HIV-1 diversity in an antiretroviral treatment naïve cohort from Bushbuckridge, Mpumalanga Province, South Africa.

BACKGROUND: South Africa has a generalized and explosive HIV/AIDS epidemic with the largest number of people infected with HIV-1 in the world. Molecular investigations of HIV-1 diversity can help enhance interventions to contain and combat the HIV/AIDS epidemic. However, many studies of HIV-1 diversity in South Africa tend to be limited to the major metropolitan centers and their surrounding provinces. Hardly any studies of HIV diversity have been undertaken in Mpumalanga Province, and this study sought to investigate the HIV-1 diversity in this province, as well as establish the occurrence and extent of transmitted antiretroviral drug resistance mutations. METHODS: HIV-1 gag p24, pol p10 and p66/p51, pol p31 and env gp41 gene fragments from 43 participants were amplified and sequenced. Quality control on the sequences was carried out using the LANL QC online tool. HIV-1 subtype was preliminary assigned using the REGA 3.0 and jpHMM online tools. Subtype for the pol gene fragment was further designated using the SCUEAL online tool. Phylogenetic analysis was inferred using the Maximum Likelihood methods in MEGA version 6. HIV-1 antiretroviral drug resistance mutations were determined using the Stanford database. RESULTS: Phylogenetic analysis using Maximum Likelihood methods indicated that all sequences in the study clustered with HIV-1 subtype C. The exception was one putative subtype BC unique recombinant form. Antiretroviral drug resistance mutations K103N and E138A were also detected, indicating possible transmission of anti-retroviral drug resistance mutations. CONCLUSIONS: The phylogenetic analysis of the HIV sequences revealed that, by 2009, patients in the Bushbuckridge, Mpumalanga were predominantly infected with HIV-1 subtype C. However, the generalized, explosive nature of the HIV/AIDS epidemic in South Africa, in the context of extensive mobility by South Africans who inhabit rural areas, renders the continued molecular monitoring and surveillance of the epidemic imperative.

Viral dynamics and CD4+ T cell counts in subtype C human immunodeficiency virus type 1-infected individuals from southern Africa.

Defining viral dynamics in natural infection is prognostic of disease progression and could prove to be important for vaccine trial design as viremia may be a likely secondary end point in phase III HIV efficacy trials. There are limited data available on the early course of plasma viral load in subtype C HIV-1 infection in Africa. Plasma viral load and CD4+ T cell counts were monitored in 51 recently infected subjects for 9 months. Individuals were recruited from four southern African countries: Zambia, Malawi, Zimbabwe, and South Africa and the median estimated time from seroconversion was 8.9 months (interquartile range, 5.7-14 months). All were infected with subtype C HIV-1 and median viral loads, measured using branched DNA, ranged from 3.82-4.02 log10 RNA copies/ml from 2-24 months after seroconversion. Viral loads significantly correlated with CD4+ cell counts (r=-0.5, p<0.0001; range, 376-364 cells/mm3) and mathematical modeling defined a median set point of 4.08 log10 (12 143 RNA copies/ml), which was attained approximately 17 months after seroconversion. Comparative measurements using three different viral load platforms (bDNA, Amplicor, and NucliSens) confirmed that viremia in subtype C HIV-1-infected individuals within the first 2 years of infection did not significantly differ from that found in early subtype B infection. In conclusion, the course of plasma viremia, as described in this study, will allow a useful baseline comparator for understanding disease progression in an African setting and may be useful in the design of HIV-1 vaccine trials in southern Africa.

Cross-reactivity of anti-HIV-1 T cell immune responses among the major HIV-1 clades in HIV-1-positive individuals from 4 continents.

