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

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
Vaccine (Impact Factor: 3.62). 07/2011; 29(40):7058-66. DOI: 10.1016/j.vaccine.2011.07.028
Source: PubMed


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.
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.
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.
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.

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    • "While PBMC are generally thawed by placing cryovials in 37 °C water baths, presently there is no consensus protocol specifying whether the washing medium should be added when the last ice crystals are visible or whether the cells should be warmed first to 37 °C, and if so, how long they can stay at 37 °C. Protocols also differ in the temperature of the washing medium used (warm vs. cold) and the speed at which the washing medium is added [1,13]; alternatively, some studies do not specify these details [14,15]. Cox and coworkers document significant variation in viability of identical PBMC samples when 11 independent laboratories were allowed to follow their own standard operating procedures (SOP) for PBMC thawing [16]; specifically, the study observed a median viability of 86% with a wide range (24.8% to 100%). "
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    ABSTRACT: Cryopreserved peripheral blood mononuclear cells (PBMC) constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments.
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