Development of an interferon-gamma ELISPOT assay to detect human T cell responses to HSV-2

Background: The need for an HSV-2 vaccine is great considering the increasing prevalence of HSV-2 despite the widespread use of antiviral drugs. Human clinical trials of HSV-2 vaccines that elicit neutralizing antibodies have proven to be only partially effective suggesting that induction of effective T cell responses to HSV-2 is also a critical component to an efficacious vaccine. A sensitive and specific assay to measure HSV-specific T cell responses is a necessary part of vaccine development and thus we undertook the development of an interferon-γ (IFN-γ) ELISPOT assay to measure T cell responses to HSV-2.

Methods: PBMC from HSV-seronegative (HSVneg) (n=35), HSV-1-seropositive (HSV-1+/2-) (n=20) and HSV-2-seropositive (HSV-2+) subjects (n=26) were screened by IFN-γ ELISPOT for T cell responses using 34 peptide pools representing 16 HSV-2 proteins including mostly virion and immediate-early (IE) proteins.

Results: Overall, 85% of HSV-2+ subjects had a positive response to the HSV-2 peptide pools and on average, HSV-2+ subjects responded to 3 peptide pools (range 1-10). The most frequent responses were to gD-2, UL39, UL46, ICP0, UL49, gB-2, and ICP4. In contrast, only 2 of 35 (6%) HSVneg subjects had detectable T cell responses and in both cases, responses were of low magnitude relative to responses in HSV-2+ subjects and were directed at a single peptide pool. The response rate to the HSV-2 peptide pools in HSV-1+/2- subjects was 40% suggesting that the HSV-2 peptide pools contain a significant number of type-common T cell epitopes. The IFN-γ ELISPOT assay detected CD4 and CD8 T cells directed at HSV-2 peptides as confirmed by intracellular cytokine staining and flow cytometry.

Conclusion: We have developed a quantitative IFN-γ ELISPOT assay that detects both CD4 and CD8 T cells to HSV-2 peptides. This assay does not require large quantities of PBMC to generate dendritic cells for T cell stimulation, making it an ideal assay for monitoring the immunogenicity of candidate HSV-2 vaccines designed to elicit T cell responses to HSV-2 specific epitopes.