Understanding the adaptive immune responses against newly emerged viruses, SARS coronavirus and avian h5n1 influenza a virus 2

TABLE OF CONTENTS SUMMARY VI LIST OF TABLES VIII LIST OF FIGURES IX ABBREVIATIONS XII CHAPTER 1: INTRODUCTION 1.1 Overview of emerging infections 1.2 Severe Acute Respiratory Syndrome (SARS) 1.2.1 Epidemiology of severe acute respiratory syndrome (SARS) Replicase genes 1.2.2.2 Structural and accessory proteins 1.2.2.3 1.3 Genome organization 1.2.2.1 1.2.2 Accessory 3a protein 15 H5N1 Influenza A 1.3.1 18 1.3.2 Genome organization 21 1.3.2.1 Structural and non-structural proteins 23 1.3.2.2 1.4 Epidemiology of H5N1 influenza A virus Hemagglutinin protein 27 Viral infection and the host immune response 1.4.1 Clinical features of SARS and H5N1 influenza 31 1.4.2 Innate immune response against viral infection 34 1.4.3 Adaptive immune response against viral infection 39 I 1.4.3.1 Humoral immune response 39 1.4.3.2 Cellular immune response 41 CHAPTER 2: MATERIALS AND METHODS 2.1 Antibodies and synthetic peptides 44 2.2 Viruses 45 2.3 Mice and immunization 46 2.4 Subjects 46 2.5 Enzyme-linked immunoabsorbent assay (ELISA) 47 2.6 Isolation and culture of splenocytes 48 2.7 Anti-mouse IFNγ ELISPOT assay 48 2.8 Isolation of PBMC and in vitro expansion of SARSspecific T cells 49 2.9 Anti-human IFNγ ELISPOT assay 50 2.10 Intracellular cytokine staining (ICS) and degranulation assays 51 2.11 MHC restriction for CD8+ T cell response 52 2.12 NP44-specific T cell clone activation with endogenous expressed NP protein by BCL 2.13 52 Western blotting 53 II 2.14 Pseudotyped particle neutralization assay 54 2.15 Proliferation assay 55 2.16 Production and screening of hybridoma 56 2.17 Fluorescent activated cell sorting (FACS) 56 2.18 Radiolabeled immunoprecipitation 57 2.19 Micro-neutralization assay 58 2.20 Hemagglutination inhibition (HI) assay 58 2.21 Epitope mapping of mAb 59 2.22 Furin cleavage assay 59 2.23 Virus binding assay 60 2.24 Post-attachment assay 60 2.25 Protease susceptibility assay 61 2.26 Passive immunization and virus challenge 61 CHAPTER 3: MEMORY T CELL RESPONSE IN BALB/C MICE AND SARS-RECOVERED INDIVIDUDALS 3.1 Immune response against SARS accessory 3a protein in Balb/c mouse model 63 III 3.2 Memory T cell responses against SARS structural nucleocapsid (NP) and accessory 3a protein in SARSrecovered patients 67 3.2.1 CD4+ and CD8+ T cell response in SARS individuals 71 3.3 Cytokine profile of CD4+ and CD8+ T cell response 78 3.4 Discussion 84 CHAPTER 4: FURTHER CHARACTERIZATION OF CD8+ T CELL REPONSE 4.1 MHC class I restriction of the CD8+ T cell response 92 4.2 NP44 T cell clone recognizes endogenous presented peptide 96 4.3 Discussion 98 CHAPTER 5: IMMUNE RESPONSE AGAINST RECOMBINANT H5N1 HEMAGGLUTININ (rHA) PROTEIN 5.1 Neutralizing humoral response against baculovirusexpressed recombinant HA in Balb/c mouse 5.2 T cell response against baculovirus-expressed recombinant HA in Balb/c mouse 5.3 102 107 Monoclonal antibody production against baculovirusexpressed recombinant HA 5.3.1 Characterization of monoclonal antibody mAb 9F4 109 5.3.2 Immunoprecipitation of mature HA protein by mAb 9F4 111 IV 5.4 Neutralizing ability of mAb 9F4 5.4.1 Neutralizing ability against homologous strain 113 5.4.2 Cross neutralization ability against other H5N1 strains and other subtypes of influenza A strains 114 5.4.3 Microneutralization assay using live H5N1 strains 5.5 Discussion 116 117 CHAPTER 6: FURTHER CHARACTERIZATION OF MONOCLONAL ANTIBODY, 9F4 6.1 Epitope mapping of 9F4 6.1.1 Binding to internal deletion and substitution mutants 6.2 120 Mechanism of inhibition 6.2.1 Virus binding and Hemagglutinin inhibition (HI) assay 6.2.2 Post-attachment and Proteolysis susceptibility assay 6.3 125 127 Protection studies of 9F4 in mouse lethal challenge model 6.3.1 Prophylactic and Therapeutic protection 6.4 131 Discussion 135 CHAPTER 7: CONCLUSION AND FUTURE WORK 7.1 Severe acute respiratory syndrome coronavirus 139 7.2 H5N1 influenza A virus 142 REFERENCES 146 APPENDICES 178 V SUMMARY With increasing opportunities for animal-to-human and human-tohuman transmission of infectious pathogens, many new strains of viruses have emerged Two recent newly-emerged viruses are the severe acute respiratory syndrome coronavirus (SARS-CoV) and the H5N1 influenza A virus Together with innate immunity, the host adaptive immune responses are crucial for the clearance of the virus during an infection In the first part of this study, the longevity and functionality of SARSspecific memory T cells were examined The SARS accessory 3a protein was first demonstrated to elicit humoral and T cell response in a mouse model The study was extended to using peripheral blood mononuclear cells (PBMCs) from 16 SARS-recovered individuals, years post-infection An unbiased approach, which is independent of HLA types, was utilized to identify putative T cell epitopes in the accessory 3a and the nucleocapsid (NP) protein The IFNγ ELISPOT and intracellular cytokine staining assays showed that approximately 50% of them had positive memory T cell responses against the two proteins tested CD4+ T and CD8+ T cell responses were observed following stimulation with a pool of overlapping peptides spanning the entire NP and 3a proteins Five potential CD8+ T cell epitopes were identified Among them, peptide NP44 was the most frequently recognized peptide and 3a2, which displayed both CD4+ and CD8+ T cell response, was the only peptide identified in the 3a protein Cytokine analysis of these T cells revealed polyfunctional activity Interestingly, all the CD8+ T cell epitopes were restricted by HLA-B subtype These data can be useful in designing VI vaccines against SARS as well as understanding memory T cell responses against novel acute infections In the second part of the study, a neutralizing monoclonal antibody (mAb) 9F4 against the hemagglutinin (HA) protein of the influenza A/chicken/hatay/2004 H5N1 virus (clade 1) was generated and characterized Firstly, the baculovirus-expressed and purified recombinant HA (rHA) was shown to elicit neutralizing humoral and T cell responses in the mouse model This was followed by the production of mAb 9F4 using the rHA MAb 9F4 binds both the denatured and native forms of HA and recognizes the HA proteins of three other heterologous strains of H5N1 viruses By using lentiviral pseudotyped particles carrying HA on the surface, mAb 9F4 was shown to effectively neutralize the homologous and other H5N1 strains belonging to clade 1, 2.1 and 2.2 but not other subtypes of the influenza A virus Epitope mapping analysis revealed that mAb 9F4 binds a previously uncharacterized and well-conserved epitope below the globular head of the HA1 subunit MAb 9F4 does not block the interaction between HA and its receptor but prevents pH-mediated conformation change of HA which is necessary for the fusion step during viral entry It was also found to be protective, both prophylactically and therapeutically, against lethal viral challenge of mice Our data suggest that mAb 9F4 could be a potential candidate for immunotherapy It also provided new information on a novel neutralizing epitope, yielding a new avenue for the design and development of a universal vaccine against H5N1 VII LIST OF TABLES Table 1.1: Summary of the SARS-CoV structural proteins Table 1.2: Summary