Breadth of CD8 T-cell mediated inhibition of replication of diverse HIV-1 transmitted-founder isolates correlates with the breadth of recognition within a comprehensive HIV-1 Gag, Nef, Env and Pol potential T-cell epitope (PTE) peptide set

PLoS One. 2021 Nov 17;16(11):e0260118. doi: 10.1371/journal.pone.0260118. eCollection 2021.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CD8-Positive T-Lymphocytes
  • Epitopes, T-Lymphocyte*
  • HIV Seropositivity
  • HIV-1*
  • Inhibition, Psychological
  • Leukocytes, Mononuclear


  • Epitopes, T-Lymphocyte

Grants and funding

This work was made possible by IAVI, which is supported by funding from many donors, including the Bill and Melinda Gates Foundation, the Ministry of Foreign Affairs of Denmark, Irish Aid, the Ministry of Finance of Japan in partnership with The World Bank, the Ministry of Foreign Affairs of the Netherlands, the Norwegian Agency for Development Cooperation, the United Kingdom Department for International Development and the US Agency for International Development. This work is made possible by the generous support of the American people through USAID. The contents are the responsibility of the authors and do not necessarily reflect the views of USAID or the United States Government. A full list of IAVI donors is available at: The funding donors had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.