CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: Use of multiple peptide sets increases the detectable breadth of the CTL response

BACKGROUND: Subtype A is a major strain in the HIV-1 pandemic in eastern Europe, central Asia and in certain regions of east Africa, notably in rural Kenya. While considerable effort has been focused upon mapping and defining immunodominant CTL epitopes in HIV-1 subtype B and subtype C infections, few epitope mapping studies have focused upon subtype A. RESULTS: We have used the IFN-γ ELIspot assay and overlapping peptide pools to show that the pattern of CTL recognition of the Gag and Nef proteins in subtype A infection is similar to that seen in subtypes B and C. The p17 and p24 proteins of Gag and the central conserved region of Nef were targeted by CTL from HIV-1-infected Kenyans. Several epitope/HLA associations commonly seen in subtype B and C infection were also observed in subtype A infections. Notably, an immunodominant HLA-C restricted epitope (Gag 296–304; YL9) was observed, with 8/9 HLA-C(W)0304 subjects responding to this epitope. Screening the cohort with peptide sets representing subtypes A, C and D (the three most prevalent HIV-1 subtypes in east Africa), revealed that peptide sets based upon an homologous subtype (either isolate or consensus) only marginally improved the capacity to detect CTL responses. While the different peptide sets detected a similar number of responses (particularly in the Gag protein), each set was capable of detecting unique responses not identified with the other peptide sets. CONCLUSION: Hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the HIV-1-specific CTL response. Interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given CTL response. Therefore, reagent choice and knowledge of the exact sequences that prime CTL responses will be important factors in experimentally defining cross-reactive CTL responses and their role in HIV-1 disease pathogenesis and validating vaccines aimed at generating broadly cross-reactive CTL responses

Variable contexts and levels of hypermutation in HIV-1 proviral genomes recovered from primary peripheral blood mononuclear cells

APOBEC-mediated cytidine cleamination of HIV-1 genomes during reverse transcription has been shown to be a potent mechanism of host restriction for HIV-1 infection ex vivo and in vitro. However, this defense system can be overcome by the viral protein Vif. Unlike other mechanisms of host restriction, the APOCEC-Vif interaction leaves an imprint on integrated proviruses in the form of G-A hypermutation. in the current work we systematically studied levels, contexts, and patterns of HIV-1 hypermutation in vivo. the analysis of 24 full-genome HIV-1 sequences retrieved from primary PBMCs, representing infections with several HIV-1 clades, and the inclusion of 7 cognate pairs of hypermutated/non-hypermutated sequences derived from the same patient sample, provided a comprehensive view of the characteristics of APOBEC-mediated restriction in vivo. Levels of hypermutation varied nearly 5-fold among the studied proviruses. GpG motifs were most frequently affected (22/24 proviruses). Levels of hypermutation varied across the genome. the reported twin peak pattern of hypermutation was observed in 18/24 hypermutants, but the remainder exhibited singular non-conforming patterns. These data suggest considerable complexity in the interplay of host restriction and viral defense during HIV-1 infection. (c) 2008 Elsevier Inc. All rights reserved.Henry M Jackson Fdn Advancement Mil Med, US Mil HIV Res Program, Rockville, MD 20850 USAUniversidade Federal de São Paulo, Paulista Sch Med, Div Infect Dis, BR-04039 São Paulo, BrazilWalter Reed Army Inst Res, Div Retrovirol, Rockville, MD 20850 USAUniversidade Federal de São Paulo, Paulista Sch Med, Div Infect Dis, BR-04039 São Paulo, BrazilWeb of Scienc

Exposure to Nonhuman Primates in Rural Cameroon

A high percentage of rural villagers are exposed to blood of nonhuman primates and risk acquiring infectious diseases

Viral Linkage in HIV-1 Seroconverters and Their Partners in an HIV-1 Prevention Clinical Trial

