Impact of Cytotoxic T Lymphocyte (CTL) escape mutations in acute/Early HIV-1 Subtype C Infection on Disease Progression

Includes abstract. Includes bibliographical references (leaves 139-162).Most HIV vaccines currently in development aim to protect people from infection or disease by eliciting strong anti-HIV cytotoxic T lymphocyte (CTL) responses. Evolved evasion mutations that undermine host immune responses pose a major challenge to the development of such vaccines. Understanding the mechanisms that selectively favour the emergence of CTL evasion mutations in vivo and the impact of these mutations on both disease progression and long-term HIV evolution will not only contribute to our understanding of HIV pathogenesis, but will also inform vaccine design strategies. This study aimed at investigating CTL escape mutations in HIV-1 Gag and Nef, during the acute and early phases of infection and the impact of these mutations on subsequent disease progression in a cohort of recently HIV-1 subtype C infected females. Of 36 women recruited into the study within 12 weeks of infection (median 6 weeks) and followed for six months, 32 were infected with single viruses. Two participants were infected with epidemiologically unlinked viruses (dual infection), and in a further two individuals the viruses were highly divergent suggestive of dual infection and/or recombination. These individuals were excluded from further analysis as it was difficult to predict CTL escape due to high degrees of diversity between sequences. In the remaining 32 study participants, there was a high frequency of CTL escape with putative escape mutations identified in 21 of 32 individuals (66%). Twelve of these 21 (33%) harboured viruses which developed escape mutations in Gag, and 17 (53%) developed escape mutations in Nef. In the conserved structural protein, p24, potential reversion mutations were more frequent than potential escape mutations. During the first six months of infection whereas potential reversion mutations occurred at low entropy sites, potential escape mutations occurred at high entropy sites. Although there was no detectable association between the timing of escape mutations and disease progression, there was an association between the degree of deviation of the p24 sequence from the subtype-C population consensus (a measure of escape mutation load) and CD4+ counts. Analysis of the earliest sampled viruses from HLA-B*57/B*5801 negative study participants for viral genetic markers associated with disease progression identified two iv polymorphisms, A146X (n = 9) and T242N (n =6), that were associated with improved viral control. The polymorphisms are well-known escape mutations in HLAB* 57/B*5801 restricted epitopes. This suggested transmission of these variants from individuals carrying these alleles. Further evidence that viruses carrying the T242N and/or A146X mutations had been previously passaged through B*57/B*5801 positive individuals came from the fact that the observed T242N mutations reverted to wild type during follow-up. There was no significant change in viral load and CD4+ counts upon reversion of the T242N mutations. In vitro replication assays using chimeric viruses containing gag sequences from one of participants showed that the virus harbouring the T242N mutation was fitter than that carrying the reversion mutation. These viruses harboured other T242N associated compensatory mutations suggesting that these compensatory mutations may themselves carry a fitness cost in the absence of the T242N mutation. This suggests that there possibly exist networks of B*57/B*5801 associated mutations and that reversion of some of these mutations in isolation does not necessarily restore viral fitness. Lastly, the kinetics of CTL escape in HLA-B*5801 positive participants (n = 6) and the impact of escape on disease progression was investigated. CTL escape within B *5801 positive individuals was found to predominantly occur within the TW10 in Gag (n = 4) and KAF9 in Nef (n = 6) epitopes. The emergence of the T242N mutation in TW10 was always preceded by mutations elsewhere in the epitope and was associated with the occurrence of previously described compensatory mutation upstream of the epitope. The targeting of TW10 and the emergence of T242N escape mutations were associated with higher CD4+ counts at 12 months postinfection in the B*5801 positive individuals (p = 0.0231 and p = 0.0282, respectively). Independent of host HLA genotypes, the presence of the A146X and T242X mutations was associated with higher CD4+ counts (p = 0.0495). This study provides some useful insights into HIV-1 subtype C pathogenesis. The notion that CTL escape mutations do not invariably result in less fit viruses is evidenced by the observation that escape was not obviously associated with disease progression in this cohort, while escape mutations in the Gag p24 region within B*5801 positive individuals v in particular, was associated with improved viral control. There is therefore evidently a complex interaction between escape and compensatory mutations and further work is required to identify the impact of compensatory mutations on viral fitness. Overall, this study provides further evidence that vaccines need to elicit responses that specifically target the functionally constrained regions of the HIV proteome

