The Antiviral Role of CD8+ T cells during Latent Immunodeficiency Virus Infections Público
McBrien, Julia (Spring 2020)
Published
Abstract
Human Immunodeficiency Virus (HIV), the infectious agent responsible for Acquired Immunodeficiency Syndrome (AIDS), infects an estimated 37 million people worldwide (UNAIDS, 2018). While antiretroviral therapy (ART) can indefinitely delay the onset of AIDS in HIV-infected individuals and limit the spread of infection, it does not eradicate the virus. The main obstacle in developing a cure for HIV infection is the presence of a population of latently infected cells, termed the viral reservoir, that is established early during infection and persists under ART (Chun et al., 1997b, Finzi et al., 1997, Wong et al., 1997, Siliciano et al., 2003a). A key paradigm in the field of HIV cure, referred to as “shock and kill” (Deeks et al., 2012, Archin et al., 2012), supposes that induction of virus expression (often referred to as “virus reactivation”) in these latently-infected cells (i.e., “shock”) followed by immune-mediated clearing (i.e., “kill”) may substantially reduce the reservoir size and possibly lead to a functional cure for HIV infection. Unfortunately, no latency-reversing agent (LRA) tested to date has successfully perturbed the viral reservoir in human clinical trials (Archin et al., 2014, Spivak et al., 2014, Rasmussen et al., 2014, Søgaard et al., 2015, Elliott et al., 2014, Elliott et al., 2015).
In this dissertation, I seek to address the antiviral role of CD8+ T cells in the maintenance of viral suppression of the latent HIV reservoir and inhibition of latency reversal using the rhesus macaque (RM) model. To this end, we assessed the stability of SIV latency after CD8+ lymphocyte depletion during long-term ART treatment in SIV-infected rhesus macaques. Specifically, we assessed interleukin-15 (IL-15) superagonist complex N-803 as an LRA alone and in combination with CD8 depletion, in addition to CD8 depletion alone. While N-803 alone did not reactivate virus production, its administration after CD8+ lymphocyte depletion induced the most robust and persistent virus reactivation ever observed in humans or macaques. Remarkably, similar results were obtained in a simian-human immunodeficiency virus (SHIV) infection model as well as ART-treated HIV-infected humanized mice, with both the CD8α- and CD8β-targeting depletion antibodies. I also detail the transcriptomic and phenotypic in vivo effects of N-803. This dissertation supports the notion that CD8+ T cells are involved in the maintenance of the HIV/SIV latent viral reservoir and encourages the development of CD8+ T cell-based therapeutic vaccine strategies targeting the latent viral reservoir in the search for a cure for HIV/AIDS.
Table of Contents
Important abbreviations......................................................................................................... 11
Chapter 1: Introduction............................................................................................................. 1
The early HIV epidemic............................................................................................................. 1
The advent of antiretroviral therapy........................................................................................ 2
Use of nonhuman primate models for studying HIV........................................................... 2
HIV transmission and clinical presentation........................................................................... 3
HIV Pathogenesis...................................................................................................................... 4
HIV Lifecycle............................................................................................................................ 6
Cellular targets of HIV/SIV....................................................................................................... 7
Loss of CD4+ T lymphocytes.................................................................................................. 8
Chronic immune activation...................................................................................................... 9
Immune evasion.................................................................................................................... 12
Natural control of HIV infection............................................................................................ 13
CD8+ T cell responses to HIV/SIV.......................................................................................... 18
General CD8+ T cell response to viral infections.................................................................. 18
Acute HIV infection................................................................................................................ 19
Chronic HIV infection............................................................................................................. 21
Expansion of CD8+ T cells and bystander activation........................................................... 21
CD8+ T cell escape............................................................................................................... 23
CD8+ T cell exhaustion......................................................................................................... 24
ART initiation and barriers to CTL-mediated elimination of the viral reservoir...................... 26
Compartmentalization and exclusion..................................................................................... 27
Cytotoxic CD8+ T cell mechanisms...................................................................................... 28
Noncytotoxic mechanisms of CD8+ T cell HIV control......................................................... 30
HIV persistence........................................................................................................................ 36
HIV Cure................................................................................................................................ 38
“Shock and Kill” strategy....................................................................................................... 39
CD8+ cells and viral reservoir maintenance......................................................................... 40
Chapter 1 summary................................................................................................................. 43
Chapter 1 Figures.................................................................................................................... 45
Figure 1: Significant CD8+ T cell events during HIV/SIV infection........................................ 45
Figure 2: Schematic representations of the association between CD8+ T cell frequency and SIV viral load. 46
Figure 3: Mechanisms of CD8+ T cell‐mediated suppression of HIV/SIV replication.............. 47
Figure 4: “Shock and Kill” HIV cure strategy schematic........................................................ 48
Table 1: Summary of noteworthy studies providing evidence of cytolytic and non-cytolytic antiviral activity of CD8+ T cells during HIV/SIV infection during different phases of infection, including treatment and natural control........................ 51
Chapter 2: IL-15 superagonist N-803 administration to SIV-infected long-term ART-treated rhesus macaques induces immunomodulatory effects without SIV reactivation..................................................... 52
Abstract.................................................................................................................................... 53
Importance............................................................................................................................... 53
Introduction............................................................................................................................. 54
Results...................................................................................................................................... 57
N-803 does not induce SIV reactivation in the plasma or cell............................................... 57
N-803 does not decrease the size of the total cell-associated SIV DNA reservoir or delay viral rebound following ART interruption 58
N-803 induces an increase in the frequency of CD8+ T cells in the peripheral blood and lymph node 58
N-803 induces expansion of the effector memory T cell compartments.............................. 59
No clear change to percentages of NK cell subpopulations following N-803 treatment....... 59