Characterization of viruses in HIV-1 transmission pairs will help identify biological determinants of infectiousness and evaluate candidate interventions to reduce transmission. Although HIV-1 sequencing is frequently used to substantiate linkage between newly HIV-1 infected individuals and their sexual partners in epidemiologic and forensic studies, viral sequencing is seldom applied in HIV-1 prevention trials. The Partners in Prevention HSV/HIV Transmission Study (ClinicalTrials.gov #NCT00194519) was a prospective randomized placebo-controlled trial that enrolled serodiscordant heterosexual couples to determine the efficacy of genital herpes suppression in reducing HIV-1 transmission; as part of the study analysis, HIV-1 sequences were examined for genetic linkage between seroconverters and their enrolled partners.We obtained partial consensus HIV-1 env and gag sequences from blood plasma for 151 transmission pairs and performed deep sequencing of env in some cases. We analyzed sequences with phylogenetic techniques and developed a Bayesian algorithm to evaluate the probability of linkage. For linkage, we required monophyletic clustering between enrolled partners' sequences and a Bayesian posterior probability of ≥ 50%. Adjudicators classified each seroconversion, finding 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) indeterminate transmissions, with linkage determined by consensus env sequencing in 91 (84%). Male seroconverters had a higher frequency of unlinked transmissions than female seroconverters. The likelihood of transmission from the enrolled partner was related to time on study, with increasing numbers of unlinked transmissions occurring after longer observation periods. Finally, baseline viral load was found to be significantly higher among linked transmitters.In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage determination process

HIV-1 recombinants with multiple parental strains in low-prevalence, remote regions of Cameroon: Evolutionary relics?

<p>Abstract</p> <p>Background</p> <p>The HIV pandemic disseminated globally from Central West Africa, beginning in the second half of the twentieth century. To elucidate the virologic origins of the pandemic, a cross-sectional study was conducted of the genetic diversity of HIV-1 strains in villagers in 14 remote locations in Cameroon and in hospitalized and STI patients. DNA extracted from PBMC was PCR amplified from HIV(+) subjects. Partial <it>pol </it>amplicons (N = 164) and nearly full virus genomes (N = 78) were sequenced. Among the 3956 rural villagers studied, the prevalence of HIV infection was 4.9%; among the hospitalized and clinic patients, it was 8.6%.</p> <p>Results</p> <p>Virus genotypes fell into two distinctive groups. A majority of the genotyped strains (109/164) were the circulating recombinant form (CRF) known to be endemic in West Africa and Central West Africa, CRF02_AG. The second most common genetic form (9/164) was the recently described CRF22_01A1, and the rest were a collection of 4 different subtypes (A2, D, F2, G) and 6 different CRFs (-01, -11, -13, -18, -25, -37). Remarkably, 10.4% of HIV-1 genomes detected (17/164) were heretofore undescribed unique recombinant forms (URF) present in only a single person. Nearly full genome sequencing was completed for 78 of the viruses of interest. HIV genetic diversity was commonplace in rural villages: 12 villages each had at least one newly detected URF, and 9 villages had two or more.</p> <p>Conclusions</p> <p>These results show that while CRF02_AG dominated the HIV strains in the rural villages, the remainder of the viruses had tremendous genetic diversity. Between the trans-species transmission of SIV_cpzand the dispersal of pandemic HIV-1, there was a time when we hypothesize that nascent HIV-1 was spreading, but only to a limited extent, recombining with other local HIV-1, creating a large variety of recombinants. When one of those recombinants began to spread widely (i.e. became epidemic), it was recognized as a subtype. We hypothesize that the viruses in these remote Cameroon villages may represent that pre-epidemic stage of viral evolution.</p

