tonsil slices (Condack et al. 2007). There, infection of both B and T lymphocytes directly segregated with expression of CD150 and targeted the CD45R0 T cell compartment, which was found to be preferentially depleted. Mechanisms other than loss of mature lymphocytes due to infection have also been proposed to contribute to lymphopenia; these were mainly studied for T cells (schematically depicted in Fig. 12.1). Thymocyte generation, viability, or differentiation into T cells can be viewed as effective targets for depletion. Consequences

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priming for apoptosis

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Fig. 12.1 Potential mechanisms for MV-induced lymphopenia. Few hematopoietic stem cells (HSCs) express CD150 (light green membrane) and remain largely unaffected by infection. Infected stroma cells can, however, release unknown components (red), which target HSC differentiation. Thymocytes express high levels of CD150 and may be depleted by infection and/or apoptosis (dark grey) induced by components released from infected thymic epithelial cells (red). Upon entry into secondary lymphatic tissues, T cells may be (1) activated normally, yet subsequently home aberrantly to peripheral tissues, (2) primed for apoptosis (dark grey), (3) lost by fusion with (dotted membrane infected dendritic cells), or (4) infection (red cells) after MV transmission from these cells

of MV interaction with CD34+ human hematopoietic stem cells (HSCs) were addressed in vitro (Manchester et al. 2002). While in mice members of the SLAM family distinguish defined HSC differentiation stages (Kiel et al. 2005), it is unknown whether the SLAM code also applies to corresponding cells in humans. MV infected with a very small subpopulation of human HSCs, however, did not affect their ability to differentiate into colony-forming cells in vitro. Interestingly, the ability of MV-infected stroma cells to support HSC development was impaired (Manchester et al. 2002). Since this study was conducted using a wild-type MV strain able to also interact with CD46, it is unclear if and how CD150-dependent wild-type viruses could access these cells. Studies on the interaction of MV with thymocytes, which express CD150 in humans (Aversa et al. 1997; Sidorenko and Clark 2003), revealed that thymocytes (and CD4 and CD8 T cells) of mice, in which transgenic expression of CD150 was driven by an Lck-promoter, expressed MV proteins after in vivo or in vitro infection (Hahm et al. 2003). Though not analyzed in this study, apoptosis of these cells likely occurs, since this was massively seen in MV-infected human thymus/liver xenografts in SCID mice where, however, thymocytes themselves remained uninfected (Valsamakis et al. 1999). Thymocyte viability and/or differentiation into functional T cells can also be affected by as yet undefined viral components released from infected thymic epithelial cells (Valentin et al. 1999). Since CD150 ligation by antibodies can also transmit inhibitory signals (Sidorenko and Clark 2003), engagement by the MV H protein could also affect thymocyte viability. Indeed, prolonged passage of avirulent MV strains in human thymic tissue enhanced their ability to cause thymocyte apoptosis, and this

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correlated with an amino acid exchange within the H protein ectodomain (Valsamakis et al. 1999). Strikingly, thymic output determined by T cell receptor excision circle frequencies was found to be even increased in measles patients, indicating that depletion of precursors possibly is not a major cause of peripheral T cell lymphopenia (Permar et al. 2003). Rather, cell loss due to infection may substantially contribute to this phenomenon. They most likely acquire MV in secondary lymphatic tissues, eventually by dendritic cells (DCs) carrying infectious virus. Indeed, infected T lymphocytes were isolated from lymphoid tissues of experimentally infected rhesus macaques (de Swart et al. 2007). As for MV acquisition from DCs, surface expression of CD150 would also be a prerequisite for loss of T cells by fusion with infected DCs, as occurs in mixed DC/T cell cultures to some extent (Fugier-Vivier et al. 1997; Grosjean et al. 1997; Servet-Delprat et al. 2000; Vidalain et al. 2000, 2001a). Giant cell formation and necrosis were reported in lymphoid tissues of MV-infected rhesus macaques, albeit to a moderate extent (McChesney et al. 1989; Kobune et al. 1996). Giant cell formation in hyperplastic lymphoid tissue was documented in a measles patient at autopsy; however, MV could not be detected in this area (Nozawa et al. 1994). Thus, T cell loss in secondary lymphoid tissues may occur, as may induction of apoptosis as seen in cocultures of infected DCs and T cells (Fugier-Vivier et al. 1997). In addition, as yet unidentified signals may prime T cells for apoptosis since peripheral T cells of measles patients revealed an enhanced sensitivity to CD3-ligation-induced cell death (Addae et al. 1995, 1998; Okada et al. 2000). Lastly, a preferential decrease in the frequency of LFA-1high T cells has been measured both in infected children and vaccinees, suggesting that these cells may aberrantly home to peripheral tissues, though activation-induced death is certainly also a possible explanation for the loss of these cells (Nanan et al. 1999).

MV Regulation of APC Viability and Function-Modulated Instruction and/or Active Lymphocyte Silencing Dendritic Cells: Maturation Program and General Functions MV-induced immunosuppression is thought to be centrally determined by its interaction with professional antigen-presenting cells (APCs), especially DCs. DCs of myeloid origin (further referred to DCs as opposed to plasmacytoid DCs [pDCs]), which can be isolated from peripheral blood or generated from precursors, are conductors of the adaptive immunity orchestrating lymphocytes to specifically recognize and handle antigens in the most effective manner. In an immature state, DCs reside in peripheral tissues and initiate a maturation program in response to danger signals such as viral components or inflammatory cytokines. This includes dramatic alteration of their receptor repertoire, acquisition of a migratory phenotype and secretion of soluble mediators to attract to T cells to which they present processed antigens (Steinman et al. 1997; Banchereau and Steinman 1998; Steinman

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2003). Immature DCs express a variety of surface and intracellular pathogen recognition receptors (PRRs), some of which (such as C-type lectin receptors) are specialized to internalize pathogens for subsequent processing and presentation, while other PRRs, such as TLRs, trigger maturation signals. Morphological changes associated with DC maturation include downregulation of actin-based ruffles and cell polarization, which allows distinction of a leading edge and an uropod (Fig. 12.2A). Surface receptors and molecules regulating cytoskeletal reorganization also polarize. Those essentially promoting directed migration (such as chemokine receptors and small GTPases Rac and Cdc42) localize to the leading edge, while the uropod concentrates adhesion receptors such as CD43 and RhoA, respectively (Sanchez-Madrid and del Pozo 1999; Arrieumerlou and Meyer 2005). Directed movement from peripheral tissues along chemokine gradients allows DC

MOCK DC

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Fig. 12.2 A, B DC/pDC functional differences and DC morphological maturation. A DCs generated from monocytes by culture in GM-CSF/IL-4 were treated with uninfected cell extract (MOCK), lipopolysaccharide (LPS) or infectious wild-type MV for 24 h, seeded onto fibronectin, and analyzed by scanning electron microscopy. Immature DCs (MOCK, upper panel) reveal large ruffled membrane protrusions while mature DCs (LPS, bottom panel) appear polarized with lamellar protrusions on their leading edge. Although overall slightly smaller, MV-infected DCs morphologically resemble LPS DCs in showing clear front-rear polarization and lamellar protrusions (MV, middle panel). B Myelopid DCs (DC) and plasmacytoid DCs (pDC) differ in their expression profiles of pattern recognition receptors (upper triangles), chemokine receptors (middle triangles) (both of which are regulated with DC maturation, e.g., CCR5 on immature, and CCR7 on mature DCs) and transcription factors, especially IRF-7 (bottom triangles). Supporting is important role in homing to secondary lymphatics, CCR7 is upregulated both on DC and pDCs. DCs and pDCs also differ substantially with regard to cytokine production

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homing to secondary lymphoid tissues where characteristic large actin based protrusions (veils) provide contact planes for scanning T cells (Shutt et al. 2000; Burns et al. 2004) (Fig. 12.2A). Antigen-processing and -presentation pathways are activated, and finally, mature DCs display and present loaded MHC class II molecules at their surface (Banchereau and Steinman 1998). Activation of MHC class I restricted CD8+ T cells by DCs relies on viral replication, or cross-priming after uptake of cell-bound or cell free antigens (for review see Wilson and Villadangos 2005). Mature DCs do not leave but rather die in lymphoid tissues thereafter and are not found in the efferent lymph. Plasmacytoid DCs (pDCs) are mainly found in blood from where they directly access secondary lymphatic tissues (Banchereau and Steinman 1998). They also mature after pathogen encounter, yet their ability to present antigen is very limited. Their distinct repertoire of PRRs, chemokine receptors, and transcription factors rather coins them as effector cells of the innate immune system particularly in viral infections, because they readily produce high amounts of type I IFN, whereas (myeloid) DCs act rather as major producers of inflammatory cytokines such as IL-12, TNF-α, IL-6, and IL-1α/β as important for effector cell activation (Asselin-Paturel and Trinchieri 2005) (Fig. 12.2B).

MV-Induced Effects on Dendritic Cells DC Infection In Vivo Direct evidence for MV infection of DCs during acute measles in humans has not yet been obtained. These cells may represent prime targets during MV transmission since they localize within and can even project through the respiratory epithelium, and in contrast to epithelial cells, can express CD150 at least in vitro (Minagawa et al. 2001; Ohgimoto et al. 2001; de Witte et al. 2006; Rethi et al. 2006). If targeted for infection, these cells would and have been suggested to be good candidates for MV transport to local lymphatic tissues. Indirect evidence for wild-type MV targeting secondary lymphoid tissues and there, determined by its H protein, DCs was obtained in experimentally infected cotton rats (Sigmodon hispidus) (Niewiesk et al. 1997; Pfeuffer et al. 2003; the chapter by S. Niewiesk, this volume). In CD46 transgenic mice, splenic DCs were found strongly activated and infected by attenuated MV (strain Edmonston), yet this required prior deletion of monocyte/macrophages (Mrkic et al. 1998, 2000). MV antigens were also detected in very limited numbers of splenic CD11c+ DCs after intravenous MV wild-type infection of mice transgenic for CD150 driven by the CD11c promotor (Hahm et al. 2004; the chapter by B. Hahm, this volume). In this system, CD11+ DCs were, however, the only cells accessible to MV entry, and thus potential infection of other cell types also expressing CD150 could not be evaluated. Though ablation of type I IFN receptor or associated signaling pathways has led to a significant improvement in detection of MV RNAs of proteins in secondary lymphoid tissues in mice, infection of CD11c+ cells in vivo was, if at all, barely detectable (Shingai et al. 2005; Welstead et al. 2005; Ohno et al. 2007).

