Rapid quantification of SIV-specific CD8 T cell responses with recombinant vaccinia virus ELISPOT or cytokine flow cytometry (2000)

The quantification of antigen-specific CD8 T cell responses has become easier with the development of MHC-peptide tetrameric peptide complexes and the development of assays that measure cytokine release after specific stimulation [1–5]. However, these assays predominantly rely on the identification of optimal epitopes and the knowledge of MHC class I alleles. Limited information is available on macaque MHC alleles or SIV-specific CD8 T cell epitopes which has hampered the assessment of candidate vaccines. Although the use of the Mamu-A*01 restricted p11C, C-M tetramer has been valuable in measuring specific responses in Mamu-A*01 animals [6], this provides limited information on the total CD8 T cell response. We have recently described a modification of the ELISPOT assay in humans, in which recombinant vaccinia viruses (rVV) are used to present HIV antigens [7]. We have now shown that we can use rVV in ELISPOT or cytokine flow cytometry (CFC) assay to quantify the number of SIV-specific CD8 T cells. These methods should allow the rapid assessment of vaccine-induced cellular immune responses in the SIV macaque model. Cryopreserved peripheral blood mononuclear cells (PBMC) from a rhesus macaque, Rh1488, infected with SIVmac251Δnef [5,8,9], were used to measure SIV-specific CD8 T cell responses using ELISPOT and CFC. This animal had previously been shown to make a response to a peptide in SIV envelope, p31 [5]. The ELISPOT assay was modified from Larsson et al. [7]. In brief, 2 × 105 PBMC were added to the wells of a microtiter plate (U-CyTech, Utrecht, the Netherlands) coated with monoclonal antibody specific for rhesus IFN-γ. PBMC had previously been infected with rVV at a multiplicity of infection of 2, expressing SIV Mac251 envelope or control (Tk−), peptide p31 (5μg/ml) or no peptide, or phytohemagglutinin for 16 h at 37°C. Cells were incubated for 5 h at 37°C. After incubation, cells were removed by washing and the wells filled with an appropriate dilution of a biotinylated detector antibody. Subsequently, a goat anti-detector antibody was added, followed by the addition of a chromogenic substrate. After color development, spots were visualized by light microscopy and adjusted to spot-forming cells/106 input PBMC. For the CFC assay, PBMC were either incubated with peptide or control, as previously described [5], or with rVV. Cells were resuspended in 200 μl of media plus 15% fetal calf serum, and incubated with rVV or control at a multiplicity of infection of 2 for 16 h at 37°C. Antibody to CD28 (1 μg/ml) (Becton–Dickenson, San Jose, CA, USA) was also added to facilitate co-stimulation. After stimulation, cells were washed and resuspended in 200 μl R15. Brefeldin A (10 μg/ml) (Sigma, St Louis, MO, USA) was added and cells were incubated for a further 5 h at 37°C. After stimulation, cells were washed in phosphate-buffered saline before fixation in 4% paraformaldehyde for 5 min at 37°C. They were then permeabilized and stained with monoclonal antibodies specific for CD8, intracellular IFN-γ and CD69 (Becton–Dickenson) [5]. After staining, cells were washed and resuspended in 1% paraformaldehyde in phosphate-buffered saline. Samples were collected by a FACS caliber flow cytometry instrument and analysed using CellQuest software. Fifty thousand total events were acquired for each sample, with sequential gating of monoclonal cells into CD8 subpopulations. The ELISPOT detected specific T cell responses to p31 and rVV-SIV Env (Fig. 1a). The response to SIV Env was higher than to p31 alone, probably because of additional non-p31 Env responses. The frequency of Env-specific spot-forming cells was approximately 1 : 2000 PBMC (Fig. 1a). The CFC assay has the advantage of being able to delineate the phenotype of responding cells. The pattern of responsiveness in the CFC assay was similar, although the frequency of the SIV Env-specific response, as a proportion of CD8 T cells, was higher (1 : 175) (Fig. 1b).Fig. 1.: Comparison of SIV Env-specific responses using (a) recombinant vaccinia virus (rVV) ELISPOT or (b) rVV cytokine flow cytometry in Rh 1488. IFN-γ-secreting cells were detected against rVV SIV Env and Env peptide p31 (a). Cytokine flow cytometry detected CD8, CD69 T cells secreting IFN-γ stimulated by rVV SIV Env and p31 in Rh 1488 (b). PBMC, Peripheral blood mononuclear cells.Our results indicate that the use of rVV as antigenic stimulants in the ELISPOT and CFC assays proves to be an efficient and quantitative tool for screening antigen-specific responses in primates, eliminating the need for specific peptide or MHC class identification. The CFC assay also has the advantage of characterizing the specific phenotype of the responding cells. The rVV ELISPOT and CFC assays can be used initially to screen cellular immune responses to viral proteins, and ultimately to identify specific viral epitopes, as well as to follow the cellular immune responses in SIV-infected or vaccinated macaques over extended periods of time. The application of these techniques will be important in the development and assessment of candidate HIV vaccines in primates. Acknowledgements The authors would like to thank the members of the Nixon Laboratory and Scott Furlan for helpful discussions, and Karin J. Metzner, Ruth I. Connor, Amara Luckay and Preston Marx for primate samples. They would also like to thank Dr G. Mazzara, Therion, for recombinant vaccinia viruses. Walter J. Moretto Lea Ann Drohan Douglas F. Nixon