Adjuvants, Vaccine Platforms, and Protection Against HIV-1 Restricted; Files Only
Yagnik, Kasey (Fall 2025)
Abstract
The costliness, inaccessibility, and difficulty in adherence to antiretroviral therapy contribute to the over 1 million new HIV-1 infections per year, and the field has not yet achieved an adequately protective HIV-1 vaccine. The overall goal of this dissertation is to contribute to the development of an effective vaccine. To this end, we investigated the effects of protein adjuvants, mRNA delivery, and host microbes on the antibody and T helper responses induced by vaccination.
Using the non-human primate model, we compared the immunogenicity and protection elicited by a cyclically permuted (cycP) gp120 protein vaccine adjuvanted with alum or double mutant heat-labile enterotoxin (dmLT). Both vaccines induced HIV-1 envelope (Env)-specific antibodies with broad V1V2-binding activity as well as antigen-specific CD4 T cell responses. However, only animals vaccinated with cycP-gp120 protein adjuvanted with dmLT were significantly protected against heterologous intrarectal SHIV challenges. Proliferating CD4 TCM cells and ICOS+ cells only increased in the dmLT group and were positively associated with protection. There was also a significant decrease in the proportion of α4β7-expressing cells among the proliferating TCM in the dmLT group, suggesting a reduction in gut-homing. V1V2-binding antibody was also positively correlated with protection, but only in the alum group.
In a separate study, we evaluated an mRNA platform expressing ConC cycP-gp120. The mRNA vaccine also resulted in a strong antibody response and increased CD4 T cells with reduced proportion of α4β7-expressing cells. Despite this, vaccinated animals were not significantly protected against heterologous challenge. These findings highlight the importance of an appropriate adjuvant and platform for eliciting protection against HIV-1.
We also sought to understand intra-host factors that determine differential vaccine responses. We devised an in vitro system to test the effects of common probiotics and gut resident microbes on T helper differentiation to an unrelated antigen. We found that Bifidobacteria reduce TH2 phenotypes and Lactobacillus induce TH1 phenotypes, and that Bifidobacteria induce B cell differentiation and preserve CXCR5 expression on B cells.
Collectively, this work illustrates vaccine-dependent differences in immunogenicity and protection against HIV-1, as well as the potential for intra-host factors to influence immune responses.
Table of Contents
CHAPTER 1: INTRODUCTION 1
1.A. Epidemiology of HIV-1 1
1.B. Virology and Pathogenesis of HIV-1 2
1.B.i. Structure and defining characteristics 2
1.B.ii. Virology 2
1.B.iii. Pathogenesis 3
1.C. Protective Immunity to HIV 4
1.C.i. Broadly neutralizing antibodies 4
1.C.ii. Non-neutralizing antibodies 5
1.C.ii. Cytotoxic T cells 8
1.C.iv. TH cells 10
1.D. HIV-1 vaccine design: Historical and Current Strategies 12
1.D.i. Protein subunit 12
1.D.i.a. Adjuvants 13
1.D.i.a.i. Alum 15
1.D.i.a.ii. dmLT 16
1.D.ii. Live attenuated viral vectors 18
1.D.iii. Nucleic acid vectors 22
1.D.iii.a. DNA vaccines 22
1.D.iii.b. mRNA vaccines 22
1.D.iii.b.i. Lipid nanoparticles used with mRNA vaccines 25
1.D.iv. Combination vaccines 26
1.E. The gut microbiome’s effect on vaccines 27
1.E.i. The gut microbiome’s effect on immunity to mucosal vaccines 27
1.E.ii. The potential of the gut microbiome to affect systemic immunity and parenteral vaccines 29
1.E.ii.a. Lessons from effects on unvaccinated systemic immunity 29
1.E.ii.b. Direct evidence in parenteral vaccines 31
1.E.iii. Potential mechanisms for the microbiome’s effect on vaccines 34
1.F. Summary of Introduction 35
CHAPTER 2: IMMUNOGENICITY AND PROTECTION MEDIATED BY DMLT AND ALUM ADJUVANTS FOR AN HIV-1 VACCINE 37
2.A. Abstract 38
2.B. Introduction 39
2.C. Results 41
2.C.i. Vaccination with dmLT but not alum provides significant protection against intrarectal SHIV.CH505 challenges 41
2.C.ii. Both adjuvants elicit a strong anti-Env and anti-V1V2 scaffold binding antibody response in serum 43
2.C.iii. The functional profile of antibody response induced by both adjuvants is comparable 46
2.C.iv. dmLT induces a higher frequency of proliferating CD4 TCM cells with lower gut migration potential, and higher potential for B cell help, which are associated with protection 47
2.C.v. Lower innate activation and higher IL-6 concentration is associated with better protection 51
2.D. Discussion 53
2.E. Materials and Methods 59
2.E.i. Study Design 59
2.E.ii. Ethics statement 60
2.E.iii. Assays to characterize antigen-specific antibody in serum and rectal secretions 61
