Development of IFN-γ secretory ELISPOT based assay for screening of ADCC responses

Abstract

Antibody dependent cell mediated cytotoxicity has been established as one of the important protective immune mechanisms against HIV making it essential to evaluate it while testing immunogenicity of emerging vaccine candidates. IFN-γ secretory ELISPOT assay, widely used for evaluation of CTL response in HIV vaccine trials, was adapted for measuring ADCC responses and the results were compared with the standard ICS based assays. IFN-γ responses elicited by plasma samples of 23 HIV infected individuals against Env and Gag peptides using granulocytes as antigen presenting cells were assessed by both the methods. Supernatants of the activated cells in ELISPOT assay were also assessed for cytokine/chemokine estimation. ELISPOT assays detected significantly more ADCC responders against HIV-Env and Gag peptide pools than ICS assay. The magnitude of IFN-γ response in both the assay correlated significantly (p=0.002). NK cells were found to be the predominant cell type secreting IFN-γ in the assay. Although IFN-γ and IL-6 levels were significantly higher in supernatants of Env peptides stimulated cells, IP-10 and MCP-1α levels were found to be more against Gag peptides. Thus, IFN-γ secretory ELISPOT assay was found to be more sensitive in detecting ADCC responders than ICS assay making it a valuable tool for screening of ADCC responses in future vaccine trials. Differences in cytokine pattern of Env versus Gag stimulated cells warrants a need for investigating their role in protection against HIV infection.