ELISPOT assay for detection of peptide specific Interferon-γ secreting cells in rhesus macaques

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Abstract

A reliable procedure to measure antigen specific T cell responses in rhesus macaques is required to determine the efficacy of vaccines and immunotherapies. The currently available T cell assays are poorly quantifiable or technically difficult to perform. Classical 51Cr-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 sensitive, but requires knowledge of MHC type and prior identification of T cell epitopes. We therefore developed a rhesus interferon-γ (IFN-γ) ELISPOT assay capable of detecting IFN-γ secretion in response to stimulation 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 incubated with pools of peptides from PkCSP. Positive responses to peptide pools and individual peptides ranging from 100 to 450 spot forming cells (SFC)/106 PBMC were detected in four of four immunized monkeys and in zero of two control monkeys. In two monkeys studied in detail, the IFN-γ response was focussed on a single 20-mer peptide, QGDGANAGQPQAQGDGANAG, and was dependent on CD4+, but not CD8+, T cells. Background responses in control monkeys and preimmunization PBMCs ranged from 10 to 50 SFC/106 PBMC. The average within assay and between assay coefficients of variation (CV) for this peptide ELISPOT were 21.9 and 24.7%, respectively. This peptide IFN-γ assay will be a useful tool for evaluation of T cell responses in rhesus macaques.

Introduction

The enumeration of antigen specific T cell responses in non-human primates is important for the evaluation of immune responses to and vaccination against a variety of infectious diseases, including HIV/SIV and malaria, in which T cells are an important component of protective immunity. Rhesus macaques are an important model for both SIV and malaria. Several approaches are available for measuring antigen specific T cell responses in rhesus monkeys. In vitro proliferation in response to antigen is technically straightforward, but is not directly related to important effector functions, such as cytotoxicity or secretion of cytokines. Classical CTL assays can be performed using a variety of approaches to in vitro stimulation of effectors and sensitization of target cells (Vowels et al., 1989, McGraw et al., 1990, Miller et al., 1990, Munroe et al., 1994, Townsend et al., 1997, Wang et al., 1998), however, these assays are technically demanding and difficult to quantify. Measurement of cytokine secretion in supernatants of antigen stimulated cultures may be complicated by consumption of the cytokine during incubation. Detection of antigen specific T cells by staining with tetrameric MHC/peptide complexes (Altman et al., 1996, Kuroda et al., 1998) is a highly sensitive, quantifiable approach, but requires the use of rhesus monkeys of known MHC alleles and presupposes the identification of immunodominant T cell epitopes for evaluation. The use of an ELISPOT assay for detection of cells which secrete interferon-γ in response to stimulation with pools of peptides from a vaccine antigen is an alternative that obviates the need for MHC typing of monkeys and knowledge of MHC restricted minimal T cell epitope(s).

The Plasmodium knowlesi/rhesus macaque system is a model for malaria vaccine development which allows the analysis of protective efficacy against challenge with infectious sporozoites. Complete sterile protection against the pre-erythrocytic stages of malaria can be induced by immunization with radiation attenuated sporozoites in both mice and humans (Nussenzweig et al., 1967, Clyde et al., 1973, Rieckmann et al., 1974), and appears dependent on secretion of IFN-γ by CD8+, and to a lesser extent, CD4+ T cells (Doolan and Hoffman, 1997). Since no reproducible sporozoite challenge model for P. falciparum malaria in primates is available, the knowlesi–rhesus model is an attractive choice to test the efficacy of pre-erythrocytic stage vaccines. As part of a study of a vaccination regimen in which macaques were primed with a mixture of DNA vaccines against both pre-erythrocytic and erythrocytic target antigens and then boosted with a mixture of recombinant canary pox expressing the same four antigens, we developed an ELISPOT assay for detection and quantification of peptide specific, IFN-γ secreting rhesus T cells.

Section snippets

Animals

Male and female rhesus macaques aged 18–33 months and weighing 3–4 kg were purchased from Three Springs Scientific, Inc. (Perkasie, PA) and maintained in the non-human primate facility of the Armed Forces Radiobiology Research Institute. Animal care and treatment were in accordance with standards approved by the Institutional Animal Care and Use Committee according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animals Resources,

Results

In order to evaluate the induction of T cell responses to malaria vaccines in the P. knowlesi/rhesus model system we developed an IFN-γ ELISPOT assay for the detection of antigen specific IFN-γ secreting cells. We chose to focus on IFN-γ responses to pooled peptides from the pre-erythrocytic stage antigen, PkCSP, because there is substantial evidence in the P. yoelii/mouse model that protection elicited by immunization with radiation attenuated sporozoites is mediated by IFN-γ (Doolan and

Discussion

Protection against the pre-erythrocytic stages of malaria is largely mediated by T cell dependent IFN-γ secretion. In order to assess the immunogenicity of malaria vaccines in the P. knowlesi/rhesus macaque model, we have developed an ELISPOT assay for the detection of peptide specific IFN-γ secretion by rhesus PBMCs. Earlier studies have described the use of ELISPOT in rhesus to detect IFN-γ secreting cells stimulated by mitogen (van Besouw et al., 1994), mixed lymphocyte reaction (Lobashevsky

Acknowledgements

The authors thank Richard Stout (Bioject, Inc., Portland, OR) for advice and the gift of Biojector® syringes for use in this trial, Daniel Carucci, for assistance with the automated counting of the ELISPOTs, Gary Brice for assistance with FACS analysis, and MAJ Mark Gold, SPC King, SPC Randolph, and PFC Cosling for assistance in care of the rhesus monkeys. This work was supported by Naval Medical Research Center Work Unit STOF 6.2.622787A.0101.870.EFX and by a Cooperative Research and

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    Present address: Center for Comparative Functional Genomics, University at Albany, SUNY, One University Place, Rensselaer, NY 12144, USA.

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