A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals.
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)-gamma secreting, virus-specific cells was achieved with an enzyme linked immuno spot (...
| Main Authors: | , , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
1999
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| _version_ | 1797098676668071936 |
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| author | Larsson, M Jin, X Ramratnam, B Ogg, G Engelmayer, J Demoitie, M Mcmichael, A Cox, W Steinman, R Nixon, D Bhardwaj, N |
| author_facet | Larsson, M Jin, X Ramratnam, B Ogg, G Engelmayer, J Demoitie, M Mcmichael, A Cox, W Steinman, R Nixon, D Bhardwaj, N |
| author_sort | Larsson, M |
| collection | OXFORD |
| description | 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)-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. |
| first_indexed | 2024-03-07T05:13:00Z |
| format | Journal article |
| id | oxford-uuid:dc37c992-ea9f-44d6-b212-417d381df522 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:13:00Z |
| publishDate | 1999 |
| record_format | dspace |
| spelling | oxford-uuid:dc37c992-ea9f-44d6-b212-417d381df5222022-03-27T09:16:08ZA recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:dc37c992-ea9f-44d6-b212-417d381df522EnglishSymplectic Elements at Oxford1999Larsson, MJin, XRamratnam, BOgg, GEngelmayer, JDemoitie, MMcmichael, ACox, WSteinman, RNixon, DBhardwaj, NOBJECTIVES: 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. |
| spellingShingle | Larsson, M Jin, X Ramratnam, B Ogg, G Engelmayer, J Demoitie, M Mcmichael, A Cox, W Steinman, R Nixon, D Bhardwaj, N A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. |
| title | A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. |
| title_full | A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. |
| title_fullStr | A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. |
| title_full_unstemmed | A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. |
| title_short | A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. |
| title_sort | recombinant vaccinia virus based elispot assay detects high frequencies of pol specific cd8 t cells in hiv 1 positive individuals |
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