Evaluation of antigen-specific T-cell responses to viral antigens is frequently performed on IFN-γ secreting cells. However, T-cells are capable of producing many more functions than just IFN-γ, some of which, like Perforin, are associated with immune protection in HIV-1 disease elite controllers. We evaluated the extent of missed T-cell functions when IFN-γ secretion is used as a surrogate marker for further evaluation of T-cell functions. Intracellular cytokine staining assay and flow cytometry were used to assess peripheral blood mononuclear cells (PBMCs) from 31 HIV-infected ART-naive individuals for the extent to which gated CD4+ and CD8+ IFN-γ producing and non-producing T-cells also secreted IL-2, Perforin, and TNF-α functions. Similarly, the extent of missed virus-specific responses in IFN-γ ELISpot assay negative T-cells from 5 HIV-1 uninfected individuals was evaluated. Cells from HIV-infected individuals were stimulated with pooled consensus group M (Con M) peptides; and those from healthy individuals were stimulated with pooled adenovirus (Ad) peptides. Overall, frequencies of virus-specific IFN-γ secreting CD4+ and CD8+ cells were low. Proportions of IFN-γ negative CD4+ expressing IL-2, Perforin, or TNF-α to Con M were significantly higher (5 of 7 functional profiles) than the corresponding IFN-γ positive CD4+ (0 of 7) T-cell phenotype, p = 0.02; Fisher’s Exact test. Likewise, proportions of CD8+ T-cells expressing other functions were significantly higher in 4 of the 7 IFN-γ negative CD8+ T-cells. Notably, newly stimulated Perforin, identified as Perforin co-expression with IL-2 or TNF-α, was significantly higher in IFN-γ negative CD8+ T-cell than in the positive CD8+ T-cells. Using SEB, lower responses in IFN-γ positive cells were most associated with CD4+ than CD8+ T-cells. These findings suggest that studies evaluating immunogenicity in response to HIV and Adenovirus viral antigens should not only evaluate T-cell responsiveness among IFN-γ producing cells but also among those T-cells that do not express IFN-γ.

Reagents for evaluating non-clade B HIV-specific T cell responses are uncommon. Peptides based on highly conserved HIV-1 consensus group M sequences that are phylogenetically closer to most circulating strains may provide potential alternative reagents in populations with diverse infections, and may be relevant for vaccine design. Recognition of such reagents in clade A1-and D-infected populations has not been previously evaluated. Interferon (IFN)-γ ELISpot assay was used to evaluate T cell recognition of Gag and Nef peptides based on consensus group M sequences in 50 treatment-naive adults predominantly infected with HIV-1 clades A1 and D. Gag-induced T cell responses were correlated with gag sequence diversity. Infecting clades were determined from gag sequences for 45 of the 50 subjects as 40% clade A1 (18/45), 45% clade D (20/45), 2% clade C (1/45), 2% A1/C recombinant (1/45), 2% A1/D (1/45), 7% CRF10_CD (3/45), and 2% U (unclassifiable) (1/45). The mean genetic divergence and diversity of clade A and D gag region compared to group M consensus sequences at synonymous and nonsynonymous nucleotide and amino acid levels were not always significant. Gag peptides were targeted at significantly higher frequency [88% (44/50)] than Nef [64% (32/50)]; p=0.014, although their mean IFN-γ magnitudes were comparable ([3703 (95% CI 2567-4839)] vs. [2120 (95% CI 478-3762)]), respectively. Measurable virus-induced IFN-γ responses were detected in 96% (48/50) individuals, primarily targeting the more conserved Gag p24 and Nef central core regions. Use of these reagents to screen for HIV-specific IFN-γ responses may mitigate the challenge of viral diversity; although this targeting is apparently biased toward a few highly conserved epitopes.

HighlightsA and D infected subjects even though they bear the same presenting HLA alleles, and live in the same environment. Escape mutations that are known to confer survival advantage were more frequent in clade A-infected subjects irrespective of host HLA alleles.There was no evidence to link this difference in outcome to the evaluated adaptive T-Cell responses (IFN-γ responses and polyfunctional responses) to those key structurally constrained Gag epitopes.However, we have demonstrated that there was significantly greater selective pressure on the Gag protein of clade A than that of clade D.The data are in line with the known faster disease progression in clade D than clade A infected individuals.The data also highlight that the current difficulties in formulating a global HIV vaccine design will be further challenged by clade associated differences in outcome.

Whilst short-term oral pre-exposure prophylaxis (PrEP) with antiretroviral drugs in men who have sex with men has shown protection against HIV-1 infection, the impact of this regimen on the in vivo foreskin transcriptome is unknown. We collected foreskin tissue after voluntary medical male circumcision from 144 young men (72 from Uganda and 72 from South Africa) randomized to one to two doses of either oral tenofovir (TFV) disoproxil fumarate (FTC-TDF) or tenofovir alafenamide (FTC-TAF) or no drug (untreated controls). This novel approach allowed us to examine the impact of short-term oral PrEP on transcriptome of the male genital tract. A single dose of FTC-TDF did not affect the foreskin transcriptome in relation to control arm, however one dose of FTC-TAF induced upregulation of four genes AKAP8, KIAA0141, HSCB and METTL17. Following two doses of either FTC-TDF or FTC-TAF, there was an increase in 34 differentially expressed genes for FTC-TDF and 15 for FTC-TAF, with nine DEGs in common: KIAA0141, SAFB2, CACTIN, FXR2, AKAP8, HSCB, CLNS1A, DDX27 and DCAF15. Functional analysis of differentially expressed genes revealed modulation of biological processes related to mitochondrial stress (KIAA0141, HSCB and METTL17), anti-viral and anti-inflammatory pathways (CACTIN and AKAP8). Our results show that short-course on-demand oral PrEP in men modulates genes in foreskin tissue which are likely unfavorable to HIV acquisition and replication. We also describe an upregulated expression of genes involved in diverse mitochondria biology which may potentially result in worsened mitochondria-related. These results warrant further studies to assess the role of short-course and prolonged oral PrEP on biological processes of the foreskin mucosa.