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However, transgenic expression of CD150 on murine DCs directly isolated or generated from bone marrow precursors conferred susceptibility to MV infection in vitro, and this was strongly enhanced if type I IFN signaling was additionally ablated, indicating that this system is one of the major restriction factors for MV in murine cells. More recently, in macaques experimentally infected with a wild-type MV encoding eGFP, strong evidence for infection of CD11c+ MHC class II-positive cells with DC morphology in secondary lymphoid tissues, the dermis, or emigrating from skin explants from these animals was obtained (de Swart et al. 2007; the chapter by R.L. de Swart, this volume). Assuming that MV tropism in this model reflects this in humans, it appears thus very likely that DCs (and/or Langerhans cells) might indeed be targeted by MV infection in vivo. Early studies in nonhuman primates found MV associated with follicular dendritic cells (FDCs) which are of nonhematopoietic origin and may, though this has not been established, serve as long-term repositories for MV (McChesney et al. 1997). MV Interaction with Receptors on DCs Langerhans cells and DCs isolated from peripheral blood or generated in vitro from monocytes or CD34+ precursor cells are susceptible to MV infection (FugierVivier et al. 1997; Grosjean et al. 1997; Schnorr et al. 1997b; Steineur et al. 1998; Klagge et al. 2000; Servet-Delprat et al. 2000; Dubois et al. 2001; Ohgimoto et al. 2001). Infection with CD46-adapted strains is not surprising since this molecule is expressed on DCs of any maturation stage. MV wild-type strains and recombinant MVs expressing wild-type-derived H proteins rely on CD150 for entry, and thus, CD150-specific antibodies inhibit MV uptake (de Witte et al. 2006) (Fig. 12.3). As detailed in Sect. 4.2.1), this molecule is either expressed at low levels on cultured immature DCs or induced upon activation by LPS exposure or proinflammatory cytokines (Kruse et al. 2001; Minagawa et al. 2001). CD150 can also be induced by MV wild-type strains themselves as shown in monocytes (and probably in DCs) since these strains act as Toll-like receptor 2 (TLR2) agonists (Minagawa et al. 2001; Bieback et al. 2002). Interestingly, their TLR2 agonistic activity seems to inversely correlate with their ability to interact with CD46. Thus, a recombinant MV expressing a wild-type H protein with aa481 reversed to that found in attenuated strains failed to activate TLR2 signaling (Bieback et al. 2002). Although the TLR2 does not support MV entry, interaction of MV wild-type strains may, besides CD150 induction, contribute to MV-induced maturation of immature DCs. MV wild-type-dependent activation of TLR2 presumably accounts for the induction of CD150, CD86, and cytokines such as IL-6, IL-12p40, and IL-1α/β (Bieback et al. 2002). Other consequences of CD150 ligation by MV on DCs remain speculative, but may include modulation of TLR4 but not TLR2 or TLR9 signaling and thereby production of IL-12, TNF-α, NO, and IL-6, as shown in macrophages (Wang et al. 2004). MV entry into DCs apparently also involves surface molecules other than CD150. Thus, MV uptake into these cells correlated with enhanced fusion rather than binding (Ohgimoto et al. 2001). Though the C-type lectin, CD209 (DC-SIGN)

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No entry receptors, yet enhance uptake (DC-SIGN) Endocytosis or virus capture for trans-infection? Maturation signals (TLR2) and/or differentiation signals (TLR2/CD209)

Fig. 12.3 Interaction of MV with cell surface receptors on DCs. Immature DCs express CD46, TR2, and CD209 to high levels and upregulate CD150 upon maturation signals. As for other cell types, CD150 is the major determinant of DC infection by all MV strains, and uptake into DCs is enhanced by CD209 (which could also involve capture and concentration of MV for transinfection of T cells). CD209 and TLR2 do not serve as entry receptors but actively trigger (TLR2) or modulate (CD209) signaling pathways in DCs. In addition to their role in entry, CD150 and CD46 may also modulate signaling pathways in DCs

does not support MV entry upon transgenic expression in CHO cells, it enhances uptake into immature DCs (de Witte et al. 2006) by an as yet unknown mechanism. Possibly, ligation of this highly abundant surface receptor locally concentrates CD150, thereby favoring viral binding and fusion. For HIV, CD209 has been shown to strongly enhance a process referred to as transinfection, i.e., transfer of virus by DCs to conjugating T cells via infectious synapses (Geijtenbeek et al. 2000; Geijtenbeek and van Kooyk 2003a), which, early after viral exposure, occurs independently of DC infection. Transmission of MV to T cells in DC/T cell conjugates has been found to be rather inefficient, yet greatly enhanced, if T cells are preactivated prior to conjugate formation (Fugier-Vivier et al. 1997; Grosjean et al. 1997). This possibly results from both enhancement of viral replication after CD40 ligation (as proposed) and induction of CD150 on T cells enabling viral fusion and entry. Whether this also affects formation of infectious synapses and what role CD209 play in this setting is currently unknown.

MV Infection and DC Viability and Maturation The efficiency of MV infection in human DCs in vitro varies between strains. In addition to their preferential uptake, wild-type strains replicate faster in DCs than attenuated strains (Schnorr et al. 1997b; Ohgimoto et al. 2001), indicating that MV proteins other than the glycoproteins (gps) favor intracellular replication in DCs. Recently, the stability of the M protein has been directly related to the ability of

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attenuated rather than wild-type strains to replicate in DCs (Ohgimoto et al. 2007), and the ability of induce formation of multinucleated giant cells in mature, but not immature DCs (and concomitantly, amplification of type I IFN production) was found restricted to a CD46-adapted strain (Herschke et al. 2007). This is in contrast to earlier findings where wild-type MV (or recombinants expressing wild-type H protein) preferentially caused fusion in DCs (Ohgimoto et al. 2001). Although viral proteins efficiently accumulate, virus production is low from immature or almost absent from LPS-DCs (Fugier-Vivier et al. 1997; Servet-Delprat et al. 2000), indicating that, as shown for monocytes (Helin et al. 1999), their differentiation stage imposes particular constraints on MV release. Ligation of CD40 on DCs caused enhancement of MV protein production and virus release (Fugier-Vivier et al. 1997; Servet-Delprat et al. 2000), yet did not affect viral replication or syncytium formation in another study (Klagge et al. 2004). Due to the accumulation of the viral gp complex on the surface of infected DCs, DC fusion with co-cultured T cells was observed (Grosjean et al. 1997). In this stud,y DC apoptosis also occurred late after infection. When cultured in the presence of a peptide inhibiting cellular fusion, DCs remained viable for at least 48 h (Schnorr et al. 1997b; Klagge et al. 2000; Dubois et al. 2001). Early after infection, MV-infected DCs strongly resemble LPS-matured cells morphologically, as determined by formation of leading edges and uropods as well as veiled protrusions (Shishkova et al. 2007; Fig. 12.2A). Indicating that rearrangements of the actin cytoskeleton were not generally affected, integrin-dependent adherence and migration on fibronectin were maintained in MV-infected DCs (Shishkova et al. 2007). Immature DCs exposed to MV rapidly mature phenotypically, as indicated by upregulation of MHC class I and II molecules, CD40 and costimulatory molecules such as CD80, CD83, and CD86 (Schnorr et al. 1997b; Klagge et al. 2000; ServetDelprat et al. 2000; Dubois et al. 2001) and cytokine production (summarized in Fig. 12.4). Infection with attenuated MV strains caused induction of IL-12p35, IL12p40, IL-23p19, IL-10, IL-1α/β, IL-1RA, and IL-6-specific transcripts in monocyte-derived DCs (Servet-Delprat et al. 2000; Klagge et al. 2004). On the protein level, IL-12p70 and IL-10 were not detectable after infection with either wild-type or attenuated strains (Klagge et al. 2004). IL-10 (both mRNA and protein) was, however, produced in MV ED-infected DCs generated from CD34+ progenitor cells (Dubois et al. 2001). More recently, induction of IL-10 transcripts in immature DCs exposed to wild-type MV was related to Raf-1 activation in response to CD209 ligation (Gringhuis et al. 2007). Interestingly, for other CD209 ligands analyzed in this study, this only occurred after TLR activation, and that MV was able to mediate this on its own possibly reflects its ability to ligate TLR2 as well (Bieback et al. 2002). Mechanisms accounting for the induction of cytokine-specific transcripts, particularly by attenuated MV strains that cannot activate TLR2 signaling, are only partially known. There is ample evidence that attenuated strains cause induction of type I IFNs in DCs, and recently this has been associated with accumulation of defective interfering RNAs (DI RNAs), the detection of which relied on RIG-I/ MDA-5 rather than onTLR3-TICAM-1-driven activation of IRF-3 (Shingai et al.

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MV contact/uptake or replication

Induction of T cell apoptosis, T cell depletion by transfer of virus or cell fusion Differentiation of Th2 cells or regulatory T cells (eg. by IL10 or IFN production?) DC function

maturation development and expansion (IFN production?)

Infection (CD150, CD46) FcγRII interaction TLR2 interaction?

Inhibition of T cell expansion by surface contact mediated signals DC viability (by fusion, CD95L induced apoptosis)

upregulation of costimulatory and MHC molecules chemotaxis? T cell attractants? IL-6, IL-1α/β, IL-12low, IL-10low, IL-18 and type I IFN T cell clustering, antigen processing?

Fig. 12.4 Impact of MV on DC maturation and differentiation. MV (dependent and independent of infection) has been found to modulate DC viability, differentiation, and functions at multiple levels ranging from development from precursors and maturation signals (which may be partially incomplete) to depletion of DCs by apoptosis or fusion. DCs can also contribute to T cell loss by fusion or transmission of infectious MV (see also Fig. 12.1), but can also actively silence T cell expansion by contact-mediated signals (as mostly evidenced by loss of allostimulatory properties). Hypothetically, MV-infected DCs could also promote expansion of regulatory T cells by production of type I IFN and/or IL-10 (this and all other mechanisms indicated which are not yet experimentally proven to occur are listed in italics)

2007). Type I IFNs were identified as important in DC maturation in terms of upregulation of CD80 and CD86, TRAIL, and TLR3 (Klagge et al. 2000; Vidalain et al. 2000; Dubois et al. 2001; Tanabe et al. 2003) while upregulation of NKG2D ligands (UL-16-binding protein 2 and retinoic acid early transcript 1G) can also occur independently of these cytokines (Ebihara et al. 2007). Moreover, acquisition of DC cytolytic activity has also been linked to IFN induction (Vidalain et al. 2000, 2001a). However, MV wild-type strains very inefficiently induced type I IFN in PBMC and DCs from which DI RNAs could not been amplified (Naniche et al. 2000; Shingai et al. 2007). Impact of MV on External Maturation/Stimulation Signals in DCs External signals triggered by TLRs, cytokines, and finally CD40 are important for activation, mobilization, and induction of terminal maturation of DCs. The ability of MV-infected DCs to switch their chemokine receptor repertoire and to migrate in response to chemokines has not been addressed in detail. Impairment of chemotactic responses to MIP3α were reported, yet were not addressed mechanistically (Dubois et al. 2001). TLR signaling can be modulated by MV infection, as evidenced in monocytes where MV exposure caused inhibition of LPS- or SACSstimulated IL-12p70 production by ligation of CD46 (Karp et al. 1996). Long-term suppression of SACS-stimulated IL-12 release in PBMCs isolated from measles

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patients supports an impaired production of this cytokine (Atabani et al. 2001). In DCs, MV interference with TLR-dependent IL-12 production in vitro is less clear. Infection of DCs isolated from peripheral blood with wild-type or attenuated strains did not interfere with release of bioactive IL-12 in response to LPS or SACS (Schnorr et al. 1997b), while inhibition of IL-12 production in MV-infected DC cultures was also reported, which correlated in a timely manner with the accumulation of apoptotic DCs (Fugier-Vivier et al. 1997). MV can also promote—rather than interfere with—TLR signaling, as shown by induction of IL-10 transcripts upon co-ligation of CD209 (Gringhuis et al. 2007). MV interference with signaling via TLR7/9 occurs evidently in pDCs that did not produce type I IFN upon infection, yet were also compromised to do so in response to TLR7 or TLR9 triggering (Schlender et al. 2005). Modulation of CD40 signaling by MV infection is very likely to be of functional importance, but this remains controversial. Enhancement of viral replication following CD40 ligation by activated T cells or antibodies suggested that CD40 signaling pathways are at least partially active. In contrast, downregulation of tyrosine-phosphorylation of cellular proteins, IL-12p40 transcripts, and IL12p70 production, induction of IL-10 transcripts, and failure of MV-infected DCs to drive proliferation of CD8+ T cells after the same stimuli suggested that CD40 signaling is blocked (Servet-Delprat et al. 2000). Enhanced production of IL-12p70 and IL10 proteins from MV-infected DCs after CD40 ligation were also reported (Dubois et al. 2001; Klagge et al. 2004). Thus, MV-mediated modulation of CD40 signaling, its targets and consequences are far from being understood and require reevaluation with virus strain, timing of stimulation, and probably the source of DCs being the critical components.

