Improving the Sensitivity of the ELISPOT Analyses of Antigen-Specific Cellular Immune Responses in Rhesus Macaques

The close similarities in hematopoietic and immune systems of humans and rhesus macaques (Macaca mulatta ) make them the desired nonhuman primate animal model for developing vaccines against infectious diseases relevant to humans. The best example is the simian immunodeficiency virus infection in macaques as a model for AIDS, resulting from infection of humans by HIV-1. Vaccine efficacy against viruses depends on priming cell-mediated immunity by the use of sensitive assays that can accurately detect even small levels of antigen-specific T-cell responses that may otherwise be missed easily. With this in mind, we developed the dendritic cell enzyme-linked immunospot (DC-ELISPOT) protocol by incorporating antigen-pulsed DCs to stimulate lymphocytes, as opposed to the conventional ELISPOT assay, which cultures mixtures of various low-level populations of indigent antigen-presenting cells and responding lymphocytes with antigens. In rhesus macaques immunized with an HIV envelope peptide cocktail vaccine, the DC-ELISPOT protocol enabled more accurate enumeration of the cellular immune responses, as antigen-specific inteferon-γ-producing cells, with up to 18-fold increase in detection sensitivity compared with conventional ELISPOT and elimination of false negative results. The increased sensitivity of DC-ELISOT protocol is further validated in tests determining recall antigen-specific responses in human volunteers after tuberculin skin testing.