Abstract
Full characterisation of functional HIV-1-specific T-cell responses, including identification of recognised epitopes linked with functional antiviral responses, would aid development of effective vaccines but is hampered by HIV-1 sequence diversity. Typical approaches to identify T-cell epitopes utilising extensive peptide sets require subjects' cell numbers that exceed feasible sample volumes. To address this, CD8 T-cells were polyclonally expanded from PBMC from 13 anti-retroviral naïve subjects living with HIV using CD3/CD4 bi-specific antibody. Assessment of recognition of individual peptides within a set of 1408 HIV-1 Gag, Nef, Pol and Env potential T-cell epitope peptides was achieved by sequential IFNγ ELISpot assays using peptides pooled in 3-D matrices followed by confirmation with single peptides. A Renilla reniformis luciferase viral inhibition assay assessed CD8 T-cell-mediated inhibition of replication of a cross-clade panel of 10 HIV-1 isolates, including 9 transmitted-founder isolates. Polyclonal expansion from one frozen PBMC vial provided sufficient CD8 T-cells for both ELISpot steps in 12 of 13 subjects. A median of 33 peptides in 16 epitope regions were recognised including peptides located in previously characterised HIV-1 epitope-rich regions. There was no significant difference between ELISpot magnitudes for in vitro expanded CD8 T-cells and CD8 T-cells directly isolated from PBMCs. CD8 T-cells from all subjects inhibited a median of 7 HIV-1 isolates (range 4 to 10). The breadth of CD8 T-cell mediated HIV-1 inhibition was significantly positively correlated with CD8 T-cell breadth of peptide recognition. Polyclonal CD8 T-cell expansion allowed identification of HIV-1 isolates inhibited and peptides recognised within a large peptide set spanning the major HIV-1 proteins. This approach overcomes limitations associated with obtaining sufficient cell numbers to fully characterise HIV-1-specific CD8 T-cell responses by different functional readouts within the context of extreme HIV-1 diversity. Such an approach will have useful applications in clinical development for HIV-1 and other diseases.
Highlights
Effective protection against human immunodeficiency virus-1 (HIV-1) infection is likely to require both humoral and cellular-mediated immune responses [1,2,3,4] with cytotoxic CD8 Tcells being a key component of the cellular immune response to HIV-1 infection [4,5,6]
For the 13 subjects living with HIV, starting with 1 vial of cryopreserved peripheral blood mononuclear cells (PBMC), 7-day bi-specific antibody expansion resulted in median 121.4 million viable cells (Table 4)
CD8 T-cells were expanded from a second PBMC vial for single peptide enzyme-linked immunospot (ELISpot) for subject 7 with insufficient cells expanded on day 7
Summary
Effective protection against human immunodeficiency virus-1 (HIV-1) infection is likely to require both humoral and cellular-mediated immune responses [1,2,3,4] with cytotoxic CD8 Tcells being a key component of the cellular immune response to HIV-1 infection [4,5,6]. Detailed mapping of individual epitopes recognised by T-cells across the HIV-1 proteome would require extensive peptide sets, typically tested in interferon gamma (IFNγ) enzyme-linked immunospot (ELISpot) assay utilising subjects’ peripheral blood mononuclear cells (PBMC) Such peptide sets have been designed based on a consensus of the most common amino acid present at each site across multiple HIV-1 protein sequences [8, 9]. One approach designed to reduce the number of peptides tested, whilst still addressing HIV-1 sequence diversity and not requiring prior knowledge of subjects’ HIV-1 sequences is the use of potential T cell epitopes (PTE) Such sets of 15 amino acid (15mer) peptides have been designed to include the most frequent naturally occurring 9 amino acid epitopes present within Gag, Nef, Env and Pol proteins within the sequences of HIV-1 circulating worldwide [11, 12] and are available from the NIH HIV reagent program. Assessment of additional functional T-cell responses in flow cytometric and HIV-1 inhibition assays would further add to cell requirements
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