THE DELAYED EFFECTS OF RESPIRATORY SYNCYTIAL VIRUS INFECTION Thesis submitted for the degree of Doctor of Philosophy in the faculty of Clinical Sciences of the University of London by Diarmuid Rodney O’Donnell Respiratory Medicine St. Mary's Hospital Medical School at Imperial College Norfolk Place London August 1996 Abstract Respiratory Syncytial (RS) virus is the most important respiratory pathogen of infants. The role of RS virus, in the pathogenesis of wheezing and asthma has been a topic of medical interest for many years. Many questions about the pathogenesis of RS virus disease remain unanswered. With new techniques, answers to some of these questions can be attempted. In this thesis, novel techniques were used in studies of RS virus infected infants, children and adults, and mice. Using a nested reverse transcriptase polymerase chain reaction (RT-PCR), it was shown that cell associated viraemia occurs in some infants hospitalised with bronchiolitis but that RS virus cannot be detected in serum or cerebrospinal fluid. RT-PCR was also used to determine, first, the frequency of RS virus in nasopharyngeal aspirates from children admitted with respiratory illnesses, and, in bronchoalveolar lavage fluid from adults infected with human immunodeficiency virus being investigated for unexplained respiratory symptoms. In mice, RT-PCR showed that RS virus persists for at least 100 days after acute infection. Almost all the isolates sequenced from these mice contained an unchanged dominant epitope for cytotoxic lymphocyte (CTL) recognition and all the mice tested had vigorous CTL recognising this epitope. A mutant epitope was also detected, but extensive studies using synthetic peptides did not show interference or inhibition of CTL responses by this mutant. The effect of RS and influenza A virus infections on the immune response to an inhaled protein antigen (ovalbumin) was studied. In the presence of viral infection, acute anaphylaxis and exaggerated TH2 responses to ovalbumin were shown, without the use of adjuvant. This comprises a novel finding of possible clinical significance. By showing viral persistence and altered responses to other antigens, these studies may in part explain the delayed effects of RS virus infection in man. 1 Acknowledgements In the last few years, there have been many people who have given me important practical and theoretical help and teaching. Many times during this work, the results confounded my initial expectations and I must particularly thank my supervisor Peter Openshaw for great patience, support and attention to detail. My thanks also go to all of my friends in the lab: Andy Georgiou for teaching and shared frustration with the flow cytometer, Tracy Hussell for her wide experience of techniques and endless enthusiasm, Lindsay Spender for her shared interest in PCR and help with Southern blotting, Serene Foster for testing viruses and other stocks for Mycoplasma spp., as well as Stephen Rapecki and Tim Sparer. Special thanks must go to Pietro Pala, who arrived in the lab at a sad and difficult time, and who bravely read a large part of this thesis and offered many valuable suggestions. Virus stocks were kindly prepared for me by Tracy Hussell and Andy Georgiou. I thank Mike McGarvey for teaching me about PCR. Antibodies for intracellular cytokine staining were a gift of Anne O'Garra at DNAX. Adrian Hill kindly gave me peptides and Steve Cobbold generously provided T cell depleting antibodies and suggested possible protocols. John Clarke gave me bronchoalveolar lavage samples from adults with HIV and Seb Johnston sent me samples of RNA extracted from nasopharyngeal aspirates. Charlotte Hetzel provided me with an IgE standard for chapter 7. Brian Seymour working, for Robert Coffman at DNAX suggested modifications for the anti-ovalbumin capture ELISA. I would very much like to thank Professor Brigitte Askonas who helped me very much in writing both my application for Wellcome funding and this thesis. My thanks also to Rodney Phillips for useful discussions about peptide inhibition studies. Thanks must go to the Wellcome Tmst for awarding me a Training Fellowship and the British Lung Foundation who supported my first two years. Most of all I thank my wife Jane, who has supported me through the many frustrations, dead ends and sometimes exciting moments that have made up the three and a half years of this PhD. n Table of Contents Abstract................................................................................................................................ i Acknowledgements.............................................................................................................. ii Table of Contents ............................................................................................................ iii List of Figures.....................................................................................................................vii List of Tables ................................................................................................................. viii Abbreviations:................................................................................................................... ix Chapter 1 Introduction..........................................................................................................................1 Features of Respiratory Syncytial Virus:................................................................4 History.......................................................................................................... 4 Epidemiology................................................................................................4 Taxonomy:....................................................................................................6 Molecular Biology.......................................................................................7 Proteins of RS virus.....................................................................................8 Infectious cycle ....................................................................................... 10 In vivo ......................................................................................... 10 In vitro......................................................................................... 11 Clinical features: ..................................................................................... 13 Pathophysiology....................................................................................... 15 The Immune response to RS virus...................................................................... 16 Humoral Immunity................................................................................... 17 Cellular Mediated Immunity .................................................................. 18 Antigen recognition by T cells................................................................ 19 MHC and peptide binding ...................................................................... 19 Avidity versus conformational change................................................... 21 Co-stimulatory molecules...........................................................................22 Mechanisms of viral clearance ..................................................................22 Formalin inactivated vaccine.....................................................................25 T cell subsets: T helper 1 and T helper 2 after vaccination ......................25 The delayed effects of respiratory syncytial virus infection ....................28 Chapter 2 Materials and m ethods.....................................................................................................33 Solutions and Chemicals Used...............................................................................33 Respiratory Syncytial Virus ................................................................................... 33 Microplaque assay for infective virus ..................................................................34 Vaccinia Recombinants..........................................................................................34 Mouse Experiments................................................................................................35 iii Intranasal infection ................................................................................................35 Injection of depleting antibodies i.v........................................................................35 Recovery of tissues from mice...............................................................................36 Exposure to ovalbumin by nebulisation................................................................36 Immunological Methods ....................................................................................... 37 Spleen cell bulk culture .............................................................................37 Preparing Targets ....................................................................................... 38 Bioassay for cytokines IL-2 and IL-4........................................................39 ELISA........................................................................................................ 39 IgE, IgGl or IgG2a to ovalbumin in mouse serum ......................39 total IgE in mouse serum ..............................................................40 Anti-RSV ELISA...........................................................................40 Flow Cytometry..........................................................................................41