The DC/T Cell Conjugate: From Attraction to Transmission and/or T Cell Activation Little is known about MV modulation of DC chemotaxis on protein or functional level, e.g., the switch from CCR5 to CCR7 expression as required for tissue exit and homing and associated signaling pathways. It is also largely unknown if the chemokine profile released by MV-infected DCs (as analyzed so far only at the transcriptional level Zilliox et al. 2006) is compatible with successful recruitment of T cells (and particular subsets thereof). In vitro, DCs infected with an attenuated MV strain can recruit co-cultured T cells into conjugates and there, both fusion and, albeit to a limited extent, transmission of virus to T cells occurs (Grosjean et al. 1997) (Fig. 12.4). For wild-type strains, transmission to T cells has not been analyzed as yet. For HIV, infection of T cells is greatly enhanced if virus is provided by DCs (Pope et al. 1995; Cameron et al. 1996), and concentration of virus and/or receptors by CD209 and transfer within an infectious synapse at the DC–T cell interface is believed to play an important role. Formation of an infectious synapse occurs independently of antigen recognition, yet relies on actin-dependent receptor clustering on the surface of the acceptor cell (Nejmeddine et al. 2005; Jolly et al. 2007). CD209-dependent trapping of MV on the DC surface or within

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endosomal compartments has not been analyzed, though there is evidence that MV proteins are present in stable DC/T cell conjugates at the interface (Shishkova et al. 2007). Receptor concentration on the conjugating T cell may be difficult to achieve since firstly, these cells, unless they are preactivated, do not express CD150, and secondly, they reveal a very limited ability to reorganize their actin cytoskeleton once they have encountered the MV gp complex (Muller et al. 2006). If following transfer impairment of T cell viability and function relies on viral replication, T cell activation would be an essential prerequisite for this to efficiently occur (Borrow and Oldstone 1995). T cell activation is also required for upregulation of FasL and sensitivity to TRAIL-dependent killing has been suggested to contribute to T cell and or DC depletion (Vidalain et al. 2000, 2001b). Thus, the ability infected DCs to promote T cell activation, which requires formation of functional immunological synapses (IS), is crucial. Interfaces formed in stable conjugates between MV-infected DCs and T cells reveal a pattern of CD3, LFA-1, or talin distribution and consistent with an activatory phenotype (Shishkova et al. 2007) (Fig. 12.5A), which is surprising given the inability of MV-infected DCs to stimulate expansion of allogenic T cells in mixed leukocyte reactions (Fugier-Vivier et al. 1997; Grosjean et al. 1997; Kaiserlian et al. 1997; Schnorr et al. 1997b; Steineur et al. 1998; Klagge et al. 2000; Servet-Delprat et al. 2000; Dubois et al. 2001; Klagge et al. 2004). As revealed by live cell imaging, the majority of conjugates between MV DCs and T cells is, however, highly unstable and does not promote T cell activation (Shishkova et al. 2007). Moreover, it appears unlikely that viral transmission occurs there since these conjugates dissolved within less than 2 min. Currently, it is unclear what determines synapse instability, yet it appears that the viral gp complex contributes to this phenomenon. This is because MGV-infected DCs (which express VSV G instead of the MV F/H proteins on their surface) at least partially retain their ability to recruit T cells into stable, stimulatory conjugates (Shishkova et al. 2007) (Fig. 12.5B).

Viral Proteins Effective at T Cell Silencing and Modulation MV infection is known to cause cell cycle arrest and to interfere with differentiation of effector functions in infected lymphocytes, including T cells. Infected PBMCs in vivo are very limited in frequency, yet these cells resist signals promoting expansion ex vivo, and thus mechanisms other than direct infection are evidently involved. These are most likely provided in vivo by contact with or factors released from infected cells which are of low abundance. Ex vivo analyses do not support deficiencies in IL-2 production or a lack of IL2R expression (Griffin and Ward 1993; Moss et al. 2002). Inhibitory soluble mediators released from in vitro infected cells may include IL-10, yet IL-10 production in vivo has not consistently been seen (Okada et al. 2001; Moss et al. 2002). Though attenuated MV strains induce type I IFN release from PBMCs, it is still unclear whether this accumulates to significant levels in vivo. Moreover, there is no evidence for type I IFN being responsible for lymphocyte arrest after mitogenic

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LPS I

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DCs seeded onto PLL

% in category

T cells loaded with Ca-sensitive fluorophore Duration and efficiency of conjugates

100 90 80 70 60 50 40 30 20 10 0 M OCK

b

I

LPS II

W TF III

IV

Fig. 12.5 A, B Conjugates formed between MV-infected DCs and T cells are mostly unstable and nonstimulatory. A Conjugates formed between Mock- (immature), LPS-, or MV-infected superantigen loaded DCs with allogenic T cells after 20 min were stained for the expression of f-actin (red) and CD3 (green). As evident in LPS-DC conjugates, CD3 is almost entirely recruited to the DC–T cell interface, sometimes centrally located (exemplied as LPS-II). The majority of LPSDCs reveal, however, a multifocal distribution of CD3 (LPS-I), and this is also predominantly observed in stable MV-DC–T cell conjugates. Contact with immature DCs (Mock) barely induces CD3 relocalization. B Duration and efficiency of conjugates formed between LPS-, mock- or MVDCs and T cells were determined by live cell imaging (left, experimental setup; the red color in some T cells indicates changes in emission of fluo-4 upon calcium fluxing). LPS DCs mainly conjugate stably with and sustain calcium mobilization in T cells (red bars), while with mock DCs, conjugates are largely unstable and do not elicit calcium fluxing (white bars). Stable, efficient conjugates were rare with MV-DCs (red bars), which mainly were dissolved within 2 min and unable to sustain calcium mobilization (which was initially successfully induced) (black bars). The last contact category (grey bar), seen almost exclusively with MV DCs, was durable, yet not associated with efficient calcium fluxing

stimulation in vitro, and as yet unidentified inhibitory factor(s) released from infected T cells were insensitive to IL-10 or type I IFN specific antibodies (Fujinami et al. 1998; Sun et al. 1998). In contrast, polyclonal MV-specific antibodies were effective at neutralizing the inability of MV-infected T cell cultures to proliferate in response to mitogenic stimulation (Sanchez-Lanier et al. 1988; Yanagi et al. 1992), indicating that viral proteins were directly involved.

The N Protein The nucleocapsid (N) protein is the major constituent of the virion, and in addition to complex the viral genome and the P protein, it interacts via its C-terminus with cellular proteins such as Hsp72 and IRF-3, thereby modulating viral replication and

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IFN-induction, respectively (tenOever et al. 2002; Zhang et al. 2002). Surprisingly, soluble N protein was found to act as a ligand for Fcγ RII on B cells to cause inhibition of antibody production and to reduce T cell rather than B cell responses in mice (Ravanel et al. 1997; Marie et al. 2001). As detailed in the chapter by C.I. Sellin and B. Horvat, this volume, N protein-mediated inhibition of T cells was indirect since the protein prevented IL-12 production and the generation of inflammatory reactions by modulating DC functions in a Fcγ RII-dependent manner (Marie et al. 2002, 2004). In vitro, soluble N protein induced calcium flux in thymic epithelial cells and interfered with proliferation of a variety of cell types, including activated, but not resting human primary T cells (Laine et al. 2003). While the ability of N protein to induce apoptosis relied on binding its core domain to the Fcγ RII, the ability to induce G1 arrest was attributed to the interaction of its carboxyterminal tail domain with an as yet unknown receptor (Laine et al. 2003, 2005). Infected cells dying from apoptosis and/or secondary necrosis such as thymic epithelial cells are considered as a source of extracellular N protein (Laine et al. 2003, 2005). In infected cells, N protein may gain access to late endosomal compartments, where it recruits Fcγ RII, and is co-transported to the cell surface (where receptor bound N protein could act in neighboring cells) and/or released into the extracellular medium (Marie et al. 2004). Whether there is a role of N protein for T cell silencing in vivo remains questionable. Firstly, the putative receptor for N protein is only found on T cells that are already activated (Laine et al. 2003) and thus would confine inhibitory signals to this particular population. Secondly, the C-terminal domain of N protein is the most variable within all MV proteins and is therefore used for MV strain genotype assignment. Indicating that this domain has significant functional activity, a single amino acid exchange at position 522 (N in the Edmonston and D prevailing in wild-type strains) caused loss of hsp72 binding and at the same time conferred fitness in vivo (Carsillo et al. 2006). It would therefore be important to determine whether particularly MV wild-type strain-derived N proteins would also efficiently inhibit T cell proliferation via their C-termini. Thirdly, a recombinant MV that expressed VSV G protein instead of the authentic MV F/H gp complex failed to induce T cell arrest in vitro and in vivo, indicating that N protein may not be essential, at least for T cell silencing.

The Glycoproteins Evidence for the ability of the MV glycoprotein (gp) complex, consisting of the hemagglutinin (H) and the proteolytically activated F1/2 heterodimer, to signal T cells came from a number of in vitro and in vivo experiments. In vitro, MV virions, but not those produced by a recombinant MV expressing VSV G as a surface protein, prevented expansion of both T cell lines and mitogen- or anti-CD3/CD28stimulated primary human or rodent T cells in a dose- and contact-dependent manner. In addition, T cell expansion in vitro was inhibited upon coculture with cells transfected to co-express F and H protein (both not with those expressing F or

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H protein alone), and transfer of these doubly transgenic cells into cotton rats significantly impaired stimulated T cell proliferation ex vivo (Schlender et al. 1996; Niewiesk et al. 1997; Schnorr et al. 1997a; Niewiesk 1999; Weidmann et al. 2000a; Avota et al. 2001). The requirement of the F/H gp complex as effector structure for T cell inhibition was confirmed for related viruses such as Rinderpest and PPRV (Heaney et al. 2002). Whereas complex glycosylation and fusogenic activity of the gp complex are not required for contact-mediated T cell arrest, proteolytic processing of the F protein is (Weidmann et al. 2000a, 2000b). Exogenous cleavage of the F0/H complex restored its inhibitory activity, indicating that the effector domain most likely resides within the F protein and is conformation-dependent. The H protein may strengthen the interaction of the complex with the target cell surface by binding to its receptor(s) or stabilizing the conformation of the F1/2 protein. T cell silencing induced by viral gps does not involve induction of apoptosis or interfere with stimulated upregulation of surface markers (including the IL-2R α chain) or cytokine release, but rather prevented S-phase entry of these cells (Schnorr et al. 1997a; Engelking et al. 1999; Niewiesk et al. 1999; Avota et al. 2001; Schneider-Schaulies and ter Meulen 2002).

