ELISPOT assay for detection of peptide specific Interferon-gamma secretingcells in rhesus macaques

A reliable procedure to measure antigen specific T cell responses in rhesus macaques is required to determine the efficacy of vaccines and immunothera pies. The currently available T cell assays are poorly quantifiable or tech nically difficult to perform. Classical Cr-51-release cytotoxic T cell (CTL ) assays are cumbersome and difficult to quantitate reproducibly. Detection of specific T-cell using MHC-peptide tetrameric complexes is highly sensit ive, but requires knowledge of MHC type and prior identification of T cell epitopes. We therefore developed a rhesus interferon-gamma (IFN-gamma) ELIS POT assay capable of detecting IFN-gamma secretion in response to stimulati on with pooled 20-mer peptides. Peripheral blood mononuclear cells (PBMCs) from rhesus monkeys immunized with a DNA vaccine and recombinant canary pox encoding the Plasmodium knowlesi circumsporozoite protein (PkCSP) were inc ubated with pools of peptides from PkCSP. Positive responses to peptide poo ls and individual peptides ranging from 100 to 450 spot forming cells (SFC) /10(6) PBMC were detected in four of four immunized monkeys and in zero of two control monkeys. In two monkeys studied in detail, the IFN-gamma respon se was focussed on a single 20-mer peptide. QGDGANAGQPQAQGDGANAG, and was d ependent on CD4(+), but not CD8(+), T cells. Background responses in contro l monkeys and preimmunization PBMCs ranged from 10 to 50 SFC/10(6) PBMC. Th e average within assay and between assay coefficients of variation (CV) for this peptide ELISPOT were 21.9 and 24.7%, respectively. This peptide IFN-g amma assay will be a useful tool for evaluation of T cell responses in rhes us macaques. (C) 2001 Elsevier Science B.V. All rights reserved.