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

Abstract

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.

Introduction

Herpes simplex virus type 2 (HSV-2) is the causative agent of genital herpes, one of the most prevalent sexually transmitted diseases worldwide. The US seroprevalence rate of HSV-2 is estimated at 17% in 14–49 yrs olds [1] but in some populations of HIV-infected subjects and female sex workers, the seroprevalence rate reaches 95% (reviewed in Ref. [2]). The high seroprevalence rates of HSV-2 in developed and developing countries, the importance of subclinical shedding on HSV-2 transmission especially in subjects unaware that they have genital herpes [3], [4], the increased risk that HSV-2 plays on HIV-1 acquisition [5], [6], [7], and the severity of neonatal herpes [7] underscore the need for an effective prophylactic and therapeutic vaccine.

Animal and human studies suggest that a robust cellular immune response in addition to neutralizing antibodies is critical in protection against HSV-2 infection and thus, current vaccine strategies are directed at eliciting both neutralizing antibody and cellular immunity against HSV-2. In order to detect vaccine-induced T cell responses in PBMC, a sensitive, specific and quantitative assay will be required. Assays previously employed to detect T cell responses elicited by candidate HSV-2 vaccines in humans include lymphoproliferation [8], [9] and standard ⁵¹Cr-release assays [9]. Both assays require cell expansion over 6–10 days which limits quantitative analysis of vaccine-induced responses, especially in HSV-2 seropositive subjects who possess positive baseline T cell responses to HSV-2. More recently, an IFN-γ ELISPOT was employed to detect CD8+ T cell responses to a vaccine comprised of a monovalent heat shock protein non-covalently associated with a gD-2 peptide [10]. While this assay was quantitative and did not require cell expansion, large numbers of PBMC were required to select CD8+ T cells and to generate dendritic cells (DC) that were used as antigen presenting cells.

In the present study, we evaluated an IFN-γ ELISPOT assay designed to detect and quantitate T cell responses in PBMC directed at HSV-2 peptides obtained from naturally infected HSV-2 seropositive subjects compared to HSV-1 seropositive subjects and subjects seronegative for HSV-1 and HSV-2. As PBMC from individual vaccines will be limiting in future vaccine trials, the IFN-γ ELISPOT assay was designed to stimulate unfractionated PBMC with pools of overlapping HSV-2 peptides without the addition of in vitro matured DC. We describe the development of this assay to measure HSV-2 peptide-specific T cell responses that should enable more detailed characterization of vaccine-induced T cell responses and assist in determining whether T cell immunity contributes to vaccine efficacy.