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The development of HIV vaccines is an urgent priority and there is need to generate reagents representing multiple subtypes that can be used to screen HIV-1-specific responses. We used Aldrithiol-2 (AT-2), a mild oxidizing reagent, to eliminate the infectivity of HIV while maintaining its structure and ability to be processed for presentation to T cells. Inactivated subtype A, B, and D viruses were evaluated for their ability to stimulate T cell responses in PBMC samples from 18 U.S. subjects infected with HIV-1 subtype B and 32 Ugandan subjects infected with subtypes A and D or recombinants AC and AD. Five HIV-1-negative samples were also analyzed. T cell responses to AT-2-inactivated viral isolates were monitored by interferon-gamma (IFN-gamma) intracellular cytokine secretion (ICS) analysis; matched microvesicle preparations served as negative controls. Among the 18 subtype B infected subjects, 39% had CD3(+) CD4 (+) IFN-gamma responses and 67% had CD3(+) CD8(+) IFN-gamma responses. Of the 32 Ugandan subjects, 34% demonstrated CD3(+) CD4(+) IFN-gamma responses and 78% demonstrated CD3(+) CD8(+) IFN-gamma responses. Both subtype-specific and cross-reactive responses were observed. Responses to the AT-2 viruses tended to be lower in magnitude than those detected by a set of overlapping gag peptides. Robust lymphoproliferative responses to AT-2 viruses were seen in a subset of subjects. In conclusion, AT-2-inactivated HIV-1 virions stimulated both CD4 and CD8 HIV-1-specific responses and may provide an additional reagent for screening HIV-1-specific responses in HIV seropositives and vaccinees.

Investigations concerning the extent and nature of subtype-specific and intersubtype immune responses in HIV-1-infected persons are necessary for the development of appropriate candidate vaccines. In the cross-sectional study described here, 26 HIV-1-positive Ugandan patients were tested for their ability to mount HIV antigen-specific cellular immune responses. Subjects were infected with either HIV-1 subtypes A, C, or D. Recombinant vaccinia virus (rVV)-based and peptide-based enzyme-linked immunospot (Elispot) assays were used to evaluate HIV-1-specific gamma-interferon (IFN-gamma) cellular responses. rVV expressing gag, pol, or env proteins derived from HIV-1 subtypes A, B, and D were evaluated for their ability to induce whole HIV-1-protein-specific IFN-gamma responses in 14 patients. A panel of previously identified HLA class I-restricted peptides based on representative sequences from HIV-1 subtypes A, B, C, and D and restricted through HLA-A2, -A29, -B42, -B53, and -B57 alleles were used to evaluate the presence of HIV-1-peptide-specific T cells in 19 patients. Using rVV, 27 of a possible 38 subtype-specific responses (71%) and 56 of a possible 110 intersubtype responses (51%) were observed. When appropriate peptides were used 18 of 39 (46.2%) subtype-specific and 13 of 39 (33.3%) intersubtype responses were observed. Peptide responses were higher quantitatively than those seen when rVV were used. In 7 patients, both rVV and specific peptides were evaluated; in 3 of 7 individuals, global responses were seen despite a lack of measurable HLA-restricted peptide-specific responses demonstrating the need to evaluate a broader range of HIV-specific immune responses.

A pilot study was undertaken with the objective of developing a simple, economical, and efficient algorithm through which to subtype HIV-1 in a large epidemiological cohort study in Uganda. A peptide enzyme immunoassay (PEIA) employing both V3 and gp41 regions and a heteroduplex mobility assay (HMA) were evaluated in comparison with DNA sequencing. Of 146 samples selected, 115 (79%) were successfully sequenced. Taking sequence data as the "gold standard," other assays were compared with these data. The HMA correctly identified 95 (83%) of the samples, and only 1 sample was wrongly identified. The V3 PEIA alone and in combination with gp41 peptides correctly identified 76 and 78% of the samples, respectively; however, the number of wrongly identified samples was four times less with the combination compared with V3 peptides alone (4 versus 16%). The sensitivity, specificity, and positive and negative predictive values for serotype A and D samples were greater for the combination than V3 peptides alone. We have described a new algorithm to segregate subtypes A and D. This algorithm uses the two peptide assays followed by HMA and then DNA sequencing for untypable samples, giving an accuracy of 95% at a cost of 37 and 21% for consumables compared with subtyping all the samples by HMA or DNA sequencing, respectively. This proposed approach is suitable for epidemiological studies in Uganda and other regions with a predominance of A and D subtypes.

The molecular epidemiology of a population-based cohort in a cluster of 15 villages in southwestern Uganda was investigated by sequencing part of the p24 gag gene and performing heteroduplex mobility assays (HMAs) of the V3 region of the env gene. Sequence and HMA data, obtained for 69 and 88 proviruses, respectively, showed that the clade A and D viruses were present at a ratio of about 0.67:1. No other clades were detected. Thirteen (22%) of 59 proviruses for which both gag and env data were obtained appeared to be recombinants. Although both clade A and D viruses were present in 13 of the villages, their distribution was unequal: for example, from env data 59% of clade A viruses were found in the eastern villages, compared with only 27% of clade D viruses. Phylogenetic (maximum likelihood) analysis of the p24 gag sequences showed a total of five clusters supported by bootstrap resampling values above or close to 75%. Four clusters were sexual partners, but there was no known sexual contact between the persons in the other cluster. The DNA sequences showed between 0.5 and 8.3% divergence from the cohort clade A or D consensus sequences. The sequences were not closely related to those published for other clade A or D proviruses.