Surface Receptors Involved in T Cell Silencing Though contact-dependent T cell arrest requires co-expression of the H protein, transmission of this particular signal does not rely on the known MV entry receptors, because murine T cells, which do not express human CD46 and CD150, are sensitive to inhibition (Dorig et al. 1993; Tatsuo et al. 2000; Erlenhoefer et al. 2001), which in turn, for human T cells, is insensitive to CD46- or CD150-specific antibodies (Erlenhoefer et al. 2001). Both molecules can modulate CD3 signals on T cells upon co-ligation in vitro; however, they have co-stimulatory rather than inhibitory action (Astier et al. 2000; Zaffran et al. 2001; Sidorenko and Clark 2003), though CD150 ligation can also confer sensitivity to CD95-mediated apoptosis in some B and T cell lines (Mikhalap et al. 2004). Most studies addressing the effects of CD46 ligation on leukocyte functions have focused on APCs rather than T cells and include modulation of production of inflammatory cytokines and enhanced sensitivity to complement-mediated lysis after downregulation from the cell surface (Schnorr et al. 1995; Karp et al. 1996; Schneider-Schaulies et al. 1996; Hirano et al. 1999; Marie et al. 2001, 2002). Since, however, firm interaction with CD46 is a property of attenuated rather than wild-type MV strains (Hsu et al. 2001; Ono et al. 2001; Yanagi et al. 2002), the relevance of these findings for immunosuppression in vivo remains to be determined. For immunosuppression, CD150 would be expected to be more important, not at least because its expression is crucial for cell and tissue targeting of wild-type MV. CD150 expression is, however, confined to activated T cells (Aversa et al. 1997), and this, together with its co-stimulatory properties, argues against a direct role in contact-dependent T cell silencing. Moreover, it does not interact with morbilliviral F proteins, which are likely to harbor the effector domains. Cell surface molecules

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interacting with MV include TLR2 (Bieback et al. 2002), CD209 (de Witte et al. 2006), and substance P receptor (Makhortova et al. 2007). These are, however, either not expressed on human B and T cells (CD209, TLR2) or not further analyzed in terms of their ligands (substance P receptors). Moesin was also found to enhance MV uptake (Dunster et al. 1994; Schneider-Schaulies et al. 1995), although it is not supposed to act as a MV binding partner. It may instead have an indirect role in coupling surface receptors to the actin cytoskeleton following MV interaction. This could be important for viral uptake, but also for clustering of signaling receptors or T cell plasticity. Present data support the view that an unknown receptor mediates signals induced by the gp complex at the cell surface of lymphoid cells. Notably, B and monocytic cell lines were also sensitive to gp-mediated inhibition, indicating that the receptor structure involved is not T cell-specific (Schlender et al. 1996).

Signaling Pathways Targeted by MV The unknown receptor for gp-dependent T cell inhibition apparently locates or is recruited into detergent resistant membrane microdomains (DRMs), which appear as extended clusters, co-staining for MV H protein at binding conditions on resting T cells (Avota et al. 2004). It is very likely that signaling interfering with T cell activation is initiated there. Apparently, activation of the phosphatidyl-inositol-3(PI3K)/Akt kinase pathway is a, if not the central target for MV interference in T cells. MV is so far the only pathogen shown to interfere with activation of this particular pathway; given its important role in conveying survival and mito- if not oncogenic signals, some viruses rather activate this particular pathway (Yu and Alwine 2002; Yuan et al. 2002; Dawson et al. 2003; Cacciotti et al. 2005). In contrast, activation of PI3/Akt kinases after IL-2R or CD3/CD28 ligation was efficiently blocked shortly after MV exposure in vivo and in vitro (Avota et al. 2001, 2004) (Fig. 12.6A). Downstream effectors of this kinase include subunits of cyclin-dependent kinases essentially involved in S-phase entry, and consequently, these were found deregulated in T cell cultures exposed to or infected with MV (Engelking et al. 1999; Naniche et al. 1999). The importance of interruption of Akt kinase activation for MV-induced T cell silencing was directly documented as transgenic expression of a catalytically membrane targeted Akt kinase largely abolished the inhibitory signal (Avota et al. 2001). The regulatory subunit of the PI3K, p85, which acts upstream Akt kinase activation, was tyrosine-phosphorylated shortly after TCR ligation in MV-exposed T cells, yet failed to redistribute to cholesterol-rich DRMs, and this correlated with a lack of TCR-stimulated degradation of Cbl-b protein (Avota et al. 2004). More recently, it has been documented that downmodulation of tonic PI3K activity (as measured under standard growth conditions of primary T cells) targets activation of cellular splice accessory proteins (both members of the STAR and the SR proteins), and causes production of an isoform of the dual phosphatase SHIP145, SIP110 (Avota et al. 2006). This protein is translated from an alternatively spliced mRNA containing intron-derived sequences (Geier et al.

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b Fig. 12.6 A, B MV interaction with T cells interferes with activation of the PI3/Akt kinase pathway and causes cell collapse. A Activation and membrane recruitment of the PI3K in response to TCR ligation is a central target for MV gp contact-mediated signaling. As a consequence polyinositol-polyphosphates do not efficiently accumulate in the plasma membrane and thus, recruitment of pleckstrin-homology containing proteins such as the Akt kinase, but also the guanosine exchange factor Vav (not shown). The importance of this pathway is illustrated by the finding that MV contacted T cells are unable to enter the cell cycle and to rearrange their actin cytoskeleton in response to stimulation. B Resting T cells when exposed to Mock cell extracts reveal a pronounced front-rear polarization when seeded onto fibronectin (left panel, Mock). Moreover, microvillar protrusions are clearly discernible by scanning EM. If exposed to MV, T cells fail to polarize on fibronectin and reveal an almost complete collapse of membrane extension (right panel, MV)

1997). Importantly, SIP110, when expressed in Jurkat or primary T cells, was a constitutively membrane-associated, active lipid phosphatase, which continuously depleted the concentration of phosphatidyl-inositol-3,4,5-phosphates, thereby raising the threshold for activation signals. Most likely also as a consequence of its ability to block PI3K activation, MV signaling interferes with activation of the guanosine exchange factor Vav and its downstream substrates, the small GTPases Rac and Cdc42. Consequently, cytoskeletal rearrangement in response to TCR ligation and formation of filopodia and lamellipodia is severely impaired (Muller et al. 2006). It is already prior to stimulation that MV causes an almost complete collapse of filopodia protrusions on T cells and this, together with its ability of induce activation of RhoA, implies that interaction of MV with T cells induces membrane signaling directly (Muller et al. 2006) (Fig. 12.6B). Although it cannot be sustained, calcium mobilization or overall activity of membrane proximal tyrosine kinases after TCR ligation or contact with MV-infected DCs is unaffected within the first 2 min (Avota et al. 2004; Shishkova et al. 2007). Thus, second messengers acting in close association with the lipid bilayer might be operative in this scenario.

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Shaping of T Cell Responses by Receptor Interactions Apparently, expression of the viral gp complex on the surface of DCs would be a potent effector mediating contact-dependent T cell inhibition, which, can be considered a physical and functional paralysis. The MV gps, in particular H proteins, could, however, also shape ensuing T cell subset differentiation by interacting with their respective receptor(s) by providing co-stimulatory signals. When cross-linked by antibodies on T cells, CD150 was suggested to act as a co-stimulator strongly enhancing IFN-γ production and thereby a Th1 response (for review see Sidorenko and Clark 2003). Studies in CD150−/− mice, however, failed to support a critical role of this molecule in IFN-γ production but rather indicated that CD150 may enhance TCR-stimulated IL-4 release (Wang et al. 2004). DCs infected in vitro with either wild-type or vaccine MV efficiently trigger expansion of IFN-γ-producing T cells from CD45RO− T cell populations, and this even if DCs were generated in a way that expectedly would promote expansion of IL-4-producing T cells (Klagge et al. 2004). Preferential expansion of IFN-γ-producing cells in these cultures relied, however, on soluble mediators released from infected DC cultures rather from direct ligation of CD150. It is also possible, if not likely, that particularly those DCs infected with attenuated strains induce expansion or differentiation of regulatory T cells (natural or adaptive, respectively). Thus, CD3/CD46 co-ligation by antibodies can induce Tr1 cell in vitro (Kemper et al. 2003, 2005), and this scenario could be envisaged for DCs expressing H proteins of CD46-adapted MV strains, although this would be limited to CD46-interacting MV strains. Moreover, incomplete or aberrant maturation and/or production of type I IFN and IL-10 rather than IL-12, as seen in infected DCs, could provide a favorable environment. So far, however, regulatory T cells have not been directly shown to be induced or expanded during measles or after vaccination.

Conclusions and Perspectives Given the role of DCs to induce and shape immune responses, it is evident that interaction with these cells is central for immunomodulation by MV, although their interaction has not been extensively studied in vivo. From in vitro data, it is clear that modulation of DC viability and function is induced by MV independently of or as a result of infection, with its pattern and efficiency being partially virus strain-dependent. DCs most likely serve as primary target cells during infection and receive their maturation signals such as PRR signaling or other as yet unknown stimuli. MV can enter into DCs via its cognate receptors, and it is not yet known whether other modes of uptake such as endocytosis (for instance via DC-SIGN (Geijtenbeek and van Kooyk 2003b; van Kooyk and Geijtenbeek 2003) are also operative. Early after infection (or, if occurring, endocytosis), presentation or of MV peptides generated by processing for subsequent MHC loading and

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presentation certainly occurs, as does cross-presentation, and this results in the efficient induction of MV-specific immune responses. Initial activation of DCs and their ability to stimulate T cells are not grossly affected early after MV encounter, since they retain phagocytic activity, reveal a normal phenotypic maturation pattern, and are perfectly able to stimulate allogenic T cell proliferation in vitro (Klagge et al. 2000, 2004; Shishkova et al. 2007), and, given the efficiency of induction of an MV-specific immunity, may reach the secondary lymphatic tissues. The extent to which loss of DC or T cells by fusion occurs needs to be determined. In lymph node material of experimentally infected rhesus macaques, giant cells were very infrequent (McChesney et al. 1997). In contrast, these Warthin-Finkeldey cells were described in hyperplastic lymphoid tissue in a measles patient at autopsy, although evidence for MV there was provided only by immunohistology, but not by electron microscopy (Nozawa et al. 1994). Incomplete T cell activation or active inhibition of T cell expansion probably mark late phases in MV DC interaction. Then these cells may transmit virus to T cells expressing CD150 (and this may also include activated CD25+ regulatory T cells (Browning et al. 2004)). Then, apoptosis, the inability to respond to external maturation signals and, in addition, accumulation of viral proteins on the cell surface, may collectively act in T cell silencing. For this, direct negative signaling of infected DCs to scanning T cells, by expression of MV N or gps, is an attractive hypothesis. Obviously, T cells do not die as a consequence of this inhibitory signal and, at least in vitro, recover and regain their mitogenic response. In agreement with this, recall responses that are suppressed during and after measles, also return to normal in vivo. As regulatory T cells can control persistent infections (reviewed in Mills 2004; Schneider-Schaulies and Dittmer 2006), they also may have a role in the establishment and/or maintenance of the persistent MV CNS infection SSPE, where no deficiencies of effector T cell functions have been found to date. Lastly, MV can trigger postinfectious encephalitis, which is generally believed to be a virusinduced autoimmune disease in which Treg cells might have a role to play. It is certainly that this hypothesis will be experimentally addressed in the near future and that the frequency and potential activation of this particular T cell subset will be evaluated ex vivo. Acknowledgements We thank The Deutsche Forschungsgemeinschaft and the Interdisciplinary Center for Clinical Research Würzburg for financial support of our work.