N-803 induces phenotypic changes in bulk CD8+ T cell, CD4+ T cell, and NK cell populations in the peripheral blood, lymph node, and rectum............................................................................................................................................... 60
N-803 induces transcriptomic changes in CD4+ T cells, CD8+ T cells, and NK cells......... 60
Discussion............................................................................................................................... 62
Methods.................................................................................................................................... 65
Animals, SIV-infection, drug administration, and sample collection/processing..................... 65
Immunophenotyping by flow cytometry................................................................................ 66
Determination of plasma viral load, cell-associated DNA, and cell-associated RNA............... 66
Flow cytometry cell sorting................................................................................................... 67
RNAseq.............................................................................................................................. 67
Statistical analyses.............................................................................................................. 68
Acknowledgments................................................................................................................. 68
Funding information............................................................................................................... 69
Chapter 2 Figures.................................................................................................................... 70
Figure 1: Treatment of SIV+ ART-treated RM with N-803..................................................... 70
Figure 2: Changes in bulk and subset T cell and NK cell frequencies after N-803 administration 71
Figure 3: Normalized enrichment scores of genes upregulated after N-803 treatment in CD4+ T cells, CD8+ T cells, and NK cells and enrichment plots of specific gene sets................................................................................................... 72
Figure 4: Effect of N-803 on CD4+ T cell signaling pathways............................................... 73
Figure S1: Additional enrichment plots for gene sets upregulated across cell types............. 74
Figure S2: Effect of N-803 on CD4+ T cell signaling pathways (continued).......................... 75
Chapter 3: Robust and persistent SIV and HIV reactivation under ART by N-803 and CD8 depletion 76
Abstract.................................................................................................................................... 77
Results...................................................................................................................................... 78
Acknowledgments.................................................................................................................. 90
Author contributions.............................................................................................................. 91
Methods.................................................................................................................................... 92
Rhesus Macaque Model........................................................................................................ 92
Animals, SIV-infection, antiretroviral therapy, CD8 depletion, and N-803 administration....... 92
CD8 depletion with N-803 in SHIV-infected, ART-treated macaques (pilot study)................. 93
Sample collection and tissue processing............................................................................. 93
Immunophenotype by flow cytometry................................................................................... 93
Determination of plasma SIV RNA, and cell-associated RNA and DNA................................ 94
In Situ RNA analysis and quantification............................................................................... 95
Fluorescence activated sorting (FACS) of live cells.............................................................. 96
RNA-Seq and data analysis................................................................................................ 96
Single genome PCR amplification of SIVmac239 env sequences........................................ 97
Sequencing of env amplicons............................................................................................. 98
Sequence analysis.............................................................................................................. 98
Humanized mouse model...................................................................................................... 99
Experimental design............................................................................................................ 99
Construction of BLT humanized mice.................................................................................. 99
Production of HIV and infection of BLT mice..................................................................... 100
Analysis of HIV infection in BLT mice................................................................................. 100
ART administration............................................................................................................ 102
N-803 and MT807R1 administration.................................................................................. 102
Immunophenotypic analysis of BLT mice........................................................................... 102
CD8 in vitro suppression assay.......................................................................................... 104
Experimental Design......................................................................................................... 104
Flow cytometry.................................................................................................................. 105
Integrated/Total/2-LTR HIV-DNA....................................................................................... 105
Statistical analysis............................................................................................................. 105
Data availability................................................................................................................. 106
Chapter 3 Figures.................................................................................................................. 107
Figure 1............................................................................................................................ 107
Figure 2............................................................................................................................ 108
Figure 3............................................................................................................................ 109
Figure 4............................................................................................................................ 110
Figure 5............................................................................................................................ 111
Supplemental Figure 1...................................................................................................... 112
Supplemental Figure 2...................................................................................................... 113
Supplemental Figure 3...................................................................................................... 114
Supplemental Figure 4...................................................................................................... 115
Supplemental Figure 5...................................................................................................... 116
Supplemental Figure 6...................................................................................................... 117
Supplemental Figure 7...................................................................................................... 118
Supplemental Figure 8...................................................................................................... 119
Supplemental Figure 9...................................................................................................... 120
Supplemental Figure 10.................................................................................................... 121
Supplemental Figure 11.................................................................................................... 122
