title: A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals.
abstract: OBJECTIVE
  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)-gamma secreting, virus-specific cells was achieved with an enzyme linked immuno 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-gamma monoclonal antibodies. Spot forming cells (SFC), i.e. antigen-specific T cells were detected 24 h later by the addition of biotinylated anti-IFN-gamma 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 10(6) 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.
authors.affiliation: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021-6399, USA.
authors.forename: M
authors.initials: M
authors.lastname: Larsson
authors.forename: X
authors.initials: X
authors.lastname: Jin
authors.forename: B
authors.initials: B
authors.lastname: Ramratnam
authors.forename: G S
authors.initials: GS
authors.lastname: Ogg
authors.forename: J
authors.initials: J
authors.lastname: Engelmayer
authors.forename: M A
authors.initials: MA
authors.lastname: Demoitie
authors.forename: A J
authors.initials: AJ
authors.lastname: McMichael
authors.forename: W I
authors.initials: WI
authors.lastname: Cox
authors.forename: R M
authors.initials: RM
authors.lastname: Steinman
authors.forename: D
authors.initials: D
authors.lastname: Nixon
authors.forename: N
authors.initials: N
authors.lastname: Bhardwaj
mesh_terms: D000328:Adult; D019380:Anti-HIV Agents / Q000627:therapeutic use; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015751:Genes, pol; D005822:Genetic Vectors; D015488:HIV Antigens / Q000276:immunology; D015658:HIV Infections / Q000188:drug therapy / Q000276:immunology* / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D011994:Recombinant Proteins; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014616:Vaccinia virus / Q000235:genetics
chemical_list: D019380:Anti-HIV Agents; D015488:HIV Antigens; D011994:Recombinant Proteins