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Chapter 13

Hostile Communication of Measles Virus with Host Innate Immunity and Dendritic Cells B. Hahm

Contents Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Host Innate Immunity Following Measles Virus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Virus Regulation of Type I IFN-Mediated Innate Immunity . . . . . . . . . . . . . . . . . Type I IFN as an Immunoregulator During Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TLR System Targeted by Measles Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TLR Signaling Pathway Influenced by Measles Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Viral Regulation of TLR-Mediated Cytokine Synthesis . . . . . . . . . . . . . . . . . . . . Interplay Between Measles Virus and Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Virus Suppression of Dendritic Cell Development . . . . . . . . . . . . . . . . . . . . . . . . Measles Virus-Induced Dendritic Cell Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Virus Interaction with Dendritic Cells at Lymphoid Organs . . . . . . . . . . . . . . . . . Modulation of Dendritic Cell Subtypes by Measles Virus . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Following measles virus (MV) infection, host innate immune responses promptly operate to purge the virus. Detection of alerting measles viral components or replication intermediates by pattern-recognizing host machinery of Toll-like receptors and RNA helicases triggers signaling to synthesize array of anti-viral and immunoregulatory molecules, including type I interferon (IFN). Diverse subtypes of dendritic cells (DCs) play pivotal roles in both host innate immunity on the primary MV-infected site and initiating adaptive immune responses on secondary lymphoid tissues. Responding to the predictable host immune responses, MV appears to have devised multiple strategies to evade, suppress, or even utilize host innate immunity and DC responses. This review focuses on versatile actions of MV-induced type I IFNs causing beneficial or deleterious influence on host immunity and the interplay between MV and heterogeneous DCs at distinct locations. B. Hahm Departments of Surgery and Molecular Microbiology and Immunology, Center for Cellular and Molecular Immunology, University of Missouri-Columbia School of Medicine, One Hospital Dr., Columbia, MO 65212, USA, e-mail: [email protected]

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Abbreviations MV IFN DC PDC iDC TLR NK ISGF3 ISRE CTL BM HSC GM-CSF Flt3-L ADAR

Measles virus Interferon Dendritic cell Plasmacytoid dendritic cell Immature dendritic cell Toll-like receptor Natural killer IFN-stimulated gene factor 3 IFN-stimulated response element Cytotoxic T lymphocyte Bone marrow Hematopoietic stem cell Granulocyte macrophage colony-stimulating factor FMS-like tyrosine kinase 3 ligand Adenosine deaminase acting on RNA

Introduction Host immunity has evolved along with continuous pathogenic invasions. During the last decade, great scientific interests have arisen in the field of host innate immunity, especially of type I interferon (IFN) synthesis and toll-like receptor (TLR) signaling, as well as diverse dendritic cell (DC) responses. DCs not only regulate host innate immune response, but also control initiation of host adaptive immunity by stimulating virus-specific naïve T lymphocytes. Upon measles virus (MV) infection, the host immune system is activated to eventually clear the virus. However, MV in return eludes or counterattacks the first line of host defense, the critical innate immune system of the type I IFN network and T cell stimulatory capacity of DCs as well. Consequently, MV could induce profound suppression of host immunity, predisposing MV-infected individuals to other microbial pathogens. Recent studies on MV immunobiology yielded interesting and astonishing results and helped us better understand how host immunity has evolved to combat pathogenic MV and more importantly how immunosuppressive MV contends with protective host innate immune response and DCs.

Host Innate Immunity Following Measles Virus Infection MV is transmitted via aerosol and thus thought to initially infect cells located in respiratory tissues, including epithelial cells and mucosal DCs. On the primary sites infected by MV, multiple host innate immune responses are swiftly operated by

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cellular machineries like macrophages, DCs, and natural killer (NK) cells. Pulmonary macrophages have phagocytotic activity engulfing viruses and apoptotic bodies to destroy them into pieces. Viral components released via cell lysis could act as specific signatures, recognizable by cellular sensors, to induce inflammatory cytokines and chemokines and type I IFN amplification. Type I IFNs secreted from infected cells activate NK cells. Subsequently, NK cells release type II interferon (IFN), IFN-γ to directly kill virus-infected cells. Although severe lymphopenia accompanies MV infection with a significant decrease in the number of T and B lymphocytes, the quantity of NK cells did not seem to decrease (Okada et al. 2001). MV was reported to induce expression of a NKG2D-ligand, UL16-binding proteins 2 on infected monocyte-derived human DCs, resulting in enhanced secretion of IFN-γ from NK cells (Ebihara et al. 2007). However, MV could infect and disturb the activity of DCs to regulate host innate and adaptive immunity.

Measles Virus Regulation of Type I IFN-Mediated Innate Immunity Following MV infection, immunoregulatory proteins are synthesized from innate immune cells to induce antiviral status. The best-known soluble antiviral protein is type I IFN (Vilcek 2006), which is a multi-gene cytokine family consisting of six classes in humans: IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-ν. Among them, IFN-α has diverse subtypes yielding over 12 separate IFN-α subtype proteins. IFN-α and IFN-β have been extensively characterized for their antiviral activities in numerous systems, ever since they were discovered by Isaacs and Lindenmann in 1957 (Isaacs and Lindenmann 1957). Type I IFN is induced through the recognition of viral components or replication intermediates by cellular sensors such as TLRs and the family of DExD/H box RNA helicases of retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA-5) (Fig. 13.1A) (Takeda and Akira 2004; Thompson and Locarnini 2007). Binding of type I IFNs to cognate receptors on the cell surface triggers JAK/STAT signaling pathway leading to a formation of IFN-stimulated gene factor 3 (ISGF3) complex comprised of STAT1, STAT2, and IRF9 (Fig. 13.1B) (Aaronson and Horvath 2002; Darnell et al. 1994). Subsequently, the ISGF3 complex translocates into the nucleus and activates the IFN-stimulated response element (ISRE)-mediated gene transcription, resulting in synthesis of numerous proteins to establish the antiviral state (Grandvaux et al. 2002). MV was reported to inhibit cellular signaling pathways for both type I IFN induction and JAK/STAT signaling (Fig. 13.1A, B). Wild-type MV was shown to induce type I IFN to a far lesser extent than vaccine strains of attenuated MV (Naniche et al. 2000). MV maintained on epithelial cells attained the ability to generate an elevated level of defective interfering RNA, which is an intense stimulator of type I IFN, through the cell culture adaptation, thereby inducing large quantities of type I IFN (Shingai et al. 2007). In addition, MV C protein appeared to inhibit

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Fig. 13.1 A–D MV induces and regulates type I IFN responses. (A) MV infection is detected by cellular sensors of RNA helicases RIG-I/MDA-5 or TLRs to induce type I IFNs via IRF3 or IRF7 activation. (B) Secreted type I IFNs bind to cognate receptors IFNAR1 and 2 complex activating tyrosine kinases JAK1/Tyk2. ISGF3 complex consisting of STAT1, STAT2, and IRF9 recognizes ISRE to activate transcription for ISGs leading to antiviral state. However, MV could inhibit signaling for both type I IFN induction (A) and JAK/STAT-mediated ISG induction (B). (C) Type I IFNs were reported to increase protective host immunity by activating NK cells to produce IFN-γ, and enhancing CTL response and DC terminal maturation. It is rarely investigated if and how MV regulates these IFN-mediated defensive host immunity, although MV-induced type I IFNs were shown to facilitate MV-induced DC maturation. (D) MV-induced type I IFNs could suppress the host immune system by disrupting lymphopoiesis of T lymphocytes in thymus and inhibiting DC development. Blockade of GM-CSF or Flt3-L-mediated DC development occurs via MV-induced type I IFN-mediated unique STAT2-specific signaling

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the production of type I IFNs, as C gene-deficient MV induced a higher level of IFNs (Nakatsu et al. 2006). Nevertheless, regardless of the MV strains, infection of humans by MV should induce synthesis of type I IFN through viral components, including 5'-triphosphate-ended viral leader transcript (Plumet et al. 2007) generated in infected cells (Berghall et al. 2006; Helin et al. 2001). Also, it is likely that viral products released from cells destroyed by MV-induced apoptosis or cellular phagocytosis stimulate type I IFN synthesis from neighboring cells. Further, cellto-cell fusion caused by MV drastically enhanced synthesis of type I IFNs (Herschke et al. 2007). As a second line of strategy to elude type I IFN-mediated host defense, MV instructs its own viral proteins to directly inhibit JAK/STAT signaling of type I IFN. Thus, even in the presence of type I IFN, MV sought a way to multiply and spread. The role of MV V, C, and P proteins in suppressing type I IFN signaling is reported (Caignard et al. 2007; Devaux et al. 2007; Palosaari et al. 2003; Takeuchi et al. 2003). Whether MV regulates antiviral activity of specific type I IFN-induced proteins as a third level of strategy for MV evasion or suppression of the type I IFN system remains elusive. Interestingly, numerous IFN-stimulated genes (ISGs) such as 2'5' oligoadenylate synthetase and Mx protein were significantly upregulated on MVinfected DCs, whereas a well-characterized antiviral gene, double-stranded RNAdependent protein kinase (PKR) was not induced on human DCs by MV infection (Zilliox et al. 2006). Among IFN-stimulated proteins, double-stranded RNA binding protein, Adenosine Deaminase Acting on RNA (ADAR) 1a is thought to display a critical activity during MV persistence (Bass et al. 1989; Cattaneo et al. 1988; Patterson et al. 2001). MV genome isolated from subacute sclerosing panencephalitis (SSPE) patients featured hypermutation of A to G and U to C in the matrix gene. The mutation is postulated to be mediated by ADAR 1a enzymatic activity. Also, ADAR1a was recently reported to bind and inhibit the kinase activity of PKR and consequently enhance host susceptibility to vesicular stomatitis virus (VSV) infection (Nie et al. 2007). The role of IFN-induced ADAR1a and PKR in MV persistence and pathogenesis requires further investigation.

Type I IFN as an Immunoregulator During Measles Blockade of virus spread by type I IFN appears to be mediated by antiproliferative or apoptotic activity concerted by IFN-induced proteins. Apoptosis of virusinfected cells or disruption of cellular proliferation before virus amplification leads to the antiviral state of neighboring cells. However, type I IFN was recently reported to exhibit pro-proliferative or anti-apoptotic actions, demonstrating the complexity of the IFN network (Gimeno et al. 2005; Tanabe et al. 2005; Yang et al. 2001). Furthermore, diverse subtypes of type I IFNs seem to retain distinctly different physiological functions (Cull et al. 2003), representing myriad IFN systems. How the complex IFN system influences host immune responses during measles is poorly understood. As a potent immunomodulator, type I IFN activates NK cells,

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enhances the expansion of cytotoxic T lymphocytes (CTLs), and prolongs the survival of stimulated T cells (Fig. 13.1C) (Biron 2001; Brinkmann et al. 1993). Uniquely, type I IFN regulates DCs in opposite paradoxical directions at two disparate stages of DCs. Type I IFN stimulates terminal maturation of committed DCs (Luft et al. 1998), which is important for stimulation of T cells. Indeed, concomitant treatment of anti-IFN antibody to MV-infected DCs, at least in part, suppressed MV-induced DC maturation consistent with the type I IFN’s capacity to stimulate DC maturation (Dubois et al. 2001). In contrast, MV-induced type I IFN severely suppressed development of DCs from bone marrow (BM) hematopoietic stem cells (HSCs) (Fig. 13.1D) (Hahm et al. 2005). The experiment was conducted by using transgenic mice bearing human SLAM receptor on DCs. MV-induced inhibition of DC generation via type I IFNs occurred in DC culture systems in vitro or ex vivo using either a granulocyte macrophage colony-stimulating factor (GM-CSF) or a fms-like tyrosine kinase 3 ligand (Flt3-L), which are decisive cytokines for DC development. Dual contradictory activities of MV-induced type I IFN on two DC stages were recapitulated using recombinant IFN-β. For instance, simultaneous administration of recombinant IFN-β into mice with Flt3-L blocked Flt3-L-mediated DC expansion in vivo. However, terminal maturation of committed DCs was enhanced by recombinant IFN-β treatment. Intriguingly, suppression of DC development caused by MV-induced type I IFN required the expression of STAT2, but not of STAT1, STAT4, or STAT6. This florid STAT1-independent but STAT2-specific type I IFN signaling resulting in DC inhibition was also reproduced in vitro and in vivo using either recombinant IFN-β or an immunosuppressive strain of lymphocytic choriomeningitis virus (LCMV) Cl 13. Currently, it is unclear why the host immune system is engineered to have type I IFN display contradictory activities on two DC stages. It is conceivable that MV utilizes the immunosuppressive function of type I IFN to inhibit DC development to survive insidiously in the host. Inhibition of DC replenishment following MV infection could enhance susceptibility of the MV-infected individual to secondary microbial invasion. Type I IFN was initially regarded as a magic bullet to purge viruses and cure multiple diseases, and was thought to be devoid of deleterious function. This idealistic concept was challenged by scientific and medical observations: 1. Neutralizing antibodies against type I IFN inhibited liver cell necrosis and growth retardation, reducing the mortality of suckling mice caused by LCMV infection (Riviere et al. 1977). The data indicates that virus-induced IFN could contribute to the exacerbation of disease and death. 2. A significantly elevated level of type I IFN (IFN-α) was detected in the sera of patients with multiple autoimmune diseases such as systemic lupus erythematosus (SLE) and insulin-dependent diabetes mellitus (IDDM) (Theofilopoulos et al. 2005). The therapeutic approach with type I IFN was reported to cause or exacerbate the autoimmune disorders of SLE, thyroiditis, arthritis, and IDDM. 3. Direct injection of experimental mice with type I IFN could cause growth inhibition, glomerulonephritis later in life, and death. Administration of IFN to humans also induces harmful effects such as fever, fatigue, myalgia, and anemia (Vilcek 2006).