of the SARS-CoV accessory proteins 12 Table 1.3: Summary of structural and non-structural proteins of the H5N1 virus 23 Table 1.4: Antiviral effects of antibody 39 Table 3.1: Summary of memory T cell response in all the 16 SARS-recovered individuals 75 Summary of CD4+ memory T cell response in SARS-recovered individuals 76 Summary of CD8+ memory T cell response in SARS-recovered individuals 77 Summary of HLA-restriction of CD8+ T cell response in SARS-recovered individuals 95 Microneutralization assay using two live H5N1 influenza A viruses belonging to clade 2.2.2 116 Hemagglutination inhibition assay using two live H5N1 influenza A viruses belonging to clade 2.2.2 127 Table 3.2: Table 3.3: Table 4.1: Table 5.1: Table 6.1: VIII LIST OF FIGURES Figure 1.1: Summary of the SARS-CoV genome organization and viral protein expression Diagram showing the phylogenetic tree for the hemagglutinin gene of highly pathogenic avian influenza A (H5N1) viruses 20 Hypothetical mechanism for membrane fusion by hemagglutinin protein 30 Figure 3.1: Expression of SARS-CoV 3a protein 65 Figure 3.2: Immune response against 3a protein in Balb/c mouse 66 ELISPOT analysis of healthy and SARSrecovered individuals 70 Summary of the positive in vitro ELISPOT results of all 16 SARS-recovered individuals 71 Figure 3.5: Example of an ELISPOT data analysis 72 Figure 3.6: Representative data of the SARS-specific CD4+ and CD8+ T cell response 74 Distribution of CD4+ T cell response within each individual with multiple CD4+ T cell responses 76 Distribution of CD8+ T cell response within each individual with multiple CD8+ T cell responses 77 Representative data of the cytokine profile of CD4+ T cell response stained with Th1/Th2 cytokines 80 Representative data of the cytokine profile of CD4+ T cell response stained with inflammatory cytokines 81 Representative data of the cytokine profile of CD8+ T cell response stained with Th1/Th2 cytokines 82 Figure 1.2: Figure 1.3: Figure 3.3: Figure 3.4: Figure 3.7: Figure 3.8: Figure 3.9: Figure 3.10: Figure 3.11: IX Figure 3.12: Figure 3.13: Figure 3.14: Figure 4.1: Figure 4.2: Figure 4.3: Figure 4.4: Figure 5.1: Figure 5.2: Figure 5.3: Figure 5.4: Figure 5.5: Figure 5.6: Figure 5.7: Representative data of the cytokine profile of CD8+ T cell response stained with inflammatory cytokines 83 Amino acid sequence alignment of the 3a protein of three different strains of the SARS corovnavirus 88 Amino acid sequence alignment of the nucleocaspid (NP) protein of three different strains of the SARS corovnavirus 89 T cell activation after incubation with correct EBV-LCL pulsed with SARS peptide 94 Summary of CD8+ T cell response for all HLAB*4001+ individuals among the 16 SARS individuals tested 95 NP44-specific T cell clone activation with endogenous peptide presented by HLA-B*4001+ EBV-LCL 97 Expression of SARS-CoV NP protein using NPexpressing vaccinia virus construct 98 Antibody response against baculovirusexpressed recombinant HA in Balb/c mouse 103 Detection of HA and p24 proteins of the Hatay04-HApp 105 Neutralizing antibody response baculovrius-expressed recombinant Balb/c mouse 106 against HA in T cell response against baculovirus-expressed recombinant HA in Balb/c mouse 108 MAb 9F4 binds both native and denatured forms of HA 110 MAb 9F4 immunoprecipitates the mature form of HA 112 MAb 9F4 prevents entry of Hatay04-HApp 113 X Figure 5.8: Figure 6.1: Figure 6.2: Figure 6.3: Figure 6.4: Figure 6.5: Figure 6.6: Figure 6.7: Figure 6.8: Figure 6.9: Figure 6.10: MAb 9F4 prevents the entry of HApp of other clades of H5N1 virus, but not that of HApp from other subtypes (H1N1 and H3N2) of influenza virus 115 A schematic diagram