The transmitted HIV-1 subtype C: characterization of the transmitted/founder full-length virus genome and the influence of early immune selective pressure on virus replication

Includes bibliographical references.The identification of targets of early immune responses associated with control of HIV-1 infection will inform immunogen design for vaccine interventions. The early evolution of transmitted/founder subtype C virus sequences was investigated to determine the location and frequency of immune selection, and the impact of early immune escape mutations on viral replicative capacity. Single-genome amplified env sequences from 26 acutely-infected women were evaluated for conformance to a model of random evolution to elucidate multiplicity of infection. Near fulllength genome sequences from the first six months of infection were generated for five women and sites evolving under immune selection were mapped. CD8+ cytotoxic Tlymphocyte escape mutations in HLA-B-restricted epitopes were introduced into infectious molecular clones of cognate transmitted/founder viruses by site-directed mutagenesis and their impact on viral replicative fitness was evaluated using parallel replication assays. In 77% of women (n=20) a single transmitted/founder variant established infection and two to five variants in the remaining 23% (n=6). Near full-length genome sequencing in five women confirmed single variant/low-diversity transmission and identified fifty-five genome regions evolving under immune selection, 40% of which was attributed to CD8+ cytotoxic Tlymphocyte pressure, 35% to antibody-mediated pressure, 16% to reversion and 9% could not be classified. The rate of sequence diversification and number of sites evolving under immune selection was highest in nef. The majority of evolving CD8+ cytotoxic T-lymphocyte epitopes (82%) contained shuffling/toggling mutations. A novel B*15:10-associated mutation, A164T, combined with a V85A Pol mutation reduced viral replication capacity in one individual. In a second individual, the attenuating HLA-B*58:01-associated mutation, T242N, enhanced viral replication capacity due to pre-existing compensatory polymorphisms in the transmitted/founder virus. A third individual, who had extremely rapid disease progression, was infected with the virus with the highest replication capacity. This thesis describes the complex nature of early immune selection and escape in transmitted/founder viruses. Although attenuating escape mutations were identified in viruses from two individuals, this was not associated with clinical benefit. The extensive variability of epitopes evolving under early selection may implicate many early immune targets as poor candidates for vaccine immunogens; however some early targets may be useful if clinical benefit is conferred through attenuating escape mutations

An investigation into the specific function of the vaccinia virus 13.8 kDa protein encoded by the N1

Includes bibliographical references.Vaccinia virus is the most extensively studied, prototype vertebrate poxvirus, which was used as a vaccine in the eradication of smallpox. The genome of this virus has characteristic variable termini encoding open reading frames that are not essential for virus replication in cell culture. One such open reading frame, N1L situated at the left terminal region of the neurovirulent Western Reserve (WR) vaccinia virus strain, encodes a protein 13.8 kDa in size. In vivo studies in mouse brains revealed that a recombinant virus, vGK5, tacking the expression of the 13.8 kDa protein was rendered replication deficient in the brain. An essential requirement of poxviruses for their replication is the energy molecule adenosine triphosphate (ATP). The supply of this molecule in the brain to support replication of a virus is limited due to the high-energy requirements and small energy reserves of this organ. The specific function of the vaccinia virus 13.8 kDa protein in relation to viral replication in the brain was investigated. The South African (SA) Lister vaccinia virus strain was confirmed to encode an identical N1L gene to that of the WR vaccinia virus by amplification, cloning and sequencing of the Lister N1L open reading frame. The Lister vaccinia virus and a 13.8 kDa deletion strain (vGK5) were cultivated and used to intracranially infect mice. Using a luciferin/luciferase bioluminescence assay system the ATP levels in Lister and vGK5 vaccinia virus-infected mouse brains were measured and found to differ significantly after a 5-day infection period. The SA vaccine Lister vaccinia virus strain was found to be a slow growing virus in the brain. Subsequently, a possible role for the vaccinia virus 13.8 kDa protein in influencing ATP levels in the brain was postulated, yet a neurovirulent wild type strain is needed for further studies to consolidate this result. The 13.8 kDa protein was successfully expressed in the P. pastoris yeast expression system and positively identified by immunodetection studies

False-negative HIV-1 polymerase chain reaction in a 15-month-old boy with HIV-1 subtype C infection

Polymerase chain reaction (PCR) testing is the gold standard for determining the HIV status in children <18 months of age. However, when clinicalmanifestations are not consistent with laboratory results, additional investigation is required. We report a 15-month-old HIV-exposed boy referredto our hospital after he had been admitted several times for infectious diseases. A rapid antibody test on the child was positive, while routinediagnostic HIV PCRs using the Roche COBAS Ampliprep/COBAS TaqMan HIV Qual Test were negative at 6 weeks, 6 months, 7 months and15 months. In addition, the same PCR test performed on the HIV-infected mother was also negative. Alternative PCR and viral load assays usingdifferent primer sets detected HIV RNA or proviral DNA in both child and mother. Gag sequences from the child and his mother classified bothinfections as HIV-1 subtype C, with very rare mutations that may have resulted in PCR assay primer/probe mismatch. Consequently, the child wascommenced on antiretroviral therapy and made a remarkable recovery. These findings indicate that more reliable PCR assays capable of detectinga wide range of HIV subtypes are desirable to circumvent the clinical problems created by false-negative PCR results

Adaptive changes in HIV-1 subtype C proteins during early infection are driven by changes in HLA-associated immune pressure

