Tetramer staining, ELISPOT analysis, and ICS after vaccination with the DNA prime-adenovirus 5 boost regimena

Animal identification no.% of CD8+ CD3+ cells positive for tetramerELISPOT (106 SFC) result for:Epitope breadth
CM9SL8GagTatRevNefCD8+CD4+CD4+ IL-4+CD4+ IFN-γ+
  • a Vaccination with the DNA prime-adenovirus 5 boost regimen engenders strong, broad immune responses to all immunogens. Responses are shown for tetramer staining, ELISPOT analysis, and ICS. The immunogens used were pools of peptides (15-mers overlapping by 11 and 10-mers overlapping by 9) or individual peptides (15-mers, 10-mers, or minimal optimal peptides). To ascertain total breadth of response, we summed ICS, ELISPOT, and tetramer unique responses in either the CD8+ (IFN-γ ELISPOT, ICS, and tetramer) or CD4+ (ICS and IL-4 ELISPOT) subsets; this is presented in the last two columns of the table. For example, if a CM9 tetramer response was present as well as a response to the Gag E pool (which contains two peptides encoding CM9 within them) and the minimal optimal for CM9 in multiple tests, this would count as only one addition to the epitope breadth. Our goal was to ascertain the number of unique responses, not how many times we tested it. Tetramer responses are presented as percentages of tetramers positive in CD3+ CD8+ lymphocyte gated cells. CD4+ responses were assessed at day 7 and day 14 post-adenovirus vaccination. Tetramer responses and ELISPOT responses were assessed throughout the postvaccination period; we only count the number of unique responses. CD4 responses are broken down into IL-4+ and IFN-γ+. Some responses are both IL-4+ and IFN-γ+.

  • b This animal is Mamu-A*02+.