Group M consensus Gag and Nef peptides are as efficient at detecting clade A1 and D cross-subtype T-cell functions as subtype-specific consensus peptides

Abstract

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.☆ This manuscript has not been published in its current form or a substantially similar form. There are no financial, consultant, institutional and other relationships that might lead to bias or a conflict of interest. Part of this work was presented as an abstract at the South African Immunology Society Conference, 5th-8th December 2010 with a title "Comparison of cross reactive epitopes using clade A, D and group M consensus peptides in an HIV-1 infected Ugandan population".

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

“…Cryopreserved PBMCs from previously defined IFN-γ ELISpot positive specimens to Con M (n = 31) and previously determined IFN-γ ELISpot negative specimens to Adenovirus (n = 5) were thawed, rested overnight and stimulated with Con M and Ad peptides, respectively, before they were further evaluated for other functions using flow cytometry. All PBMCs were stimulated with their respective antigens for 6 h at 37 °C in a 5% CO 2 incubator; and stained with specified fluorochrome antibodies, as previously described [25]. Briefly, trypan blue exclusion counting was used to select groups of 500,000 live PBMCs.…”

“…Evaluation of other cellular immune functions is commonly performed only among those T-cells initially identified to be IFN-γ secreting using the ‘back-gating’ procedure of flow cytometry analysis [20], or using ELISpot assay screening for individuals with positive IFN-γ secretion [21], [22], [23], [24], [25]. Proportions of CD4+ and CD8+ T-cells that lack expression of IFN-γ (IFN-γ negative) but secrete other T-cell functions in response to viral antigens have not been well studied.…”