A recombinant vaccinia virus based ELISPOT assay detects high frequencies of pol-specific CD8 T cells in HIV-1-positive individuals

Marie Larsson, Xia Jin, Bharat Ramratnam, Graham S. Ogg, Jose Engelmayer, Marie Ange Demoitie, Andrew J. McMichael, William I. Cox, Ralph M. Steinman, Douglas Nixon, Nina Bhardwaj

Research output: Contribution to journalArticlepeer-review

198 Scopus citations


Objectives: HIV-1-specific CD8 T cells are considered to be critical in anti-HIV responses. It is important to quantify these cells and to determine their antigenic targets. Here quantification of interferon (IFN)-γ secreting, virus-specific cells was achieved with an enzyme linked immune spot (ELISPOT) assay. Methods: Peripheral blood mononuclear cells (PBMC) were infected with recombinant vaccinia vectors expressing HIV-1 genes (gag, pol, env or nef) and added to wells precoated with anti-IFN-γ monoclonal antibodies. Spot forming cells (SFC), i.e. antigen-specific T cells were detected 24 h later by the addition of biotinylated anti-IFN-γ monoclonal antibodies, followed by avidin-bound biotinylated horseradish peroxidase. Results: In a cohort of 19 patients, of whom 15 were on highly active antiretroviral therapy, 18 had primed T cells directed against one or more HIV-1 antigens (P < 0.0001). Pol-specific T cells routinely dominated the CD8 response with frequencies up to 2000 SFC per 106 PBMC. In HLA A(*)0201-positive patients, the vaccinia vectors detected much higher frequencies of SFC than haplotype-restricted peptides. Elimination of CD8 T cells resulted in > 90% loss of antigen-specific SFC when vaccinia virus was used as a vector. The number of CD8 SFC exceeded the number of memory cells detected in limiting dilution assays by > 1 log10, whereas a correlation was found between the frequency of effector cells detected by both ELISPOT and MHC class I peptide tetramer assays. Conclusions: Vaccinia virus vectors used in ELISPOT assays are useful for determining the frequency and specificity of CD8 T cells for individual HIV-1 gene products. The dominance of cytolytic T lymphocytes (CTL) recognizing pol proteins suggests that this antigen should be considered in vaccine strategies.

Original languageEnglish
Pages (from-to)767-777
Number of pages11
Issue number7
StatePublished - 1999
Externally publishedYes


  • Canarypox
  • Cytotoxic T lymphocytes
  • ELISPOT assay
  • HIV-1
  • Vaccinia


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