Quantitative and qualitative differences in the T cell response to HIV in uninfected Ugandans exposed or unexposed to HIV-infected partners.

HIV-exposed and yet persistently uninfected individuals have been an intriguing, repeated observation in multiple studies, but uncertainty persists on the significance and implications of this in devising protective strategies against HIV. We carried out a cross-sectional analysis of exposed uninfected partners in a Ugandan cohort of heterosexual serodiscordant couples (37.5% antiretroviral therapy naive) comparing their T cell responses to HIV peptides with those of unexposed uninfected individuals. We used an objective definition of exposure and inclusion criteria, blinded ex vivo and cultured gamma interferon (IFN-γ) enzyme-linked immunospot assays, and multiparameter flow cytometry and intracellular cytokine staining to investigate the features of the HIV-specific response in exposed versus unexposed uninfected individuals. A response rate to HIV was detectable in unexposed uninfected (5.7%, 95% confidence interval [CI] = 3.3 to 8.1%) and, at a significantly higher level (12.5%, 95% CI = 9.7 to 15.4%, P = 0.0004), in exposed uninfected individuals. The response rate to Gag was significantly higher in exposed uninfected (10/50 [20.%]) compared to unexposed uninfected (1/35 [2.9%]) individuals (P = 0.0004). The magnitude of responses was also greater in exposed uninfected individuals but not statistically significant. The average number of peptide pools recognized was significantly higher in exposed uninfected subjects than in unexposed uninfected subjects (1.21 versus 0.47; P = 0.0106). The proportion of multifunctional responses was different in the two groups, with a higher proportion of single cytokine responses, mostly IFN-γ, in unexposed uninfected individuals compared to exposed uninfected individuals. Our findings demonstrate both quantitative and qualitative differences in T cell reactivity to HIV between HESN (HIV exposed seronegative) and HUSN (HIV unexposed seronegative) subject groups but do not discriminate as to whether they represent markers of exposure or of protection against HIV infection.

Interferon gamma (IFN-γ) negative CD4+ and CD8+ T-cells can produce immune mediators in response to viral antigens.

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.

HIV-1 superinfection can occur in the presence of broadly neutralizing antibodies.

BACKGROUND: Superinfection of individuals already infected with HIV-1 suggests that pre-existing immune responses may not adequately protect against re-infection. We assessed high-risk female sex workers initially infected with HIV-1 clades A, D or A/D recombinants, to determine if HIV-1 broadly neutralizing antibodies were lacking prior to superinfection. METHODS: Six superinfected female sex workers previously stratified by HIV-1 high-risk behavior, infecting virus clade and volunteer CD4 counts were evaluated at baseline (n = 5) and at 350 days post-superinfection (n = 6); one superinfected volunteer lacked pre-superinfection plasma. Retrospective plasmas were assessed for neutralization of a multi-clade panel of 12 HIV-1 viruses before superinfection, and then at quarterly intervals thereafter. Similarly stratified singly infected female sex workers were correspondingly assessed at baseline (n = 19) and 350 days after superinfection (n = 24). Neutralization of at least 50% of the 12 viruses (broad neutralization), and geometric means of the neutralization titers (IC50) were compared before and after superinfection; and were correlated with the volunteer HIV-1 superinfection status, CD4 counts, and pseudovirus clade. RESULTS: Preexisting broad neutralization occurred in 80% (4/5) of the superinfected subjects with no further broadening by 350 days after superinfection. In one of the five subjects, HIV-1 superinfection occurred when broad neutralization was lacking; with subsequent broadening of neutralizing antibodies occuring within 9 months and plateauing by 30 months after detection of superinfection. Clade B and C pseudoviruses were more sensitive to neutralization (13; [87%]); and (12; [80%]) than the locally circulating clades A (10; [67%]) and D (6; [40%]), respectively (p = 0.025). Low antibody titers correlated with clade D viruses and with >500 CD4 T cell counts, but not with the superinfection status. CONCLUSION: These data demonstrate that HIV-1 superinfection can occur both in the presence, and in the absence of broadly neutralizing antibodies.

Frequencies of Gag-restricted T-cell escape "footprints" differ across HIV-1 clades A1 and D chronically infected Ugandans irrespective of host HLA B alleles.

OBJECTIVE(S): We evaluated relationships between critical Gag T-cell escape mutations and concomitant T-cell responses to determine whether HLA-restricted Gag mutations that confer protection, occur at similar rates in a population infected with mixed HIV-1 clades A1 and D viruses. METHODS: Assessment of Gag selective pressure, and adaptive T-cell functions to KAFSPEVIPMF (KF11), ISPRTLNAW (ISW9) and TSTLQEQIGW (TW10) Gag epitopes were combined with host HLA to assess correlations with rates of critical epitope escape mutations in clades A1- (n=23) and D- (n=21) infected, untreated subjects. Infecting clades and selection pressure were determined from the gag sequences. RESULTS: Overall, Gag escape mutations A163X in KF11 were detected in 61% (14/23) A1- infected compared to 5% (1/21) in D-infected subjects (p=0.00015). Gag mutations I147X in the ISW9 epitope were seen in 43%: (10/23) clade A compared to 5%: (1/21) clade D infected subjects, p=0.007, Fisher's Exact test. Both mutations were more frequent in clade A1 infection. Frequencies of the measured epitope-specific T-cell responses were comparable across clades. Peptide binding affinities for the restricting HLA alleles did not differ across clades. Overall, selection pressure on the Gag protein was significantly greater in clade A than in clade D sequences. CONCLUSIONS: These findings imply that HIV-1 vaccine strategies designed to target structurally constrained T-cell epitopes may be further challenged by clade-driven outcomes in specific HLA-restricted Gag epitopes. Equally, the data are line with slower HIV-1 disease progression in clade A infection; and raise hope that increased selective pressure on Gag may be protective irrespective of host HLA alleles.