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4. Highly virulent influenza viruses such as the reconstructed 1918 pandemic influenza virus induce a cytokine storm, which includes hyperactivation of the type I IFN system (Kash et al. 2006; Kobasa et al. 2007), suggesting possible roles of type I IFN in pathogenic immune response, i.e., viral immunopathology. It is possible that MV-induced type I IFNs exhibit harmful immune responses in certain locations, e.g., intense suppression of DC development at the site of DC commitment. Also, MV caused terminal differentiation and apoptosis of thymic epithelial cells (Auwaerter et al. 1996) via MV-induced type I IFNs, which could lead to disruption of T cell lymphopoiesis in the thymus (Fig. 13.1D) (Vidalain et al. 2002). The data were confirmed by the direct application of IFNs. While the ordinary host immune response is turned off effectively after its purpose is accomplished, perhaps via a host regulatory circuit mechanism, excessive stimulation of the immune system at specific locations might be too strong to be controlled and could consequently turn into a detrimental and pathogenic response. It is also plausible that MV is clever enough to utilize the pathogenic function of host immunity by causing a more hostile environment.

TLR System Targeted by Measles Virus TLR Signaling Pathway Influenced by Measles Virus TLRs belong to pattern-recognizing receptors detecting molecular signatures with specificity (Bowie and Haga 2005; Reis e Sousa 2004). Triggering the TLR system activates molecules for two main pathways of (1) type I IFN induction and (2) synthesis of inflammatory cytokines. While TBK-1/IKK-e-induced IRF-7/IRF-3 activation is crucial for TLR-mediated type I IFN induction (Honda et al. 2005), IRF-5 activation of NF-κB and AP-1 signaling operates the production of inflammatory cytokines, including IL-12, IL-6, and tumor necrosis factor (TNF)-α (Takaoka et al. 2005). It remains unclear how MV induces or regulates TLR-mediated expression of inflammatory cytokines and chemokines. Stimulation of immature DCs with ligands for TLR2 and/or TLR4 significantly increased MV amplification in DCs, which directly correlates with upregulation of SLAM, the MV receptor (Murabayashi et al. 2002). Wild-type MV hemagglutinin (H) protein was reported to activate TLR2, leading to induction of proinflammatory cytokines such as IL-6 and enhancement of surface expression level of SLAM (Fig. 13.2A) (Bieback et al. 2002). The increase in its own receptor expression via TLR2 attachment of MV might be an effective viral immunoregulatory strategy for the spread of the virus (Bieback et al., 2002). Laboratory-adapted MV upregulated TLR3 expression via type I IFN regulation, which could contribute to host innate immunity (Berghall et al. 2006; Tanabe et al. 2003). However, MV displayed inhibitory activity on type I IFN induction signaling mediated by TLR7 or TLR9 on plasmacytoid DCs (PDCs) (Schlender et al. 2005).

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Fig. 13.2 Schematic model for the interplay between MV and diverse DCs. MV infects DCs via cellular receptor SLAM or CD46. Attachment proteins of TLR2 and DC-SIGN are known to interact with MV to influence DC phenotype or function. MV HA binding to TLR2 enhances the level of SLAM receptor on DCs [A]. MV inhibits TLR4-mediated IL-12 synthesis, which could enhance permissiveness of individuals to the infection by secondary microbe Y activating TLR4 [B]. Cross-talk of DCs with other immune cells such as NK cells affects host immunity to MV infection [C]. MV-infected DCs could express inflammatory cytokines including type I IFNs, TNF-α, IL-6, and IL-10 on the infected site. Such cytokines and chemokines may display crucial roles in either protective host immunity or MV-induced immunosuppression [D]. MV interferes with GM-CSF or Flt3-L-dependent DC development from BM HSCs via type I IFN-mediated STAT2-specific signaling. Inhibition of DC amplification and replenishment contributes to the suppression of host immunity [E]. MV infection or attachment to DCs leads to phenotypic maturation of DCs [F], which may cause migration of MV-infected DCs to secondary lymphoid tissues such as mediastinal lymph node [G]. MV-infected DCs could prevent T cells from proliferating via diverse mechanisms, including direct contact of MV glycoproteins to T cells, inhibition of

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Measles Viral Regulation of TLR-Mediated Cytokine Synthesis Among inflammatory cytokines induced by the TLR system, IL-12 is a crucial cytokine for eliciting cellular immunity by skewing cytokine responses toward Th1 phenotype, whereas IL-10 displays immune suppressive activity and one of the core Th2 type cytokines. During measles, Th2 form cytokine response prevailed, contributing to the suppression of cellular immunity (Moss et al. 2002); these phenomena could increase host susceptibility to other microbial infections. Indeed, prolonged detection of upregulated IL-10 was observed in the plasma of measles patients for several weeks following measles-specific rash (Moss et al. 2002). MV was reported to stimulate the Raf-1-acetylation-dependent pathway to enhance IL10 production with or without TLR4 ligation (Gringhuis et al. 2007). The role of IL-10 in measles pathogenesis and mechanism for viral regulation of this immunosuppressive cytokine remains to be determined. In multiple experimental settings, IL-12 inhibition was observed in human monocytes (Karp et al. 1996) and DCs (Servet-Delprat et al. 2000b) and murine DCs (Hahm et al. 2007; Marie et al. 2001) when they were infected with MV followed by stimulation of TLR4 (Fig. 13.2B) or CD40 ligand. In contrast, TLR4-mediated IL-12 synthesis was not inhibited on epithelial cells infected by MV, suggesting the existence of cell type specificity (Indoh et al. 2007). On the virus side, MV nucleoprotein was shown to cause IL-12 inhibition (Marie et al. 2001). Also, the presence of human SLAM or CD46 on murine cells facilitated MV-induced IL-12 inhibition, indicating that HA interaction with CD46 or SLAM facilitates IL-12 interference (Hahm et al. 2007; Marie et al. 2001). However, whether the receptors for MV simply acted as a door for efficient MV entrance or MV HA interaction with the receptor affected specific CD46 or SLAM signaling cascade to influence IL-12 synthesis remains undetermined. It is likely that MV HA binding to the receptors in part mediates IL-12 suppression, as this inhibition does not necessitate direct MV infection of DCs (Hahm et al. 2007; Karp et al. 1996). Notably, SLAM was reported to affect TLR4 signaling and subsequent cytokine secretion, including IL-12 in an animal model of SLAM-deficient mice (Wang et al. 2004), providing evidence for the involvement of SLAM signaling in TLR-mediated cytokine generation. MV P protein upregulated ubiquitin-modifying enzyme A20, which is a host negative feedback regulator of NF-κB in infected monocytic cells to suppress NF-κB-mediated TLR4 signaling (Yokota et al., 2008). However, MV V and C proteins were not responsible for TLR4-mediated IL-12 inhibition, as recombinant MV deficient in V or C gene displayed potent IL-12 inhibitory activity, as did wild-type MV (Hahm et al. 2007).

Fig. 13.2 (Continued) CD40–CD40L signaling, or induction of apoptosis of DCs and T cells by FAS or TRAIL synthesized in DCs [H]. In contrast, certain DCs stimulated by viral components may display ordinary DC activity to stimulate virus-specific T cells leading to eventual clearance of MV. Transfer of antigenic stimuli from migrated DCs to lymph node-resident DCs could be blocked by MV regulation of infected DCs, but not studied yet [I]

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On the other hand, MV appears to inhibit IL-12 specifically upon TLR4 stimulation, as other cytokines of IL-6 and TNF-α were not diminished on monocytes and DCs upon TLR4 stimulation (Hahm et al. 2007; Karp et al. 1996). Additionally, IL-12 inhibition was barely observed when MV-infected murine DCs expressing SLAM were treated with other TLR ligand such as peptidoglycan (PGN, TLR2L), the synthetic analog of double-stranded RNA poly(I:C) (TLR3L), unmethylated CpG DNA (CpG, TLR9L) or guanosine analog loxoribine (TLR7L) (Hahm et al. 2007). The data indicate that MV targets the signaling of TLR4-mediated IL-12 synthesis selectively. MV suppressed production of IL-12 after TLR4 engagement in DCs generated by two different DC culture systems utilizing either Flt3-L or GM-CSF. Moreover, MV-induced IL-12 inhibition via TLR4 activation is dominantly intensive. Indeed, TLR9-mediated IL-12 synthesis in MV-infected DCs was suppressed by simultaneous treatment with TLR4 agonist LPS. Thus, MV could specifically target TLR4 signaling to inhibit IL-12 expression even in the presence of other molecular signatures stimulating diverse types of TLR signaling.

Interplay Between Measles Virus and Dendritic Cells DCs are ideally scattered throughout the entire body as a cellular detector of pathogens invading the host. With multiple subtypes at different locations, DCs have diverse activities, including type I IFN synthesis, cross-talk with various immune cells (Fig. 13.2C), inflammatory response (Fig. 13.2D), antigen presentation to stimulate naïve T cells, and immune tolerance. Investigation on the MV–DC interaction revealed that MV could modulate multiple functions of DCs.

Measles Virus Suppression of Dendritic Cell Development DCs are initially thought to be nondividing end-stage cells (half-life 1.5–2.9 days) (Kamath et al. 2002). However, recent studies revealed that DCs could undergo a limited number of cell divisions over a 10- to 14-day period in peripheral lymphoid organs in situ and replenished from blood-borne precursors (Liu et al. 2007). Synthesized GM-CSF or Flt3-L following virus infection in tissues should increase the number of DCs for effective host immunity. However, MV was shown to suppress GM-CSF or Flt3-L-dependent DC amplification, which is a strategy of immunosuppression employed by MV infection (Fig. 13.2E). As described above, MV-induced type I IFN triggers STAT2-specific signaling to suppress DC development. In addition, MV was reported to inhibit hematopoiesis of HSCs indirectly by infecting BM stromal cells (Manchester et al. 2002), which provides an explanation of the long-term immunosuppression seen in measles patients. MV might disturb regeneration of certain types of immune cells, including DCs, leading to long-term suppression of host immune system.

