Role of Polyfunctional and Proliferative CD8+ T cell Responses in HIV-1 Infection

248 Pages·2014·14.54 MB·English

Role of Polyfunctional and Proliferative CD8+ T cell Responses in HIV-1
Infection

by

Meika Elizabeth Ivy Richmond

A Thesis submitted to the Faculty of Graduate Studies of

The University of Manitoba

in partial fulfilment of the requirements of the degree of

Doctor of Philosophy

Department of Medical Microbiology and Infectious Diseases

University of Manitoba

Winnipeg

Copyright © 2013 by Meika Richmond

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Abstract 1
The limited success of HIV vaccine candidates to date highlights our need to better 2
characterize protective cell-mediated immunity. Understanding correlates CD8+ T cell 3
protection against HIV infection and progressive disease is essential for informing 4
effective vaccine development, design and evaluation. CD8+ T cell responses with a 5
robust polyfunctional and proliferative component are strongly linked to better disease 6
outcomes. However, the specificity of polyfunctional and proliferative CD8+ T cell 7
responses has not been thoroughly investigated. Additionally, the specificity of memory 8
subsets and their connection to polyfunctionality and proliferation responses has not been 9
adequately assessed. We address these gaps in knowledge and provide a better 10
understanding of the fine specificity of HIV-specific CD8+ T cell responses. We 11
hypothesize that the epitopes recognized by central memory (T ) and effector memory 12
CM
(T ) CD8+ T cells, defined by functional attributes, differ in chronic HIV-1 infection. 13
EM
Additionally, we hypothesize that polyfunctional and proliferative responses will better 14
correlate with protection in HIV disease progression. 15
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The qualities of CD8+ T cell responses were evaluated using polyfunctional flow 17
cytometry measuring both functional and phenotypic attributes of both T and T 18
EM CM
subsets in HIV infected individuals. We evaluated the quality and evolution of CD8+ T 19
cell responses in HIV infected individuals shortly after seroconversion through to the 20
chronic phase of infection, finding that early polyfunctional responses may result in 21
better HIV disease outcomes. Additionally, we show that epitope-specificity differs 22
between short-term cytokine/chemokine secretion and long-term proliferative assays. 23
Importantly, we show that, at a cohort level, particular epitopes preferentially elicit 24
specific qualities of CD8+ T cell responses in preference to others. 25
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This research improves our understanding of HIV pathogenesis and indicates that we can 27
identify specific epitopes that can elicit protective responses and that early polyfunctional 28
responses may slow HIV disease progression. Understanding the polyfunctional and 29
proliferative capacities of HIV-specific effector and memory cells at various stages of 30
HIV infection is of critical importance to the design of vaccines intended to elicit 31
protective cell-mediated responses. 32
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! I!
Acknowledgments 35
I am honoured to have been part of a great research community and am very thankful my 36
co-supervisor, Dr. Plummer, accepted me into his lab. I am eternally grateful to have 37
added Dr. Ball as co-supervisor when I transitioned to my PhD. His guidance, feedback, 38
encouragement and support, both academically and emotionally, allowed me to find the 39
light at the end of the PhD tunnel. He is an amazing man and I am blessed to have him 40
by my side all these years. 41
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I am also forttunate and thankful to have the mentorship of Lyle. He is an undisputable 43
genius and continually pushed me to expand my knowledge, not only in science, but all 44
aspects of life. His guidance and friendship during my studies, particularly while in 45
Nairobi, was invaluable. I’m equally grateful to my fellow students and colleagues in the 46
Plummer, Ball, Fowke and Luo labs for their friendship and support. I’m particularly 47
thankful for the wonderful lifelong friendships I developed, often under the intense stress, 48
working and living together in Nairobi. Thank you Melissa, Aida, Catherine, Jen, Jill, 49
Caitlin, Sandy and Nadine for the friendship and encouragement. 50
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I would like to thank my committee members, Dr. Fowke, Dr. Uzonna and Dr. Ho for 52
their advice and patience. I would particularly like to thank Keith for his leadership in 53
the collective HIV group and the Department of Medical Microbiology. Thank you Dr. 54
Micheal Grant for surving as my external examiner. 55
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I thank all the staff in the Department for helping me through my studies, Dr. Embree, 57
Jude, Eva, Cheryl, Lynn and Carol. In particular, thanks to Angela for guiding me 58
through the required paperwork and deadlines to keep me in good standing in the Grad 59
Studies program. Additionally, I thank Brenden Dufault for his statistical support. 60
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I acknowledge the amazing work of my colleagues at the University of Nairobi. The lab 62
and clinic staffs show unwavering determination to help the lives of many wonderful 63
Majengo cohort participants. I was honoured to have met many of the great women of 64
the cohort during clinic visits and at yearly Barazas. These meetings were very 65
meaningful to me and I’m grateful for their continued participation in the cohort. 66
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On a more personal note I am tremendously grateful to my wonderful family and friends 68
who have supported me every step of the way. Their encouragement and willingness to 69
listen to my “Science rants” even when they have no idea what I am talking about, is an 70
enormous blessing. My friends Andrea, Evonne and Michaela have been the best friends 71