It is unresolved whether recently transmitted human immunodeficiency viruses (HIV) have genetic features that specifically favour their transmissibility. To identify potential "transmission signatures", we compared 20 full-length HIV-1 subtype C genomes from primary infections, with 66 sampled from ethnically and geographically matched individuals with chronic infections. Controlling for recombination and phylogenetic relatedness, we identified 39 sites at which amino acid frequency spectra differed significantly between groups. These sites were predominantly located within Env, Pol and Gag (14/39, 9/39 and 6/39 respectively) and were significantly clustered (33/39) within known immunoreactive peptides. Within 6 months of infection, we detected reversion-to-consensus mutations at 14 sites and potential CTL escape mutations at seven. Here we provide evidence that frequent reversion mutations probably allows the virus to recover replicative fitness which, together with immune escape driven by the HLA alleles of the new hosts, differentiate sequences from chronic infections from those sampled shortly after transmission

Decreased incidence of dual infections in South African subtype C-infected women compared to a cohort ten years earlier.

Previously, we determined the incidence of dual infections in a South African cohort and its association with higher viral setpoint. Ten years later, we compare the incidence and impact of dual infections at transmission on viral setpoint in a geographically similar cohort (n=46) making use of both the heteroduplex mobility assay (HMA) and the more recent single genome amplification (SGA) approach. HIV incidence was lower in this cohort (7% compared to 18%), and we find a similar reduction in the number of dual infections (9% compared to 19%). Unlike the previous study, there was no association between either dual infection (n=4) or multivariant transmission (n=7) and disease progression. This study emphasized the importance of monitoring changes in the HIV epidemic as it may have important ramifications on our understanding of the natural history of disease

Addressing an HIV cure in LMIC

HIV-1 persists in infected individuals despite years of antiretroviral therapy (ART), due to the formation of a stable and long-lived latent viral reservoir. Early ART can reduce the latent reservoir and is associated with post-treatment control in people living with HIV (PLWH). However, even in post-treatment controllers, ART cessation after a period of time inevitably results in rebound of plasma viraemia, thus lifelong treatment for viral suppression is indicated. Due to the difficulties of sustained life-long treatment in the millions of PLWH worldwide, a cure is undeniably necessary. This requires an in-depth understanding of reservoir formation and dynamics. Differences exist in treatment guidelines and accessibility to treatment as well as social stigma between low- and-middle income countries (LMICs) and high-income countries. In addition, demographic differences exist in PLWH from different geographical regions such as infecting viral subtype and host genetics, which can contribute to differences in the viral reservoir between different populations. Here, we review topics relevant to HIV-1 cure research in LMICs, with a focus on sub-Saharan Africa, the region of the world bearing the greatest burden of HIV-1. We present a summary of ART in LMICs, highlighting challenges that may be experienced in implementing a HIV-1 cure therapeutic. Furthermore, we discuss current research on the HIV-1 latent reservoir in different populations, highlighting research in LMIC and gaps in the research that may facilitate a global cure. Finally, we discuss current experimental cure strategies in the context of their potential application in LMICs

Replication capacity of viruses from acute infection drives HIV-1 disease progression.

CAPRISA, 2017.Abstract available in pdf

CAPRISA 004 tenofovir microbicide trial: no impact of tenofovir gel on the HIV transmission bottleneck.

Alterations of the genital mucosal barrier may influence the number of viruses transmitted from a human immunodeficiency virus–infected source host to the newly infected individual. We used heteroduplex tracking assay and single-genome sequencing to investigate the effect of a tenofovir-based microbicide gel on the transmission bottleneck in women who seroconverted during the CAPRISA 004 microbicide trial. Seventy-seven percent (17 of 22; 95% confidence interval [CI], 56%–90%) of women in the tenofovir gel arm were infected with a single virus compared with 92% (13 of 14; 95% CI, 67%–>99%) in the placebo arm (P = .37). Tenofovir gel had no discernable impact on the transmission bottleneck

Rapid, complex adaption of transmitted HIV-1 full-length genomes in subtype C-infected individuals with differing disease progression.

CAPRISA 2013.Objective(s): There is limited information on full-length genome sequences and the early evolution of transmitted HIV-1 subtype C viruses, which constitute the majority of viruses spread in Africa. The purpose of this study was to characterize the earliest changes across the genome of subtype C viruses following transmission, to better understand early control of viremia. Design: We derived the near full-length genome sequence responsible for clinical infection from five HIV subtype C-infected individuals with different disease progression profiles and tracked adaptation to immune responses in the first 6 months of infection. Methods: Near full-length genomes were generated by single genome amplification and direct sequencing. Sequences were analyzed for amino acid mutations associated with cytotoxic T lymphocyte (CTL) or antibody-mediated immune pressure, and for reversion. Results: Fifty-five sequence changes associated with adaptation to the new host were identified, with 38% attributed to CTL pressure, 35% to antibody pressure, 16% to reversions and the remainder were unclassified. Mutations in CTL epitopes were most frequent in the first 5 weeks of infection, with the frequency declining over time with the decline in viral load. CTL escape predominantly occurred in nef, followed by pol and env. Shuffling/toggling of mutations was identified in 81% of CTL epitopes, with only 7% reaching fixation within the 6-month period. Conclusion: There was rapid virus adaptation following transmission, predominantly driven by CTL pressure, with most changes occurring during high viremia. Rapid escape and complex escape pathways provide further challenges for vaccine protection