BACKGROUND: The genetic diversity of human immunodeficiency virus type 1 (HIV-1) raises the question of whether vaccines that include a component to elicit antiviral T cell immunity based on a single viral genetic clade could provide cellular immune protection against divergent HIV-1 clades. Therefore, we quantified the cross-clade reactivity, among unvaccinated individuals, of anti-HIV-1 T cell responses to the infecting HIV-1 clade relative to other major circulating clades. METHODS: Cellular immune responses to HIV-1 clades A, B, and C were compared by standardized interferon- gamma enzyme-linked immunospot assays among 250 unvaccinated individuals, infected with diverse HIV-1 clades, from Brazil, Malawi, South Africa, Thailand, and the United States. Cross-clade reactivity was evaluated by use of the ratio of responses to heterologous versus homologous (infecting) clades of HIV-1. RESULTS: Cellular immune responses were predominantly focused on viral Gag and Nef proteins. Cross-clade reactivity of cellular immune responses to HIV-1 clade A, B, and C proteins was substantial for Nef proteins (ratio, 0.97 [95% confidence interval, 0.89-1.05]) and lower for Gag proteins (ratio, 0.67 [95% confidence interval, 0.62-0.73]). The difference in cross-clade reactivity to Nef and Gag proteins was significant (P<.0001). CONCLUSIONS: Cross-clade reactivity of cellular immune responses can be substantial but varies by viral protein.

Novel and promiscuous CTL epitopes in conserved regions of Gag targeted by individuals with early subtype C HIV type 1 infection from southern Africa.

Characterization of optimal CTL epitopes in Gag can provide crucial information for evaluation of candidate vaccines in populations at the epicenter of the HIV-1 epidemic. We screened 38 individuals with recent subtype C HIV-1 infection using overlapping consensus C Gag peptides and hypothesized that unique HLA-restricting alleles in the southern African population would determine novel epitope identity. Seventy-four percent of individuals recognized at least one Gag peptide pool. Ten epitopic regions were identified across p17, p24, and p2p7p1p6, and greater than two-thirds of targeted regions were directed at: TGTEELRSLYNTVATLY (p17, 35%); GPKEPFRDYVDRFFKTLRAEQATQDV (p24, 19%); and RGGKLDKWEKIRLRPGGKKHYMLKHL (p17, 15%). After alignment of these epitopic regions with consensus M and a consensus subtype C sequence from the cohort, it was evident that the regions targeted were highly conserved. Fine epitope mapping revealed that five of nine identified optimal Gag epitopes were novel: HLVWASREL, LVWASRELERF, LYNTVATLY, PFRDYVDRFF, and TLRAEQATQD, and were restricted by unique HLA-Cw*08, HLA-A*30/B*57, HLA-A*29/B*44, and HLA-Cw*03 alleles, respectively. Notably, three of the mapped epitopes were restricted by more than one HLA allele. Although these epitopes were novel and restricted by unique HLA, they overlapped or were embedded within previously described CTL epitopes from subtype B HIV-1 infection. These data emphasize the promiscuous nature of epitope binding and support our hypothesis that HLA diversity between populations can shape fine epitope identity, but may not represent a constraint for universal recognition of Gag in highly conserved domains.

Hierarchical targeting of subtype C human immunodeficiency virus type 1 proteins by CD8+ T cells: correlation with viral load.

An understanding of the relationship between the breadth and magnitude of T-cell epitope responses and viral loads is important for the design of effective vaccines. For this study, we screened a cohort of 46 subtype C human immunodeficiency virus type 1 (HIV-1)-infected individuals for T-cell responses against a panel of peptides corresponding to the complete subtype C genome. We used a gamma interferon ELISPOT assay to explore the hypothesis that patterns of T-cell responses across the expressed HIV-1 genome correlate with viral control. The estimated median time from seroconversion to response for the cohort was 13 months, and the order of cumulative T-cell responses against HIV proteins was as follows: Nef > Gag > Pol > Env > Vif > Rev > Vpr > Tat > Vpu. Nef was the most intensely targeted protein, with 97.5% of the epitopes being clustered within 119 amino acids, constituting almost one-third of the responses across the expressed genome. The second most targeted region was p24, comprising 17% of the responses. There was no correlation between viral load and the breadth of responses, but there was a weak positive correlation (r = 0.297; P = 0.034) between viral load and the total magnitude of responses, implying that the magnitude of T-cell recognition did not contribute to viral control. When hierarchical patterns of recognition were correlated with the viral load, preferential targeting of Gag was significantly (r = 0.445; P = 0.0025) associated with viral control. These data suggest that preferential targeting of Gag epitopes, rather than the breadth or magnitude of the response across the genome, may be an important marker of immune efficacy. These data have significance for the design of vaccines and for interpretation of vaccine-induced responses.