2.E.iv. Neutralization Assays 63
2.E.v. Antibody Dependent Cell Mediated Virus Inhibition (ADCVI) 63
2.E.vi. Antibody-dependent cellular phagocytosis (ADCP) 64
2.E.vii. Antibody-dependent cell-mediated cytotoxicity (ADCC) 65
2.E.viii. IgG subclass 66
2.E.ix. Flow cytometry-based phenotypic characterization and intracellular cytokine staining (ICS) assay 67
2.E.x. Innate cytokine analysis using Mesoscale Discovery (MSD) 69
2.E.xi. Statistical Analysis 69
2.F. Acknowledgements 70
2.G. Statements 71
2.G.i. Funding 71
2.G.ii. Author contributions 71
2.G.iii. Competing interests 72
2.G.iv. Disclaimer 72
2.H. Main Figures and Tables 73
Figure 1 73
Figure 2 75
Figure 3 77
Figure 4 79
Figure 5 81
Figure 6 83
Table 1 84
Table 2 85
2.I. Supplemental Figures and Tables 86
Figure S1 86
Figure S2 87
Figure S3 88
Figure S4 89
Figure S5 91
Figure S6 93
Figure S7 95
Table S1 96
Table S2 97
Table S3 98
Table S4 99
Table S5 100
CHAPTER 3: IMMUMOGENICITY AND PROTECTION ELICITED BY AN MRNA PLATFORM HIV-1 VACCINE 101
3.A. Abstract 102
3.B. Introduction 103
3.C. Results 105
3.C.i. Vaccination with mRNA expressing ConC CycP-gp120 induces Env and V1V2 binding in serum and rectal secretions 105
3.C.ii. Vaccination with mRNA/ConC CycP-gp120 induces antigen-specific functional activity 106
3.C.iii. Vaccination with mRNA/LNP-CycPgp120 induces an increase in non-target proliferating CD4 T cells and cells with a TH1 phenotype 107
3.C.iv. CD80 and CD86 expression increases on most innate subsets post-mRNA vaccination 110
3.C.v. Animals vaccinated with ConC-CycP-gp120-expressing mRNA were not significantly protected against infection 112
3.D. Discussion 114
3.E. Materials and Methods 117
3.E.i. Study Design 117
3.E.ii. Ethics statement 118
3.E.iii. Development of mRNA/LNP vaccine expressing CycPgp120 118
3.E.iv. Characterization of CycP-gp120 expressed by mRNA 119
3.E.v. Assays to characterize antigen-specific antibody in serum and rectal secretions 119
3.E.vi. Neutralization Assays 121
3.E.vii. Antibody Dependent Cell Mediated Virus Inhibition (ADCVI) 122
3.E.viii. Antibody-dependent cell-mediated cytotoxicity (ADCC) 123
3.E.ix. IgG subclass 124
3.E.x. Flow cytometry-based phenotypic characterization and intracellular cytokine staining (ICS) assay 125
3.E.xi. Innate cytokine analysis using Mesoscale Discovery (MSD) 126
3.E.xii. Statistical Analysis 127
3.F. Acknowledgements 128
3.G. Statements 128
3.G.i. Funding 128
3.G.ii. Author contributions 129
3.G.iii. Competing interests 129
3.G.iv. Disclaimer 129
3.H. Main Figures and Tables 130
Figure 1 130
Figure 2 132
Figure 3 134
Figure 4 136
Figure 5 138
Table 1 140
Table 2 141
Table 3 142
3.I. Supplemental Figure Captions 143
Figure S1 143
Figure S2 144
Figure S3 145
Figure S4 147
Figure S5 148
Table S1 150
Table S2 151
Table S3 152
Table S4 153
Table S5 154
CHAPTER 4: THE MICROBIOME’S INFLUENCE ON NON-MICROBIOME SPECIFIC IMMUNITY 155
4.A. Abstract 156
4.B. Introduction 157
4.C. Results 159
4.C.i. Development of an in vitro assay to investigate microbiota influence on non-specific TH differentiation 159
4.C.ii. Bifidobacteria reduce TH2 cytokines and L. lactis strains induce TH1 cytokines 161
4.C.iii. Changes in IFNγ production occur in earlier divisions, but IL-4 and IL-2 occur in later divisions 163
4.C.iv. B cell proliferation increases with microbe treatment, especially with B. longum and L. reuteri 165
4.C.v. SCFA and B. longum treatment preserves CXCR5 expression on dividing B cells 167
4.D. Discussion 167
4.E. Methods 169
4.E.i. Bacteria details and preparation for stimulations 169
4.E.ii. Mice details and splenocyte preparation 170
4.E.iii. Splenocyte preparation and CellTrace Violet Staining 170
4.E.iv. Stimulations 171
4.E.v. Restimulation and ICS 172
4.E.vi. Statistics 173
4.F. Acknowledgements 173
4.G. Main Figures 174
Figure 1 174
Figure 2 176
Figure 3 178
Figure 4 179
Figure 5 181
Figure 6 182
4.I. Supplemental figures 183
Figure S1 183
Figure S2 184
Figure S3 186
Figure S4 187
Figure S5 188
Table S1 189
Table S2 190
CHAPTER 5: DISCUSSION 191
5.A. HIV-1 vaccine adjuvants and platforms 191
5.A.i Summary of findings 191
5.A.ii. Broader applications and future directions 194
5.B. Commensal microbe and metabolite effects on adaptive responses 200
5.B.i. Summary of findings 200
5.B.ii. Broader applications and future directions 201
5.C. Closing discussion 203
REFERENCES 205
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File download under embargo until 12 January 2028 | 2025-12-15 17:09:58 -0500 | File download under embargo until 12 January 2028 |
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