showing the C-terminal truncation, internal deletion, single and double substitution mutants of Hatay04-HA 122 Residues 260 to 263 in HA are important for the binding of MAb 9F4 123 Internal deleted mutant HAs can be cleaved by furin 124 MAb 9F4 does not inhibit cell binding of Hatay04-HApp 126 MAb 9F4 acts by inhibiting the fusion step of viral entry 129 MAb 9F4 inhibits the acquisition of protease sensitivity of the HA protein at pH 130 Prophylactic protection against Smew06 by MAb 9F4 133 Therapeutic protection against Smew06 by MAb 9F4 134 Protein sequence alignment for residues 254 to 270 in HA of the H5N1 influenza A viruses 136 A ribbon representation of the trimeric VN04 HA protein 136 XI ABBREVIATIONS ACE2 Ad5 ADP AIDS ARDS BSA CD CFA CTL DAD DC EBV ELISA ELISPOT ER FBS FITC GM-CSF GST HA HApp Hatay04 HI HIV HLA HPAI HRP ICS IFA IFN IFNAR1 IL IND06 Indo05 mAb MAPK MBL MCP MDCK MHC MHV MIP mRNA NA NF-κB angiotensin-converting enzyme adenovirus adenosine diphosphate acquired immunodeficiency syndrome acute respiratory distress syndrome bovine serum albumin cluster of differentiation complete Freund’s adjuvant cytotoxic T lymphocytes diffuse alveolar damage dendritic cell Epstein-Barr virus enzyme-linked immunoabsorbent assay enzyme-linked immunospot endoplasmic reticulum fetal bovine serum fluorescein isothiocyanate granulocyte macrophage colony-stimulating factor glutathione-S-transferase hemagglutinin lentiviral pseudotyped virus expressing hemagglutinin A/chicken/hatay/2004 (H5N1) hemagglutination inhibition human immunodeficiency virus human leukocyte antigen highly pathogenic avian influenza horseradish peroxidase intracellular cytokine staining incomplete Freund’s adjuvant interferon IFN alpha-receptor subunit interleukin A/Chicken/India/NIV33487/06 (H5N1) A/Indonesia/5/2005 (H5N1) monoclonal antibody mitogen-activated protein kinase mannose-binding lectin monocyte chemoattractant protein Madin-Darby canine kidney major histocompatibility complex murine hepatitis coronavirus macrophage inflammatory protein messenger ribonucleic acid neuraminidase nuclear factor kappa B XII NHBE NK Nsp Nt ORF PA PB PBMC PBS PCR PHA PKR RBD rHA RNA RT-PCR SARS SARS-CoV SDS-PAGE SEB SFU Swan06 Smew06 TCID TCR TNF UTR VLP VN04 vRNP WHO normal human bronchial epithelial natural killer non-structural protein nucleotide open reading frame polymerase acidic polymerase basic peripheral blood mononuclear cells phosphate buffered saline polymerase chain reaction phytohemagglutinin protein kinase R receptor binding domain recombinant hemagglutinin ribonucleic acid reverse transcriptase polymerase chain reaction severe acute respiratory syndrome severe acute respiratory syndrome coronavirus sodium dodecyl sulfate polyacrylamide gel electrophoresis staphylococcal entertoxin B spot forming unit A/MuteSwan/Sweden/V827/06 (H5N1) A/Smew/Sweden/V820/06 (H5N1) tissue culture infective dose T cell receptor tumor necrosis factor untranslated region virus-like particle A/Viet Nam/1203/2004 (H5N1) viral ribonucleoproteins World Health Organization XIII ... Epitope mapping of mAb 59 2. 22 Furin cleavage assay 59 2. 23 Virus binding assay 60 2. 24 Post-attachment assay 60 2. 25 Protease susceptibility assay 61 2. 26 Passive immunization and virus challenge... coronavirus (SARS- CoV) and the H5N1 influenza A virus Together with innate immunity, the host adaptive immune responses are crucial for the clearance of the virus during an infection In the first... VI vaccines against SARS as well as understanding memory T cell responses against novel acute infections In the second part of the study, a neutralizing monoclonal antibody (mAb) 9F4 against the