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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., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Evaluating HIV-1 specific T-cell response in African populations is sometimes compromised by extensive virus diversity and paucity of non-clade B reagents. We evaluated whether consensus group M (ConM) peptides could serve as comparable substitutes for detecting immune responses in clade A and clade D HIV-1 infection., Methods: Frequencies, breadths and polyfunctionality (≥ 3 functions: IFN-γ, IL-2, TNF-α and Perforin) of HIV-specific responses utilizing ConM, ConA and ConD Gag and Nef peptides was compared., Results: Median genetic distances of infecting gag sequences from consensus group M were (8.9%, IQR 8.2-9.7 and 9%, IQR 3.3-10) for consensus A and D, respectively. Of 24 subjects infected with A and D clade virus, Gag responses were detected in comparable proportions of subjects when using ConM peptides 22/24, ConA peptides 17/24, and ConD peptides 21/24; p=0.12. Nef responses were also detected at similar proportions of subjects when using ConM peptides 15/23, ConA peptides 19/23, and ConD peptides 16/23, p=0.39. Virus-specific CD4+ and CD8+ T-cell polyfunctionality were also detected in similar proportions of infected individuals when using different peptide sets., Conclusions: These data support the use of consensus group M overlapping peptide sets as reagents for detecting HIV-specific responses in a clade A and D infected population, but underscore the limitations of utilizing these reagents when evaluating the breadth of virus-specific responses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)