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

I TABLE OF CONTENTS CHAPTER 1: INTRODUCTION 1.2 Severe Acute Respiratory Syndrome SARS 1.2.1 Epidemiology of severe acute respiratory 1.2.2.2 Structural and accessory proteins 8 1.3 H5N1

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TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION

1.2 Severe Acute Respiratory Syndrome (SARS)

1.2.1 Epidemiology of severe acute respiratory

1.2.2.2 Structural and accessory proteins 8

1.3 H5N1 Influenza A

1.3.1 Epidemiology of H5N1 influenza A virus 18

1.3.2.1 Structural and non-structural proteins 23

1.4 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

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1.4.3.2 Cellular immune response 41

CHAPTER 2: MATERIALS AND METHODS

2.5 Enzyme-linked immunoabsorbent assay (ELISA) 47

2.6 Isolation and culture of splenocytes 48

2.8 Isolation of PBMC and in vitro expansion of

2.10 Intracellular cytokine staining (ICS) and degranulation

2.11 MHC restriction for CD8+ T cell response 52

2.12 NP44-specific T cell clone activation with endogenous

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2.14 Pseudotyped particle neutralization assay 54

2.16 Production and screening of hybridoma 56

2.17 Fluorescent activated cell sorting (FACS) 56

2.20 Hemagglutination inhibition (HI) assay 58

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

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3.2 Memory T cell responses against SARS structural

nucleocapsid (NP) and accessory 3a protein in

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

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

CHAPTER 5: IMMUNE RESPONSE AGAINST RECOMBINANT H5N1

HEMAGGLUTININ (rHA) PROTEIN 5.1 Neutralizing humoral response against

baculovirus-expressed recombinant HA in Balb/c mouse 102 5.2 T cell response against baculovirus-expressed

5.3 Monoclonal antibody production against

baculovirus-expressed recombinant HA

5.3.1 Characterization of monoclonal antibody mAb

5.3.2 Immunoprecipitation of mature HA protein by

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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

CHAPTER 6: FURTHER CHARACTERIZATION OF MONOCLONAL

ANTIBODY, 9F4 6.1 Epitope mapping of 9F4

6.1.1 Binding to internal deletion and substitution mutants 120 6.2 Mechanism of inhibition

6.2.1 Virus binding and Hemagglutinin inhibition (HI) assay 125 6.2.2 Post-attachment and Proteolysis susceptibility assay 127 6.3 Protection studies of 9F4 in mouse lethal challenge model

6.3.1 Prophylactic and Therapeutic protection 131

CHAPTER 7: CONCLUSION AND FUTURE WORK

7.1 Severe acute respiratory syndrome coronavirus 139

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SUMMARY

With increasing opportunities for animal-to-human and human-to-human 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 SARS-specific 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, 4 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

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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

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LIST OF TABLES

Table 1.1: Summary of the SARS-CoV structural proteins 9 Table 1.2: Summary of the SARS-CoV accessory proteins 12 Table 1.3: Summary of structural and non-structural

Table 3.1: Summary of memory T cell response in all the

Table 3.2: Summary of CD4+ memory T cell response in

Table 3.3: Summary of CD8+ memory T cell response in

Table 4.1: Summary of HLA-restriction of CD8+ T cell

response in SARS-recovered individuals 95

Table 5.1: Microneutralization assay using two live H5N1

influenza A viruses belonging to clade 2.2.2 116

Table 6.1: Hemagglutination inhibition assay using two

live H5N1 influenza A viruses belonging to

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LIST OF FIGURES

Figure 1.1: Summary of the SARS-CoV genome

organization and viral protein expression 7

Figure 1.2: Diagram showing the phylogenetic tree for the

hemagglutinin gene of highly pathogenic avian

Figure 1.3: Hypothetical mechanism for membrane fusion

Figure 3.1: Expression of SARS-CoV 3a protein 65 Figure 3.2: Immune response against 3a protein in Balb/c

Figure 3.3: ELISPOT analysis of healthy and

Figure 3.4: 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+

Figure 3.7: Distribution of CD4+ T cell response within

each individual with multiple CD4+ T cell

Figure 3.8: Distribution of CD8+ T cell response within each

individual with multiple CD8+ T cell responses 77

Figure 3.9: Representative data of the cytokine profile of

CD4+ T cell response stained with Th1/Th2

Figure 3.10: Representative data of the cytokine profile of

CD4+ T cell response stained with inflammatory

Figure 3.11: Representative data of the cytokine profile of

CD8+ T cell response stained with Th1/Th2

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Figure 3.12: Representative data of the cytokine profile of

CD8+ T cell response stained with inflammatory

Figure 3.13: Amino acid sequence alignment of the 3a

protein of three different strains of the SARS

Figure 3.14: Amino acid sequence alignment of the

nucleocaspid (NP) protein of three different

Figure 4.1: T cell activation after incubation with correct

Figure 4.2: Summary of CD8+ T cell response for all

HLA-B*4001+ individuals among the 16 SARS

Figure 4.3: NP44-specific T cell clone activation with

endogenous peptide presented by HLA-B*4001+

Figure 4.4: Expression of SARS-CoV NP protein using

NP-expressing vaccinia virus construct 98 Figure 5.1: Antibody response against

baculovirus-expressed recombinant HA in Balb/c mouse 103 Figure 5.2: Detection of HA and p24 proteins of the

Figure 5.3: Neutralizing antibody response against

baculovrius-expressed recombinant HA in

Figure 5.4: T cell response against baculovirus-expressed

Figure 5.5: MAb 9F4 binds both native and denatured forms

Figure 5.6: MAb 9F4 immunoprecipitates the mature form

Figure 5.7: MAb 9F4 prevents entry of Hatay04-HApp 113

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Figure 5.8: 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

Figure 6.1: A schematic diagram showing the C-terminal

truncation, internal deletion, single and double substitution mutants of Hatay04-HA 122 Figure 6.2: Residues 260 to 263 in HA are important for the

Figure 6.3: Internal deleted mutant HAs can be cleaved by

Figure 6.4: MAb 9F4 does not inhibit cell binding of

Figure 6.5: MAb 9F4 acts by inhibiting the fusion step of

Figure 6.6: MAb 9F4 inhibits the acquisition of protease

sensitivity of the HA protein at pH 5 130

Figure 6.7: Prophylactic protection against Smew06 by

Figure 6.8: Therapeutic protection against Smew06 by MAb

Figure 6.9: Protein sequence alignment for residues 254 to

270 in HA of the H5N1 influenza A viruses 136 Figure 6.10: A ribbon representation of the trimeric VN04

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ABBREVIATIONS

ARDS acute respiratory distress syndrome

ELISA enzyme-linked immunoabsorbent assay

GM-CSF granulocyte macrophage colony-stimulating

factor

HApp lentiviral pseudotyped virus expressing

hemagglutinin

HPAI highly pathogenic avian influenza

IFNAR1 IFN alpha-receptor subunit 1

MAPK mitogen-activated protein kinase

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NHBE normal human bronchial epithelial

PBMC peripheral blood mononuclear cells

RT-PCR reverse transcriptase polymerase chain reaction SARS severe acute respiratory syndrome

SARS-CoV severe acute respiratory syndrome coronavirus SDS-PAGE sodium dodecyl sulfate polyacrylamide gel

electrophoresis

TCID tissue culture infective dose