Origin and Epidemiological History of HIV-1 CRF14_BG

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Users must also make clear the license terms under which the work was published. CC BY Licence: http://creativecommons.org/licenses/by/4.0/Background: CRF14_BG isolates, originally found in Spain, are characterized by CXCR4 tropism and rapid disease progression. This study aimed to identify the origin of CRF14_BG and reconstruct its epidemiological history based on new isolates from Portugal.Methodology/Principal Findings: C2V3C3 env gene sequences were obtained from 62 samples collected in 1993–1998 from Portuguese HIV-1 patients. Full-length genomic sequences were obtained from three patients. Viral subtypes, diversity, divergence rate and positive selection were investigated by phylogenetic analysis. The molecular structure of the genomes was determined by bootscanning. A relaxed molecular clock model was used to date the origin of CRF14_BG. Geno2pheno was used to predict viral tropism. Subtype B was the most prevalent subtype (45 sequences; 73%) followed by CRF14_BG (8; 13%), G (4; 6%), F1 (2; 3%), C (2; 3%) and CRF02_AG (1; 2%). Three CRF14_BG sequences were derived from 1993 samples. Near full-length genomic sequences were strongly related to the CRF14_BG isolates from Spain. Genetic diversity of the Portuguese isolates was significantly higher than the Spanish isolates (0.044 vs 0.014, P,0.0001). The mean date of origin of the CRF14_BG cluster was estimated to be 1992 (range, 1989 and 1996) based on the subtype G genomic region and 1989 (range, 1984–1993) based on the subtype B genomic region. Most CRF14_BG strains (78.9%) were predicted to be CXCR4. Finally, up to five amino acids were under selective pressure in subtype B V3 loop whereas only one was found in the CRF14_BG cluster.Conclusions: CRF14_BG emerged in Portugal in the early 1990 s soon after the beginning of the HIV-1 epidemics, spread to Spain in late 1990 s as a consequence of IVDUs migration and then to the rest of Europe. CXCR4 tropism is a general characteristic of this CRF that may have been selected for by escape from neutralizing antibody response

High-Throughput High-Resolution Class I HLA Genotyping in East Africa

HLA, the most genetically diverse loci in the human genome, play a crucial role in host-pathogen interaction by mediating innate and adaptive cellular immune responses. A vast number of infectious diseases affect East Africa, including HIV/AIDS, malaria, and tuberculosis, but the HLA genetic diversity in this region remains incompletely described. This is a major obstacle for the design and evaluation of preventive vaccines. Available HLA typing techniques, that provide the 4-digit level resolution needed to interpret immune responses, lack sufficient throughput for large immunoepidemiological studies. Here we present a novel HLA typing assay bridging the gap between high resolution and high throughput. The assay is based on real-time PCR using sequence-specific primers (SSP) and can genotype carriers of the 49 most common East African class I HLA-A, -B, and -C alleles, at the 4-digit level. Using a validation panel of 175 samples from Kampala, Uganda, previously defined by sequence-based typing, the new assay performed with 100% sensitivity and specificity. The assay was also implemented to define the HLA genetic complexity of a previously uncharacterized Tanzanian population, demonstrating its inclusion in the major East African genetic cluster. The availability of genotyping tools with this capacity will be extremely useful in the identification of correlates of immune protection and the evaluation of candidate vaccine efficacy

Distinct Human Immunodeficiency Virus Type 1 Subtype A Virus Circulating in West Africa: Sub-Subtype A3

Phylogenetic analyses demonstrate significant diversity in worldwide circulating strains of human immunodeficiency virus type 1 (HIV-1). Detailed studies have revealed a complex pattern of intersubtype recombinations, as well as evidence of sub-subtypes circulating in various populations. In this study, we characterized an HIV-1 strain that had previously been identified as a distinct subcluster within the subtype A radiation based on partial sequence data. These viruses were of particular interest given that we recently found that their prevalence was significantly higher in dually infected individuals compared to women who were singly infected with HIV-1. Five viruses isolated from commercial sex workers in Dakar, Senegal, were full-length PCR amplified and sequenced. Phylogenetic analyses indicated that, whereas three of these viruses were closely related and clustered overall within the HIV-1 subtype A radiation, they were distinct from previously characterized sub-subtype A1 and A2 viruses. The clustering pattern was maintained in the individual gag, pol, and env regions of the genome. Distance calculations between these viruses, which we termed A3, and other reference sub-subtype A1 and A2 viruses fell in the range of distances between previously characterized sub-subtype groups. In addition, we found evidence of two A3-containing recombinants in our cohort. These recombinants are mosaics composed of sequence from both sub-subtype A3 and CRF02_AG, the major circulating recombinant form in this West African population. Based on phylogenetic analyses, we propose that the group of viruses found in the Dakar sex worker cohort, previously referred to as HIV-1 A subcluster 2, be referred to as HIV-1 sub-subtype A3