a girl could ask for and I am entirely indebted to them for their friendship and laughter. 72
Similarly my Mom, Alanna, Dad, John, and Sister, Aidan, are like air to me and without 73
them I wouldn’t float! Thanks to close relatives (Cydney, Chuck, Galen, Marne, Craig, 74
Ross, Lee and Amy) for providing laughter over dinner and keeping the “how’s the PhD” 75
questions to a minimum. Last but not least, thanks to Chicklet for sitting on me or next to 76
me during the entire thesis writing process. Thesis writing is a lot less scary and stressful 77
when you can take cuddle breaks. 78
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! II!
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Dedicated to the memory of my Grandpa,
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Mr. Sydney McMurray
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! III!
Table of Contents
107
Abstract(........................................................................................................................................(I! 108
Acknowledgments(...................................................................................................................(II! 109
List(of(Tables(........................................................................................................................(VIII! 110
List(of(Figures(............................................................................................................................(X! 111
1.(Chapter(1.(Introduction(....................................................................................................(1! 112
1.1. The HIV Pandemic ................................................................................................. 1! 113
1.1.1.!Kenya!........................................................................................................................................................!2! 114
1.1.2.!History!of!the!HIV/AIDS!Pandemic!.............................................................................................!3! 115
1.2. HIV Transmission, Prevention and Treatment ................................................... 4! 116
1.2.1.!HIV!Transmission!................................................................................................................................!4! 117
1.2.2.!HIV!Prevention:!Behavioural!and!Biomedical!interventions!...........................................!5! 118
1.2.3.!HIV!Prevention!and!Treatment:!Antiretroviral!Therapy!...................................................!8! 119
1.2.4.!HIV!Prevention:!HIV!Microbicide,!PrEP!and!Vaccine!development:!..........................!10! 120
1.3. HIV Virology ........................................................................................................ 14! 121
1.3.1.!HIV!Classification!and!Structure!................................................................................................!14! 122
1.3.2.!HIV!Life!Cycle!.....................................................................................................................................!16! 123
1.3.3.!Origin!and!diversity!of!the!HIV/AIDS!Pandemic!................................................................!17! 124
1.3.4.!HIV!Genetic!Diversity!and!Evolution!.......................................................................................!20! 125
1.4. HIV Pathogenesis ................................................................................................. 21! 126
1.4.1.!HIV!Disease!Progression!...............................................................................................................!21! 127
1.4.2.!Models!of!Natural!Protection!......................................................................................................!24! 128
1.5. Basic Immunology ................................................................................................ 25! 129
1.5.1.!Innate!Immune!System!..................................................................................................................!25! 130
1.5.2.!Adaptive!Immune!System!.............................................................................................................!26! 131
1.5.3.!B!cells!and!humoral!immunity!...................................................................................................!27! 132
1.5.4.!T!cells:!...................................................................................................................................................!27! 133
1.5.5.!CD4+!T!cell!immunology!...............................................................................................................!31! 134
1.5.6.!CD8+!T!cell!immunology!...............................................................................................................!32! 135
1.5.7.!T!cell!memory!paradigms!.............................................................................................................!36! 136
1.5.8.!T!cell!receptor!usage!of!CD8+!T!cell!responses!...................................................................!42! 137
1.6. HIV Immunology .................................................................................................. 45! 138
1.6.1.!Innate!response!to!HIV!infection!...............................................................................................!45! 139
1.6.2.!HIVUspecific!T!cells!..........................................................................................................................!47! 140
1.6.3.!Escape!from!CD8+!T!cell!responses!and!CrossUreactivity:!.............................................!48! 141
1.6.4.!Evidence!for!protective!HIVUspecific!CD8+!T!cells!in!HIV!infection:!..........................!50! 142
1.6.5.!Evidence!for!insufficient!CD8+!T!cell!activity!in!HIV!infection:!...................................!56! 143
1.7. Thesis Rationale .................................................................................................... 58! 144
1.7.1.!Global!Hypotheses!...........................................................................................................................!61! 145