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Measles Virus-Induced Dendritic Cell Maturation DCs are developed from BM HSCs and exist as an immature form in the periphery. MV-infected immature DCs (iDC) might mature phenotypically, which could facilitate migration of MV-infected DCs to lymphoid tissues. MV-induced phenotypic maturation of iDCs was judged by an increased level of DC marker proteins, including co-stimulatory molecules CD80, CD86, and CD40 (Fig. 13.2F) (Klagge et al. 2000; Schnorr et al. 1997). It is unclear whether MV-induced DC maturation simply represents prompt host immune response or is a strategy designed by the virus for its effective spread. It is conceivable that MV wants to quickly move toward lymphoid tissues (Fig. 13.2G), where major cellular receptor SLAM is widely expressed, before intense host innate immunity occurs on initial target tissues. The idea is supported by reports demonstrating that MV interacts with DCSIGN (de Witte et al. 2006) or TLR2, which activates the cells by enhancing the synthesis of inflammatory cytokines and SLAM receptor (Bieback et al. 2002) (Fig. 13.2A). Efficient transport of HIV bound to DC-SIGN from the infected place to the lymphoid tissues, where viral target CD4+ T cells reside, was well documented (Geijtenbeek et al. 2000; Kwon et al. 2002). It is also possible that matured DCs via the attachment of MV to TLR2 or DC-SIGN express an increased amount of SLAM receptor, as SLAM is highly expressed upon maturation, and thereby become more permissive to MV infection. How does MV infection cause phenotypic DC maturation? MV-induced type I IFNs appear to play a role in enhancing phenotypic maturation of iDCs, as described earlier. However, type I IFN-deficient DCs from mice bearing human SLAM receptor on DCs reached a similar level of maturation to the wild-type DCs expressing SLAM when infected by MV (Hahm et al., unpublished data), suggesting that type I IFN is not an essential component for the phenotypic DC maturation induced by MV.

Measles Virus Interaction with Dendritic Cells at Lymphoid Organs Upon maturation, DCs migrate to secondary lymphoid tissues such as mediastinal lymph node, a major site for naïve T cell stimulation upon pulmonary infection, transmitting the danger signal to T cells and inducing adaptive immunity. We do not clearly understand how MV, following respiratory infection, induces and regulates DC responses in the lung as well as LN in vivo. Another respiratory pathogen of influenza virus was reported to activate DCs in the lung and certain subtype, but not all, of these mature myeloid DCs migrate efficiently to MLN not only to directly induce virusspecific T cells (Fig. 13.2H), but also to stimulate lymph node-resident CD8α+ DCs (Fig. 13.2I) (Belz et al. 2004). A murine model study revealed that when MV-infected BM-derived DCs were adoptively transferred, the virus was found in secondary lymphoid organs, suggesting that DCs could disseminate MV into secondary lymphoid

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tissues (Shingai et al. 2005). Studies with proper animal models for MV infection should further clarify host DC responses to MV infection at distinct locations. MV-infected DCs fail to induce expansion of T cells (Fig. 13.2H) (Dubois et al. 2001; Fugier-Vivier et al. 1997; Grosjean et al. 1997; Hahm et al. 2004; Schnorr et al. 1997). Moreover, DCs infected by MV could actively inhibit uninfected T cell proliferation by cell-to-cell contact or TRAIL-mediated T cell apoptosis. Indeed, replication-incompetent UV-inactivated MV or MV glycoproteins expressed on DCs inhibited proliferation of uninfected T cells upon antigenic or mitogenic stimulation (Dubois et al. 2001; Klagge et al. 2000; Schlender et al. 1996). Co-culture of MV-infected DCs with T cells induced Fas-mediated DC apoptosis with enhanced viral replication (Servet-Delprat et al. 2000a). MV interfered with CD40ligand-dependent differentiation of DCs upon cross-talk with T cells (ServetDelprat et al. 2000b). Consequently, blockade of CD40-to-CD40L interaction could perturb DC-mediated signal transmission to T cells. Mouse genetics studies showed that individual expression of human SLAM MV receptor, type I IFN receptor, TNFα, lymphotoxin (LT)-α or LT-β from T cells was not required for MV-infected DCs to inhibit the proliferation of T cells (Hahm et al. 2004). Additionally, antibodies against IL-10 or type I IFN could not block MV-infected human DC-mediated T cell suppression (Dubois et al. 2001).

Modulation of Dendritic Cell Subtypes by Measles Virus PDCs were identified as a major cell type producing type I IFNs. MV was reported to retain the capability of inhibiting TLR-dependent or -independent type I IFN productions from PDCs (Schlender et al. 2005). Yet, there are multiple questions that remain unanswered regarding the protective role of PDCs in host innate immunity to MV, expression of MV receptors on PDCs, and the mechanism of PDC regulation by MV. On the other hand, several reports showed that type I IFNs are released from MV-infected conventional myeloid DCs including, monocyte-derived DCs, although controversy exists. Accumulated data indicates that CD8α+ draining lymph node-resident lymphoid DCs are the most efficient in priming naïve CD8+ T cells (Allan et al. 2003) upon pulmonary infection of mouse with influenza virus (Belz et al. 2004). Currently, no data are available to address the specific interaction mode of lymphoid DCs in MLNs with MV. Most research on the interplay between MV and human DCs was conducted using myeloid DCs, perhaps due to the availability of DCs derived from human blood in the GM-CSF-dependent culture system and limited sources of human DCs isolated from tissues. Recent development of animal models might help to advance the studies on MV-DC subtype interplay. Interestingly, MV suppressed the upregulation of co-stimulatory molecules and MHC proteins on splenic DCs from mice expressing human SLAM receptor (Hahm et al. 2004), whereas the virus enhanced the phenotypic maturation of DCs derived from the BM of the mice. The data suggest that MV may regulate DC maturation differentially depending on the locations and subtypes of DCs.

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Conclusions Because it is highly contagious and immunosuppressive, MV continues to cause outbreaks and threaten humans. Recent studies demonstrate that MV modulates host immune signaling pathways involving DC activation by targeting specific host molecules. Surprisingly, research on measles virus immunobiology uncovered novel mechanisms for host immunity–virus interaction, which were recurrently observed in a situation of another pathogenic invasion. Identification of both generalized and MV-specific immune signaling mechanisms would advance our understanding of relationship between viruses and the host immune system. Consequently, mechanistic studies to reveal how MV regulates host innate immunity and DC responses should help us develop novel therapeutics for the treatment of diseases caused by immunosuppressive viruses like MV. Acknowledgements I thank the editors for giving me the opportunity to contribute to the CTMI book on measles virus. I greatly appreciate Drs. Sung Key Jang (Pohang University of Science and Technology) and Michael B.A. Oldstone (The Scripps Research Institute) for their excellent support and mentorship. My current measles virus research is supported by the Departments of Surgery and Molecular Microbiology and Immunology and Center for Cellular and Molecular Immunology at University of Missouri-Columbia School of Medicine.

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Index

A Actin cytoskeleton, 255 Activation-induced death, 247 ADAR. See Adenosine deaminase acting on RNA ADAR1a knockout mice, 50 Adenosine deaminase acting on RNA, 40, 42, 43, 49, 50, 275 Adenovirus, 114 Adjuvants, 201 Adverse outcomes, 192 Aerosol, 57, 60, 61 Aerosol administration, 197 Age for measles vaccination, 197 of vaccination, 196 Alpha/beta interferon receptor, 118 Alphavirus, 201 Alphavirus replicon particle vaccines, 203 Altered basolateral targeting signals, 94 Alternate routes of delivery, 197 Alu1 hypermutated M genes, 42 Anergy, 244 Animal model(s), 112, 199 Antibody, 156, 193, 200 modulation, 43 responses, 198 Antibody to F, 195 Antibody to H, 195 Antibody to MV, 42, 43, 46 Antivirals, 90, 91, 99, 100 A to G (U to C) base changes, 49 Attenuation, 123 Atypical measles, 57–58, 67, 156, 194, 195, 200 Autism, 183 Avidity, 198

B B95a cells, 61 Basic reproductive number R, 181 Biased hypermutation, 34, 37–42, 45, 50 Bioterrorism, 185 Bouteille, 33 C Cationic lipids, 201 Cbl-b protein, 259 CD46, 34, 35, 41, 43, 49, 113 CD209 (DC-SIGN), 250 CD 150 or SLAM, 119 CD40 signaling, 254 CD4+ T cells, 118 CD8+ T cells, 118, 194 Cell cycle retardation, 99 Cell targeting, 245 Cellular immune response acute measles, 157 IFN-γ, 157 IL-12, 157 T-helper type 1, 157 T-helper type 2, 157 vaccination, 157 Cellular immune responses, 196, 200 Cellular splice accessory proteins, 259 Challenged, 58, 60 Chemokines, 253 Cholesterol-rich DRMs, 259 CNS complications MIBE, 8, 9 PIE, 7, 8, 13 SSPE, 7–9, 12–14, 20, 22 CNS disease, 34, 37–39, 44, 47 Collapse, 260 Complement fixing (CF) antibodies, 194

289

290 Contact-dependent T cell arrest, 258 Contact-dependent T cell silencing, 258 Cotton rat(s), 89–105, 199 C-type lectin, 250 Cynomolgus macaques, 61–63 Cytokine IL-10, 279 IL-12, 279–280 Cytoplasmic inclusion bodies in neurons, 33 Cytoskeletal rearrangement, 260 D Dahlem Conference on Disease Eradication, 177 DC-SIGN, 281 DC/T cell conjugate, 254 Deficiencies of cellular immunity, 197 Dendritic cell (DC) development, 276, 277, 280 maturation, 276, 281, 282 Plasmacytoid DC (PDC), 277, 282 Dendritic cells, 121, 243, 246, 247, 249, 250 Differentiation of regulatory T cells, 261 Disease elimination, measles, 130 DNA vaccines, 200, 201 Dose, 198 dsRNA adenosine deaminase, 49 DTH model, 97 Dual-Hit Hypothesis, 44–46 Dynein, 13, 15, 17, 19 E Edmonston B virus, 195 Effector structure, 258 EGFP. See Enhanced green fluorescent protein Endemic transmission, measles, 137, 141 Enhanced disease, 57, 58 Enhanced green fluorescent protein (EGFP), 61, 63–67 EPI, 176 Epitopes, 200 Expanded Programme on Immunization, 174 Extracellular virus, 7, 20 F Fcγ receptor, 115 Fcγ RII, 257 Follicular dendritic cells, 250 Formalin-inactivated measles vaccine, 192 F Protein, 193 Francis Payne, 33 Fusion, 5, 16, 17, 20, 21

Index G Genetic polymorphisms, 159 CD46, 158 HLA, 158 IL-2, 158 IL-10, 158 IL-12b, 158 SLAM, 158 Global Immunization Vision and Stratergy (GIVS), 178 Glutamate receptor editing, 40, 49, 50 Goldberger and Anderson, 32, 41 GTPases Rac and Cdc42, 260 H Heat shock protein 72, 95 Hemagglutination inhibiting (HI) antibodies, 194 Herpesvirus, 6, 114 H glycoprotein, 193 High-titer vaccines, 198 HIV, 184, 198 HIV-1, 197 HIV-infected infants, 197 Horta-Barbosa, 33 H protein, 194, 202 Human tonsil slices, 245 Humoral immunity, 155 affinity-maturation, 156 avidity, 156 IgG3, 156 isotype selection, 156 Hydroxymethylglutaryl coenzyme A reductase, 114 I IFN-γ, 118 IFN induction, 253 IL-10, 252 IL-12, 115 Immature DCs, 248 Immune activation, 154 Immune complex, 195 Immune suppression, 90, 96, 97, 99 Immunity, 56, 59, 60, 63, 67 Immunocompromised hosts children, 161 giant-cell pneumonia, 161 HIV, 161 Immunopathological, 67 Immunosuppression, 6, 10, 11, 41, 44, 45, 61, 63, 64, 67, 115, 154, 244, 245 Inactivated MV vaccines, 194