Molecular characterization of XvlNO1, a myo-inositol 1-phosphate synthase gene from Xerophyta viscosa

Includes bibliographical references.Myo-inositol I-phosphate synthase (INO 1) catalyses the conversion of glucose-6-phosphate to myo-inositol I-phosphate, which is subsequently dephosphorylated to myo-inositol. Myo-inositol is a precursor for a number of important metabolites that include membrane components, storage molecules, phytohormones and a variety of osmoprotectants. Xerophyta viscosa Baker (Family Velloziaceae) is a monocotyledonous angiosperm which has the ability to resume full physiological function after desiccation. The full-length cDNA for INO1 from X viscosa was isolated using the RACE technique

Exploring the trends in prevalence of human immunodeficiency virus drug resistance in South Africa over the course of the HIV epidemic

Magister Public Health - MPHBackground: Antiretroviral therapy (ART) was rolled out in South Africa in the public sector in 2004 and the treatment coverage has increased over the years to 56% in 2016. The increased treatment coverage has the potential to increase the level of HIV drug resistance. Drug resistance presents a major challenge to the management of HIV infection through antiretroviral therapy at the population level. The aim of this study was to determine the impact of the public sector antiretroviral therapy rollout on the prevalence of HIV drug resistance in South Africa and the factors associated with drug resistance. Methodology: A cross-sectional analytical study was used to determine the prevalence of drug resistance before and after ART rollout. The study population was HIV infected South Africans (infected between 1996 and 2011) who were not on antiretroviral therapy. The study sample was therapy naïve HIV infected South Africans who participated in published studies conducted between 1996 and 2011. HIV DNA sequences and associated data (participants’ age, gender, geographic location and estimated year of HIV infection) were accessed through the Los Alamos HIV Database. The database contains all HIV DNA sequences and associated data from all published studies and the data was freely accessible. A descriptive analysis was carried out on the data to determine characteristics of the study sample. Drug resistance mutations were detected using Calibrated Population Resistance Program on the Stanford University HIV Drug Resistance database. The output from the Calibrated Population Resistance Program analysis were used to determine the prevalence of drug resistance mutations. Results: There were 1701 DNA sequences obtained from the Los Alamos HIV Database for the three gene regions targeted by ART (reverse transcriptase, protease and integrase). Of these, 604 (35,5%) were for reverse transcriptase, 794 (46,7%) were for protease and 303 (17,8%) were for integrase. There was overrepresentation of DNA sequences from female participants (91%). There was no significant difference in the prevalence of drug resistance mutations between 1996-2004 (before ART rollout) and 2005-2011 (after ART rollout) in all the drug classes. There was also no association between drug resistance and age as well as gender. Conclusion: The data from this study suggest that the public sector rollout of ART did not result in an increase in the prevalence of drug resistance mutations in therapy naïve HIVinfected South Africans. There is need for further studies, which have a wider coverage of the South African population

No evidence for selection of HIV-1 with enhanced Gag-Protease or Nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial

BACKGROUND: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. Methods and RESULTS: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. CONCLUSION: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness

How to improve research capacity strengthening efforts: learning from the monitoring and evaluation of four research consortia in Africa

Recent efforts to shift the control and leadership of health research on African issues to Africa have led to increased investments for scientific research capacity strengthening (RCS) on the continent and a greater demand for accountability, value for money and demonstration of return on investment. There is limited literature on monitoring and evaluation (M&E) of RCS systems and there is a clear need to further explore whether the M&E frameworks and approaches that are currently used are fit for purpose. The M&E approaches taken by four African RCS consortia funded under the Developing Excellence in Leadership, Training and Science in Africa (DELTAS) I initiative were assessed using several methods, including a framework comparison of the M&E approaches, semi-structured interviews and facilitated discussion sessions. The findings revealed a wide range in the number of indicators used in the M&E plans of individual consortium, which were uniformly quantitative and at the output and outcome levels. Consortia revealed that additional information could have been captured to better evaluate the success of activities and measure the ripple effects of their efforts. While it is beneficial for RCS consortia to develop and implement their own M&E plans, this could be strengthened by routine engagement with funders/programme managers to further align efforts. It is also important for M&E plans to consider qualitative data capture for assessment of RCS efforts. Efforts could be further enhanced by supporting platforms for cross-consortia sharing, particularly when trying to assess more complex effects. Consortia should make sure that processes for developmental evaluation, and capturing and using the associated learning, are in place. Sharing the learning associated with M&E of RCS efforts is vital to improve future efforts. Investing and improving this aspect of RCS will help ensure tracking of progress and impact of future efforts, and ensure accountability and the return on investment. The findings are also likely applicable well beyond health research