2.(Chapter(2.(Materials(and(Methods(.............................................................................(62! 146
2.1. General Reagents .................................................................................................. 62! 147
2.1.1.!Solutions!...............................................................................................................................................!62! 148
2.2. General Methods .................................................................................................. 63! 149
2.2.1.!Study!population!..............................................................................................................................!63! 150
2.2.2.!Samples!.................................................................................................................................................!64! 151
! IV!
2.2.2.1.!Blood!Sample!collection!and!processing!........................................................................................!64! 152
2.2.2.2.!HIV!Testing!and!Confirmation!............................................................................................................!64! 153
2.2.2.3.!CD4+!T!cell!enumeration!.......................................................................................................................!65! 154
2.2.2.4.!Plasma!viral!load!determination!........................................................................................................!65! 155
2.2.2.5.!HLA!Typing!..................................................................................................................................................!65! 156
2.2.3.!Stimulations!........................................................................................................................................!66! 157
2.2.3.1.!HIV!gag!Consensus!sequence!..............................................................................................................!66! 158
2.2.3.2.!Antigen!used!for!stimulation!...............................................................................................................!68! 159
2.2.3.3.!Increasing!detection!of!functional!CD8+!T!Cell!responses!.....................................................!68! 160
2.2.3.4.!Stimulation!Protocol!...............................................................................................................................!71! 161
2.2.4.!Flow!Cytometry!.................................................................................................................................!71! 162
2.2.4.1.!Surface!staining!.........................................................................................................................................!71! 163
2.2.4.2.!Intracellular!cytokine!staining!............................................................................................................!73! 164
2.2.4.3.!CFSE!dilution!proliferation!assay!......................................................................................................!73! 165
2.2.4.4.!Flow!cytometry!Compensations!........................................................................................................!74! 166
2.2.4.5.!Data!acquisition!and!analysis!..............................................................................................................!75! 167
2.2.4.6.!Statistical!analysis!....................................................................................................................................!76! 168
3.(Chapter(3.(Memory(phenotypes(of(HIVFspecific(CD8+(T(cell(responses(are( 169
independent(of(functional(activity(as(defined(by(cytokine(output(and( 170
proliferative(capacity.(.........................................................................................................(77! 171
3.1. Rationale ............................................................................................................... 77! 172
3.2. Hypothesis ............................................................................................................. 79! 173
3.3. Objectives .............................................................................................................. 79! 174
3.4. Methods ................................................................................................................. 79! 175
3.4.1.!Subjects!.................................................................................................................................................!79! 176
3.4.2.!In!vitro!stimulation!..........................................................................................................................!81! 177
3.4.3.!Flow!cytometry!.................................................................................................................................!81! 178
3.4.4.!Statistical!analysis!............................................................................................................................!83! 179
3.5. Results ................................................................................................................... 83! 180
3.5.1.!Phenotype!of!HIVU!and!CEFUspecific!CD8+!T!cell!responses:!........................................!83! 181
3.5.2.!AntigenUspecific!proliferating!CD8+!T!cells!are!of!diverse!memory!phenotypes!.!91! 182
3.5.3.!CD8+!T!cells!expressing!different!functions!have!distinct!memory!phenotypes!.!95! 183
3.5.4.!Polyfunctionality!does!not!directly!associate!with!specific!memory!phenotypes99! 184
3.5.5.!Phenotype!of!HIV!and!CEFUspecific!CD8+!T!cell!responses!stratified!by!disease! 185
status!...............................................................................................................................................................!102! 186
3.6. Summary ............................................................................................................. 104! 187
3.7. Implications ......................................................................................................... 104! 188