Index Infant immunity maternal antibody, 160 vaccination, 160 vulnerability, 160 Infants, 199 Infectious synapses, 251, 254 Infiltration of B and T cells, CNS, 44 Inflammation, 115 Innate immune response, 154 Innate immunity, 272, 273, 277, 281, 282 Interferon-γ, 47 Iscoms, 202 Isolation, 192 J John Enders, 32 K Kinesin, 13, 17, 18 L Lck promoter, 119 LCMV Cl 13, 44–46 Live-attenuated vaccines, 57, 59 Longevity, 159 Louis Pasteur, 32 Low-avidity antibody, 195 Lymphopenia, 243–247 M Macaques, 6, 12, 200 Maternal antibody, 90, 101–105, 193, 196, 199, 200 Matrix protein M, 119 Mature DCs, 249 Measles, 154 Measles and Rubella Laboratory Network (LabNet), 177 Measles immunity CTL, 159 vaccination, 159 Measles Initiative, 174 Measles rash, 157 Measles-specific antibody hemagglutinin, 155 hemagglutinin-inhibition, 155 IgM, 155 neutralization, 155 plaque-reduction neutralizing, 155 waning, 160 Measles virus, 32–50 genome structure, 131 genotypes, 131–133, 136, 137, 140, 144

291 molecular biology, 132 molecular epidemiology, 129–144 proteins, 131, 136 subacute sclerosing panencephalitis, 132, 136–137 transmission pathways, 131, 134, 135, 143, 144 vaccination programs, 130, 136, 141, 142, 144 Mediastinal lymph node (MLN), 281 M gene, 34, 37–42, 44–46, 49 Mice, 202 Microtubules, 13, 17–19 Mixed leukocyte reactions, 255 MMR (measles, mumps, and rubella), 196 Moesin, 259 Monkeys, 56, 57, 59 Moraten, 195 Morbillization, 192 More attenuated vaccines, 195 Morphological changes associated with DC maturation, 248 Mortality, 192, 193, 198 MV antibody-induced antigen modulation, 43 MV Glycoproteins, 94, 98 MV-infected DCs, 252 MV postinfection encephalitis, 33 MV receptor, 34, 41, 44 MV receptor CD46, 34 MV replication, 6, 11, 12, 14, 18, 20–22 MV spread, 9, 12, 14–17, 19–22 N Neurokinin, 20, 21 Neurokinin 1, 116 Neurons, 33–41, 43–47, 49, 50 Neuron-specific promoter, 115 Neutralization, 156 Neutralizing antibody, 193, 196, 199–201 New-generation vaccines, 57, 59–60 NF-κB, 277, 279 NK cells, 273, 275 Non-human primates, 56, 59, 199 N protein, 257 NSE-CD46, 35 Nucleocapsids, 33, 37, 38, 43, 45 P Pan American Health Organization (PAHO), 176 Pasteur-Chamberland filters, 32 Pathogenesis, 57, 58, 61, 63–65, 67, 112 Pathogen recognition receptors, 248 Pathology, 56, 64

292 Phenotypic maturation, 262 Phosphatidyl-inositol-3-(PI3K)/Akt kinase pathway, 259 PKR. See Protein kinase Plasmacytoid DCs, 249 Pneumonitis, 194 Polio, 182–184 Polioviruses, 181, 185 Polymicrobial disease, 90 Population immunity, 198 Primary neurons, 10, 12, 20 Primates, 195 Progressive infection, 198 Protection, 60, 61, 63, 64 Protective immunity, 192, 198, 199 Protein kinase, 275 R Rash, 194 Receptors, 34, 35, 40, 41, 43, 44, 49, 50 Recombinant viruses, 119 Reverse genetics, 3, 14, 38, 41 Rhesus macaques, 58, 62, 63, 199, 201, 202 Robert Koch, 32 Rodent-adapted MV, 11, 20 S Schwarz, 195 Secondary vaccine failure, 198 Second dose, 199 Seroconversion, 196 SHIP145, 259 SIA, 180, 183 Sigmodon hispidus, 90 Silencing, 243, 247, 255, 257–259, 262 SLAM, 35 as receptor, 195 Smallpox, 181, 186 Smallpox virus, 178 S-phase entry, 259 STAT-1, 121 STAT-2, 121 Stem cells, 246 Subacute sclerosing encephalitis, 116 Subacute sclerosing panencephalitis (SSPE), 31–50 Supplementary immunization activities, 176 Supplementary immunization campaigns, 199 Suppression of primary and secondary humoral and cell-mediated immune responses, 36

Index Surveillance, 177 Synapse instability, 255 T T cell epitopes, 194 T cells, 243, 245–247, 249, 251–262 Thymocytes, 246 TLR (toll-like receptor) TLR2, 277, 281 TLR4, 277, 279, 280 Toll-like receptor 2, 250 Toll-like receptors (TLRs), 121 Transgenic animals, 113 Transgenic mice, 199 Transgenic mouse, 3, 10, 11, 19 Transgenic (tg) mouse model, 34, 46 Transinfection, 251 Two-dose strategy, 198 Type I IFN (interferon) antiviral activity, 275 deleterious function, 276 ISRE, 273 STAT, 273, 275, 276, 280 V Vaccine(s), 91, 95, 96, 99, 100, 105, 192 Vaccinia virus, 202 Viremia, 200 Vitamin A, 176 von Pirquet, 41 V protein, 94 W Window of vulnerability, 192 World Health Assembly, 178 World Health Organization African Region, 130, 139 alternative sampling techniques, 143 Eastern Mediterranean region, 130 Laboratory manual, 130–131 Measles and Rubella Laboratory Network, 130 measles nomenclature, 130–132 recommended protocols, 132 Region of the Americas, 130, 134 Southeast Asian Region, 130 Western Pacific Region, 130 Y YAC-CD46, 34–40, 42, 45–50 Yeast artificial chromosome, 117

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Vol. 294: Caligaris-Cappio, Federico, Dalla Favera, Ricardo (Eds.): Chronic Lymphocytic Leukemia. 2005. 25 figs., VIII, 187 pp. ISBN 3-540-25279-7

Vol. 295: Sullivan, David J.; Krishna Sanjeew (Eds.): Malaria: Drugs, Disease and Post-genomic Biology. 2005. 40 figs., XI, 446 pp. ISBN 3-540-25363-7 Vol. 296: Oldstone, Michael B. A. (Ed.): Molecular Mimicry: Infection Induced Autoimmune Disease. 2005. 28 figs., VIII, 167 pp. ISBN 3-540-25597-4 Vol. 297: Langhorne, Jean (Ed.): Immunology and Immunopathogenesis of Malaria. 2005. 8 figs., XII, 236 pp. ISBN 3-540-25718-7 Vol. 298: Vivier, Eric; Colonna, Marco (Eds.): Immunobiology of Natural Killer Cell Receptors. 2005. 27 figs., VIII, 286 pp. ISBN 3-540-26083-8 Vol. 299: Domingo, Esteban (Ed.): Quasispecies: Concept and Implications. 2006. 44 figs., XII, 401 pp. ISBN 3-540-26395-0 Vol. 300: Wiertz, Emmanuel J.H.J.; Kikkert, Marjolein (Eds.): Dislocation and Degradation of Proteins from the Endoplasmic Reticulum. 2006. 19 figs., VIII, 168 pp. ISBN 3-540-28006-5 Vol. 301: Doerfler, Walter; Böhm, Petra (Eds.): DNA Methylation: Basic Mechanisms. 2006. 24 figs., VIII, 324 pp. ISBN 3-540-29114-8

Vol. 308: Honjo, Tasuku; Melchers, Fritz (Eds.): Gut-Associated Lymphoid Tissues. 2006. 24 figs. XII, 204 pp. ISBN 3-540-30656-0 Vol. 309: Polly Roy (Ed.): Reoviruses: Entry, Assembly and Morphogenesis. 2006. 43 figs. XX, 261 pp. ISBN 3-540-30772-9 Vol. 310: Doerfler, Walter; Böhm, Petra (Eds.): DNA Methylation: Development, Genetic Disease and Cancer. 2006. 25 figs. X, 284 pp. ISBN 3-540-31180-7 Vol. 311: Pulendran, Bali; Ahmed, Rafi (Eds.): From Innate Immunity to Immunological Memory. 2006. 13 figs. X, 177 pp. ISBN 3-540-32635-9 Vol. 312: Boshoff, Chris; Weiss, Robin A. (Eds.): Kaposi Sarcoma Herpesvirus: New Perspectives. 2006. 29 figs. XVI, 330 pp. ISBN 3-540-34343-1 Vol. 313: Pandolfi, Pier P.; Vogt, Peter K. (Eds.): Acute Promyelocytic Leukemia. 2007. 16 figs. VIII, 273 pp. ISBN 3-540-34592-2 Vol. 314: Moody, Branch D. (Ed.): T Cell Activation by CD1 and Lipid Antigens, 2007, 25 figs. VIII, 348 pp. ISBN 978-3-540-69510-3

Vol. 302: Robert N. Eisenman (Ed.): The Myc/Max/Mad Transcription Factor Network. 2006. 28 figs., XII, 278 pp. ISBN 3-540-23968-5

Vol. 315: Childs, James, E.; Mackenzie, John S.; Richt, Jürgen A. (Eds.): Wildlife and Emerging Zoonotic Diseases: The Biology, Circumstances and Consequences of Cross-Species Transmission. 2007. 49 figs. VII, 524 pp. ISBN 978-3-540-70961-9

Vol. 303: Thomas E. Lane (Ed.): Chemokines and Viral Infection. 2006. 14 figs. XII, 154 pp. ISBN 3-540-29207-1

Vol. 316: Pitha, Paula M. (Ed.): Interferon: The 50th Anniversary. 2007. VII, 391 pp. ISBN 978-3-540-71328-9

Vol. 304: Stanley A. Plotkin (Ed.): Mass Vaccination: Global Aspects – Progress and Obstacles. 2006. 40 figs. X, 270 pp. ISBN 3-540-29382-5

Vol. 317: Dessain, Scott K. (Ed.): Human Antibody Therapeutics for Viral Disease. 2007. XI, 202 pp. ISBN 978-3-540-72144-4

Vol. 305: Radbruch, Andreas; Lipsky, Peter E. (Eds.): Current Concepts in Autoimmunity. 2006. 29 figs. IIX, 276 pp. ISBN 3-540-29713-8

Vol. 318: Rodriguez, Moses (Ed.): Advances in Multiple Sclerosis and Experimental Demyelinating Diseases. 2008. XIV, 376 pp. ISBN 978-3-540-73679-9

Vol. 306: William M. Shafer (Ed.): Antimicrobial Peptides and Human Disease. 2006. 12 figs. XII, 262 pp. ISBN 3-540-29915-7

Vol. 319: Manser, Tim (Ed.): Specialization and Complementation of Humoral Immune Responses to Infection. 2008. XII, 174 pp. ISBN 978-3-540-73899-2

Vol. 307: John L. Casey (Ed.): Hepatitis Delta Virus. 2006. 22 figs. XII, 228 pp. ISBN 3-540-29801-0

Vol. 320: Paddison, Patrick J.; Vogt, Peter K. (Eds.): RNA Interference. 2008. VIII, 273 pp. ISBN 978-3-540-75156-4