Supplemental Table 1....................................................................................................... 123
Supplemental Table 2....................................................................................................... 124
Supplemental Table 3....................................................................................................... 125
Chapter 4: Sustained SHIV Reactivation by N-803 in ART-Treated CD8-Depleted Macaques 126
Abstract.................................................................................................................................. 127
Importance............................................................................................................................. 128
Introduction........................................................................................................................... 129
Results.................................................................................................................................... 132
Study design........................................................................................................................ 132
N-803 induces an increase in plasma viremia in CD8-depleted, ART-treated macaques. 132
Intervention with anti-CD8 depleting antibody MT807R1 with N-803 changes the frequency of immune cell subsets in the periphery, lymph node, and rectum................................................................................................................. 133
Immunologic characterization of CD4+ T cells reveals upregulation of PD-1 on peripheral cells and increased proliferation of lymph node and rectal cells............................................................................................................................ 134
N-803 administration in CD8-depleted macaques results in a decrease in the size of the peripheral CD4+ T cell reservoir 135
Discussion............................................................................................................................. 135
Materials and Methods:........................................................................................................ 138
Study approval.................................................................................................................. 138
Animals, SIV-infection, and Antiretroviral therapy............................................................... 138
CD8 depletion and N-803 administration........................................................................... 139
Sample collection and tissue processing........................................................................... 139
Immunophenotype by flow cytometry................................................................................. 139
Determination of plasma SIV RNA, and cell-associated RNA and DNA.............................. 140
Acknowledgements............................................................................................................. 140
Author contributions............................................................................................................. 141
Chapter 4 Figures.................................................................................................................. 142
Figure 1: Study Design...................................................................................................... 142
Figure 2: N-803 induces an increase in plasma viremia when administered in the absence of CD8+ T cells. 143
Figure 3: Peripheral blood cell frequencies pre- and post-intervention............................... 144
Figure 4: Peripheral blood CD4+ T cell phenotypes and subsets pre-ART, before intervention, and one week after 145
Figure 5: Levels of cell-associated SHIV decrease in peripheral CD4+ T cells following treatment with N-803 in CD8-depleted animals 146
Supplemental Figure 1: Plasma viral loads with CD8+ T cell count during antiretroviral therapy. 147
Supplemental Figure 2: Plasma viral loads with the percentage of NK cells in the periphery during antiretroviral therapy 148
Supplemental Figure 3: Efficacy of CD8+ lymphocyte depletion day 7 post-intervention.... 149
Supplemental Figure 4: Lymph node cell subsets pre-ART, before intervention, and one week after 150
Supplemental Figure 5: Rectal biopsy cell subsets pre-ART, before intervention, and one week after 151
Supplemental Figure 6: Lymph node CD4+ T cell phenotypes and subsets pre-ART, before intervention, and one week after. 152
Supplemental Figure 7: Rectal biopsy CD4+ T cell phenotypes and subsets pre-ART, before intervention, and one week after. 153
Supplemental Figure 8: Peripheral blood NK subsets and proliferation pre-ART, before intervention, and one week after. 154
Chapter 5: Combination of CD8β depletion and IL-15 superagonist N-803 induces virus reactivation in SHIV-infected, long-term ART-treated rhesus macaques............................................................................... 155
Abstract.................................................................................................................................. 156
Importance............................................................................................................................. 157
Introduction........................................................................................................................... 158
Results.................................................................................................................................... 160
Study design........................................................................................................................ 160
N-803 administration in CD8β-depleted, ART-treated, SHIV-infected macaques results in modest virus reactivation 161
CD8+ T cell depletion using the anti-CD8β antibody was suboptimal compared to the anti-CD8α antibody 162
Level of virus reactivation is correlated with efficacy of CD8+ T cell depletion and post-depletion viral loads are correlated between sequential depletions............................................................................................................................. 163
Discussion............................................................................................................................. 163
Materials and Methods......................................................................................................... 166
Study approval.................................................................................................................. 166
Acknowledgments............................................................................................................... 169
Author contributions............................................................................................................. 169
Chapter 5 Figures.................................................................................................................. 170
Figure 1............................................................................................................................ 170
Figure 2............................................................................................................................ 171
Figure 3............................................................................................................................ 172
Figure 4............................................................................................................................ 173
Chapter 6: Discussion.......................................................................................................... 174
The HIV epidemic today....................................................................................................... 174
Hope for a cure...................................................................................................................... 175
“Shock and Kill” approach................................................................................................... 179
CD8+ T cells thwart an HIV cure.......................................................................................... 180
Unlock, Shock, and Kill........................................................................................................ 181
Conclusion............................................................................................................................. 184
References:............................................................................................................................ 186
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