4.(Chapter(4.(Epitope(mapping(of(HIVFspecific(CD8+(T(cell(responses(reveals( 189
distinct(specificities(defined(by(function.(...................................................................(106! 190
4.1. Rationale ............................................................................................................. 106! 191
4.2. Hypothesis ........................................................................................................... 107! 192
4.3. Objectives ............................................................................................................ 107! 193
4.4. Methods ............................................................................................................... 108! 194
4.4.1.!Subjects!..............................................................................................................................................!108! 195
4.4.2.!In!vitro!stimulation!.......................................................................................................................!110! 196
4.4.3.!Flow!cytometry!..............................................................................................................................!111! 197
4.4.4.!Statistical!analysis!.........................................................................................................................!113! 198
4.5. Results ................................................................................................................. 113! 199
! V!
4.5.1.!Epitope!mapping!of!p24!using!parallel!measurement!of!five!CD8+!TUcell! 200
functions!........................................................................................................................................................!113! 201
4.5.2.!LTNPs!maintain!a!higher!degree!of!HIVUspecific!CD8+!T!cell!functionality!.........!113! 202
4.5.3.!Different!functional!readouts!define!divergent!specificities!of!CD8+!T!cell! 203
responses!......................................................................................................................................................!121! 204
4.5.4.!The!epitopeUspecificity!of!CD8+!T!cells!responses!differs!based!on!functional! 205
readout!...........................................................................................................................................................!127! 206
4.6. Summary ............................................................................................................. 138! 207
4.7. Implications ......................................................................................................... 139! 208
5.(Chapter(5.(Characterization(of(novel(clade(A(HIV(epitopes:(measuring( 209
polyfunctional(responses(and(proliferative(capacity(in(CD8+(T(cells(..............(140! 210
5.1. Rationale ............................................................................................................. 140! 211
5.2. Hypothesis ........................................................................................................... 141! 212
5.3. Objectives ............................................................................................................ 141! 213
5.4. Methods ............................................................................................................... 142! 214
5.4.1.!Subjects!..............................................................................................................................................!142! 215
5.4.2.!In!vitro!Stimulation:!.....................................................................................................................!142! 216
5.4.3.!Flow!cytometry!..............................................................................................................................!142! 217
5.4.4.!Statistical!analysis!.........................................................................................................................!144! 218
5.5. Results ................................................................................................................. 144! 219
5.5.1.!Selection!of!Epitopes!of!interest!for!characterization!...................................................!144! 220
5.5.2.!Proliferative!capacity!of!CD8!T!cells!in!response!to!eleven!epitopes!of!interest!148! 221
5.5.3.!Epitope!Characterization!measuring!five!CD8+!T!cell!functions!..............................!155! 222
5.5.4.!Epitope!Characterization!measuring!responding!CD8+!T!cell!Polyfunctionality 223
!...........................................................................................................................................................................!158! 224
5.5.5.!Characterization!of!putative!restricting!HLA!....................................................................!166! 225
5.5.6.!Characteristics!of!Peptide!88!...................................................................................................!167! 226
5.6. Summary ............................................................................................................. 168! 227
5.7. Implications ......................................................................................................... 169! 228
6.(Chapter(6.(Evolution(of(polyfunctional(and(proliferative(CD8+(T(cell( 229
responses(from(early(to(chronic(HIVF1(infection(.....................................................(170! 230
6.1. Rationale ............................................................................................................. 170! 231
6.2. Hypothesis ........................................................................................................... 171! 232
6.3. Objective ............................................................................................................. 171! 233
6.4. Methods ............................................................................................................... 171! 234
6.4.1.!Subjects!..............................................................................................................................................!171! 235