Subtle Longitudinal Alterations in Env Sequence Potentiate Differences in Sensitivity to Broadly Neutralizing Antibodies following Acute HIV-1 Subtype C Infection

Broadly neutralizing antibodies (bNAbs) for HIV-1 prevention or cure strategies must inhibit transmitted/founder and reservoir viruses. Establishing sensitivity of circulating viruses to bNAbs and genetic patterns affecting neutralization variability may guide rational bNAbs selection for clinical development. We analyzed 326 single env genomes from nine individuals followed longitudinally following acute HIV-1 infection, with samples collected at ~1 week after the first detection of plasma viremia; 300 to 1,709 days postinfection but prior to initiating antiretroviral therapy (ART) (median = 724 days); and ~1 year post ART initiation. Sequences were assessed for phylogenetic relatedness, potential N- and O-linked glycosylation, and variable loop lengths (V1 to V5). A total of 43 env amplicons (median = 3 per patient per time point) were cloned into an expression vector and the TZM-bl assay was used to assess the neutralization profiles of 15 bNAbs targeting the CD4 binding site, V1/V2 region, V3 supersite, MPER, gp120/gp41 interface, and fusion peptide. At 1 μg/mL, the neutralization breadths were as follows: VRC07-LS and N6.LS (100%), VRC01 (86%), PGT151 (81%), 10-1074 and PGT121 (80%), and less than 70% for 10E8, 3BNC117, CAP256.VRC26, 4E10, PGDM1400, and N123-VRC34.01. Features associated with low sensitivity to V1/V2 and V3 bNAbs were higher potential glycosylation sites and/or relatively longer V1 and V4 domains, including known "signature" mutations. The study shows significant variability in the breadth and potency of bNAbs against circulating HIV-1 subtype C envelopes. VRC07-LS, N6.LS, VRC01, PGT151, 10-1074, and PGT121 display broad activity against subtype C variants, and major determinants of sensitivity to most bNAbs were within the V1/V4 domains. IMPORTANCE Broadly neutralizing antibodies (bNAbs) have potential clinical utility in HIV-1 prevention and cure strategies. However, bNAbs target diverse epitopes on the HIV-1 envelope and the virus may evolve to evade immune responses. It is therefore important to identify antibodies with broad activity in high prevalence settings, as well as the genetic patterns that may lead to neutralization escape. We investigated 15 bNAbs with diverse biophysical properties that target six epitopes of the HIV-1 Env glycoprotein for their ability to inhibit viruses that initiated infection, viruses circulating in plasma at chronic infection before antiretroviral treatment (ART), or viruses that were archived in the reservoir during ART in subtype C infected individuals in South Africa, a high burden country. We identify the antibodies most likely to be effective for clinical use in this setting and describe mutational patterns associated with neutralization escape from these antibodies

Early evolution of HLA-associated escape mutations in variable Gag proteins predicts CD4+ decline in HIV-1 subtype C infected women.

CAPRISA, 2017.Abstract available in pdf

Identification of effective subdominant anti-HIV-1 CD8+ T cells within entire post-infection and post-vaccination immune responses

