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

• Published in: AIDS (London) Vol. 13; no. 7; pp. 767 - 777
• Main Authors: LARSSON, M, XIA JIN, BHARDWAJ, N, RAMRATNAM, B, OGG, G. S, ENGELMAYER, J, DEMOITIE, M.-A, MCMICHAEL, A. J, COX, W. I, STEINMAN, R. M, NIXON, D
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD Lippincott Williams & Wilkins 07.05.1999
• Subjects: Adult; AIDS/HIV; Anti-HIV Agents - therapeutic use; Biological and medical sciences; CD8-Positive T-Lymphocytes - immunology; Enzyme-Linked Immunosorbent Assay - methods; Genes, pol; Genetic Vectors; HIV Antigens - immunology; HIV Infections - drug therapy; HIV Infections - immunology; HIV Infections - virology; HIV-1 - genetics; HIV-1 - immunology; Human immunodeficiency virus 1; Human viral diseases; Humans; Infectious diseases; Male; Medical sciences; Middle Aged; Recombinant Proteins; T-Lymphocytes, Cytotoxic - immunology; Vaccinia virus; Vaccinia virus - genetics; Viral diseases; Viral diseases of the lymphoid tissue and the blood. Aids; Human; Immunopathology; Chordopoxvirinae; Immune response; HIV-1 virus; Recombinant virus; Orthopoxvirus; Vaccination; Retroviridae; AIDS; Immune deficiency; Lentivirus; Infection; Virus; Vaccinia virus; Specificity; Poxviridae; Efficiency; Viral disease; T-Lymphocyte; Human immunodeficiency virus
• ISSN: 0269-9370; 1473-5571
• DOI: 10.1097/00002030-199905070-00005

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. 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. 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. 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.