6.4.2.!In!vitro!stimulation!.......................................................................................................................!172! 236
6.4.3.!Flow!cytometry!..............................................................................................................................!172! 237
6.4.4.!Statistical!analysis!.........................................................................................................................!175! 238
6.5. Results ................................................................................................................. 175! 239
6.5.1.!CrossUsectional!analysis!of!CD8+!T!cells!responses!during!early,!earlyUchronic! 240
and!chronic!infection!reveal!a!distinct!response!pattern!........................................................!175! 241
6.5.2.!Longitudinal!analysis!of!CD8+!T!cell!responses!over!time!..........................................!180! 242
6.6. Summary ............................................................................................................. 184! 243
6.7. Implications ......................................................................................................... 186! 244
7.(Chapter(7.(Discussion(...................................................................................................(187! 245
7.1. Concluding Remarks and Future Directions ................................................... 208! 246
! VI!
7.1.1.!Major!findings!of!thesis!..............................................................................................................!208! 247
7.1.2.!Hypothetical!Vaccine!design!....................................................................................................!210! 248
7.1.3.!Limitations!and!Future!Directions!.........................................................................................!214! 249
Chapter(8.(References(.......................................................................................................(216! 250
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! VII!
List of Tables
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Chapter 1 254
Table 1.1. Prominent HIV-1 subtypes and their global distribution……………………..19 255
Chapter 3 256
Table 3.1. Clinical and demographic data for subjects in this study…………………….80 257
Table 3.2. Panel of fluorochrome-conjugated antibodies to assess CD8+ T cell memory 258
phenotype and polyfunctionality…………………………………………………………82 259
Table 3.3. Panel of fluorochrome-conjugated antibodies to assess CD8+ T cell memory 260
phenotype and proliferation……………………………………………………………...82 261
Table 3.4. Categories of CD8+ T cells on the basis of surface phenotypic marker 262
expression………………………………………………………………………………..84 263
Table 3.5. The percentage of cells responding with each function correlated with the 264
proportion of responding cells expressing each of the eight phenotypic categories.…….98 265
Table 3.6. The percentage of cells responding with each level of polyfunctionality was 266
correlated with the proportion of responding cells expressing each of the eight phenotypic 267
categories……………………………………………………………………………….101 268
Chapter 4 269
Table 4.1. Study subjects’ demographic data…………………………………………..109 270
Table 4.2. Panel of fluorochome conjugated antibodies to assess CD8+ T cell memory 271
phenotypes and polyfunctionality………………………………………………………112 272
Table 4.3. Panel of fluorochrome conjugated antibodies to assess CD8+ T cell memory 273
phenotype and proliferation…………………………………………………………….112 274
Table 4.4. Correlations between short-term parameters stratified by disease status. 275
Table 4.5. Correlation between short-term parameters and proliferation stratified by 276
disease status……………………………………………………………………………119 277
Table 4.6. Comparison of breadth and magnitude of responses in LTNP and NP……..119 278
Table 4.7. Comparison of breadth of response with CD4 counts and viral loads………120 279
Table 4.8. Comparison of mean response magnitudes with CD4 counts and viral 280
loads….............................................................................................................................120 281
Table 4.9. p24 pool responses, showing disconnect between IFNγ and other short-term 282
parameters………………………………………………………………………………122 283
Table 4.10. 54 identified epitopes and responses that were elicited……………….128-130 284
Table 4.11. Comparison of B consensus sequence with recognized peptides from A1 285
library used in the current study…………………………………………………...135-137 286
Chapter 5 287
Table 5.1. Panel of fluorochrome-conjugated antibodies to assess CD8+ T cell 288
polyfunctionality………………………………………………………………………..143 289
Table 5.2. Panel of fluorochrome-conjugated antibodies to assess CD8+ T cell 290
proliferation……………………………………………………………………………..143 291
Table 5.3. Eleven epitopes of interest and HLA associations…………………………..147 292
Table 5.4. Demographic data, HLA type and peptide responses of study 293
participants…………………………………………………………………………149-151 294
Table 5.5. Specific peptides are associated with dual-functional CD8+ T cell responses 295
but not mono-functional responses……………………………………………………163 296
Table 5.6. Specific peptides are more likely to induce dual-functional CD8+ T cell 297
responses than others…………………………………………………………………...165 298
! VIII!
Table 5.7. Specific peptides are associated with tri-functional CD8+ T cell responses but 299
not 4+ or 5+ responses………………………………………………………………….165 300
Table 5.8. Peptide 88 was more likely to induce Tri-functional CD8+ T cell responses 301
than others………………………………………………………………………………15 302
Chapter 6 303
Table 6.1. Subjects’ disease status and demographic data……………………………...173 304
Table 6.2. Panel of fluorochrome-conjugated antibodies to assess CD8+ T cell 305
polyfunctionality………………………………………………………………………..174 306
Table 6.3. Panel of fluorochrome-conjugated antibodies to assess CD8+ T cell 307
proliferation…………………………………………………………………………….174 308
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! IX!