Ajuts: R01/R56 NIH Grant AI-52779 (GDT), NIH F31 Fellowship (1F31AI106519-01)(TLP), Center for AIDS Research (P30 AI 64518) i Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, grant number UM1-AI100645-01 (AM)Abstract.Defining the components of an HIV immunogen that could induce effective CD8+ T cell responses is critical to vaccine development. We addressed this question by investigating the viral targets of CD8+ T cells that potently inhibit HIV replication in vitro, as this is highly predictive of virus control in vivo. We observed broad and potent ex vivo CD8+ T cell-mediated viral inhibitory activity against a panel of HIV isolates among viremic controllers (VC, viral loads <5000 copies/ml), in contrast to unselected HIV-infected HIV Vaccine trials Network (HVTN) participants. Viral inhibition of clade-matched HIV isolates was strongly correlated with the frequency of CD8+ T cells targeting vulnerable regions within Gag, Pol, Nef and Vif that had been identified in an independent study of nearly 1000 chronically infected individuals. These vulnerable and so-called "beneficial" regions were of low entropy overall, yet several were not predicted by stringent conservation algorithms. Consistent with this, stronger inhibition of clade-matched than mismatched viruses was observed in the majority of subjects, indicating better targeting of clade-specific than conserved epitopes. The magnitude of CD8+ T cell responses to beneficial regions, together with viral entropy and HLA class I genotype, explained up to 59% of the variation in viral inhibitory activity, with magnitude of the T cell response making the strongest unique contribution. However, beneficial regions were infrequently targeted by CD8+ T cells elicited by vaccines encoding full-length HIV proteins, when the latter were administered to healthy volunteers and HIV-positive ART-treated subjects, suggesting that immunodominance hierarchies undermine effective anti-HIV CD8+ T cell responses. Taken together, our data support HIV immunogen design that is based on systematic selection of empirically defined vulnerable regions within the viral proteome, with exclusion of immunodominant decoy epitopes that are irrelevant for HIV control

Magnitude and Kinetics of CD8+ T Cell Activation during Hyperacute HIV Infection Impact Viral Set Point

CD8[superscript +] T cells contribute to the control of HIV, but it is not clear whether initial immune responses modulate the viral set point. We screened high-risk uninfected women twice a week for plasma HIV RNA and identified 12 hyperacute infections. Onset of viremia elicited a massive HIV-specific CD8[superscript +] T cell response, with limited bystander activation of non-HIV memory CD8[superscript +] T cells. HIV-specific CD8[superscript +] T cells secreted little interferon-γ, underwent rapid apoptosis, and failed to upregulate the interleukin-7 receptor, known to be important for T cell survival. The rapidity to peak CD8[superscript +] T cell activation and the absolute magnitude of activation induced by the exponential rise in viremia were inversely correlated with set point viremia. These data indicate that rapid, high magnitude HIV-induced CD8[superscript +] T cell responses are crucial for subsequent immune control of acute infection, which has important implications for HIV vaccine design.Bill & Melinda Gates FoundationCollaboration for AIDS Vaccine DiscoveryWitten Family FoundationDan and Marjorie SullivanUrsula BrunnerGary and Loren CohenMark and Lisa Schwartz Foundation,International AIDS Vaccine Initiative (UKZNRSA1001)National Institute of Allergy and Infectious Diseases (U.S.) (R37AI067073)Center for AIDS Research (P30 AI060354

How to improve research capacity strengthening efforts: learning from the monitoring and evaluation of four research consortia in Africa

Recent efforts to shift the control and leadership of health research on African issues to Africa have led to increased investments for scientific research capacity strengthening (RCS) on the continent, and a greater demand for accountability, value for money and demonstration of return on investment. There is limited literature on monitoring and evaluation (M&E) of RCS systems and there is a clear need to further explore whether the M&E frameworks and approaches that are currently used are fit for purpose. The M&E approaches taken by four African RCS consortia funded under the Developing Excellence in Leadership, Training and Science in Africa (DELTAS) I initiative were assessed using several methods including: a framework comparison of the M&E approaches; semi-structured interviews; and facilitated discussion sessions. The findings revealed a wide range in the number of indicators used in the M&E plans of individual consortia, which were uniformly quantitative and at the output and outcome level. Consortia revealed that additional information could have been captured to better evaluate the success of activities and measure the ripple effects of the efforts. While it is beneficial for RCS consortia to develop and implement their own M&E plans, this could be strengthened by routine engagement with funders/programme managers to further align efforts. It is also important for M&E plans to consider qualitative data capture for assessment of RCS efforts. Efforts could be further enhanced by supporting platforms for cross-consortia sharing, particularly when trying to assess more complex effects. Consortia should make sure that processes for developmental evaluation, and capturing and using the associated learning, are in place. Sharing the learning associated with M&E of RCS efforts is vital to improve future efforts. Investing and improving this aspect of RCS will help ensure tracking of progress and impact of future efforts, and ensure accountability and the return on investment. The findings are also likely applicable well beyond health research