Viral Determinants of Subtype C HIV-1 Transmission in Zambian Heterosexual Couples Pubblico

Abstract

Heterosexual transmission of subtype C HIV-1 is the predominant route of infection worldwide. A dramatic loss in HIV-1 genetic diversity occurs during HIV-1 transmission by sexual exposure, where approximately 80% of new infections are established by single viral variants. These breakthrough transmitted/founder viruses may have unique properties that confer a higher capacity to transmit, or may in contrast be selected at random from the quasispecies of the HIV infected source partner. Appreciating the balance of random and natural selection pressures operating during transmission, by determining transmitted virus properties, could help inform the rational design of vaccines and enhance our understanding of the molecular details of mucosal transmission. 

Characteristics of genome-length authentic primary viruses from an infected individual's quasispecies and across transmission in acutely infected partners was lacking prior to the investigations undertaken here. We applied novel molecular virological and immunological techniques to study viruses from a cohabiting heterosexual couples cohort in Zambia to investigate the genetic and phenotypic properties of viruses from the plasma of infected donors, along with viruses that transmitted to their epidemiologically linked acutely infected partners.

The results presented here demonstrate transmitting viruses harbor more consensus residues across the entire genome, and are slightly more sensitive to concurrent donor plasma antibody neutralization, indicative of selection pressures acting on virus fitness at the transmission bottleneck. However, we observed that newly infected recipients were permissive to infection with viruses that have a range of phenotypic properties. Transmitted viruses had variable replicative capacities, particle infectivities, interferon resistance profiles, HLA-I downregulation signatures, and susceptibility to NK cells. These data provide the first characterization of authentic genome-length virus clones from transmission pairs, and will serve as useful tools in the study of HIV infection.

Table of Contents

Chapter I: Introduction...................................................................................................... 1

Figures.......................................................................................................................... 32

Fig 1. Model of HIV infection from mucosal exposure............................................................................. 32

Fig 2.  Analysis of sequences from a transmission pair.......................................................................... 34

Fig 3. Viral traits that may be advantageous during transmission................................................. 35

References.................................................................................................................... 37

Chapter II: Heterosexual transmission of subtype C HIV-1 selects consensus-like variants without increased replicative capacity or interferon-α resistance......................................................................................................................... 88

Abstract........................................................................................................................ 89

Introduction................................................................................................................. 91

Results.......................................................................................................................... 94

Discussion.................................................................................................................. 103

Materials and Methods.............................................................................................. 111

Figures........................................................................................................................ 119

Fig. 1. HIV-1 Full-length genome phylogenetic analysis of six epidemiologically-linked heterosexual transmission pairs    120

Fig. 2. Transmission selects for more consensus-like TF variants............................................. 121

Fig. 3. Particle infectivity of TF and NT infectious molecular clones.......................................... 122

Fig. 4. TF are more sensitive to neutralization by donor plasma than NT.............................. 123

Fig. 5.  replication of TF and NT viruses in PBMC.................................................................................... 125

Fig. 6. Interferon-α resistance of TF and NT viruses............................................................................. 126

S1 Fig. Particle infectivity from 293T and PBMC derived virus..................................................... 128

S2 Fig. Particle infectivity correlates with replicative capacity.................................................... 129

S3 Fig. Replication of TF and NT viruses in monocyte derived dendritic cells.................... 130

S4 Fig. TF and NT resistance to IFN-α............................................................................................................. 131

S5 Fig. Replication of TF and 6-month consensus infectious molecular clones.................. 133

References................................................................................................................. 134

Chapter III: HLA class I downregulation by HIV-1 Variants From Subtype C Transmission Pairs                 145

Abstract...................................................................................................................... 146

Introduction............................................................................................................... 148

Results........................................................................................................................ 152

Discussion.................................................................................................................. 159

Materials and Methods.............................................................................................. 165

Figures........................................................................................................................ 173

Fig. 1. Diversity of Nef and Vpu in characterized viruses................................................................... 173

Fig. 2. HLA downregulation of infected cells .............................................................................................. 175

Fig. 3. Relationship of HLA class I molecules on infected cells........................................................ 177

Fig. 4. Quasispecies level HLA class I downregulation and clustering by phenotype...... 179

Fig. 5. Amino acids associated with HLA-A and HLA-B downregulation................................ 180

Fig. 6. NK cell suppression of virus growth ................................................................................................ 181

Fig. 7. Relationship between NK cell suppression and HLA-C downregulation.................. 183

Fig. 8. Replicative capacity and NK cell suppression............................................................................. 184

S1 Fig. Supplemental Figure Nef and Vpu amino acid alignment of viruses phenotyped in this study                185

References................................................................................................................. 186

Chapter IV: Discussion................................................................................................... 207

References................................................................................................................. 221

Appendix........................................................................................................................ 235

Appendix A: Patient Characteristics......................................................................... 237

Fig. 1. Viral load, CD4 counts, and HLA ligand groups for NK KIR binding of transmission pairs        239

Appendix B: Virus Traits........................................................................................... 240

Fig. 2. Plasma isolation of viruses from patient plasma....................................................................... 244

Fig. 3. Rate of viral titer decay over time  for primary viruses from transmission pairs 246

Fig. 4. Donor plasma neutralization of  transmitted/founder and non-transmitted viruses one year after transmission      250

Fig. 5. Induction of replication from resting cells in vitro.................................................................. 254

Fig. 6. CD4 and CD62L expression on infected cells in vitro............................................................... 259

Appendix C: Technical Findings................................................................................ 261

Fig. 7. Infection of CEM cell line with MJ4, Gag-MJ4 chimeras, and full-length primary viruses from four individuals               262

Fig. 8. HIV relevant phenotypic markers on CD4 T cells during culture following stimulation           264

Fig. 9. Reverse transcriptase assay versus TZM-bl titer to measure virus in supernatant 267

Fig. 10. HLA downregulation short in vitro time course.................................................................... 269

Fig. 11. Nef deleted virus does not downregulate CD4 or HLA class I........................................ 271

Fig. 12. Nef reading frame analysis and its impact on HLA-A2 downregulation.................. 273

References................................................................................................................. 274

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Microbiology Biology, Virology Health Sciences, Immunology
Parola chiave	HIV transmission
Committee Chair / Thesis Advisor	Hunter, Eric, Emory University
Committee Members	Derdeyn, Cynthia, Emory University Spearman, Paul, Emory University Paiardini, Mirko, Emory University Lowen, Anice, Emory University Suthar, Mehul, Emory University

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