Validation of IFN-γ/IL-2 FluoroSpot assay for monitoring HPV L1-specific immuneresponses in Cervarix™ vaccinees

Objective:

HPV (Human Papillomavirus) vaccines are adopted worldwide. To monitor T cellular immune responses after vaccination, a quick and simple assay is needed. IFN-γ and IL-2 are robust sentinel cytokines secreted by antigen-specific T cells. A multiplexed fluorogenic ELISpot assay, based on IFN-γ/IL-2 double staining, to detect T cell responses after Cervarix™ vaccination was evaluated.

Methods:

Six women (age > 18) were vaccinated with 3 doses of Cervarix™. Blood samples were drawn before 1^st, and 1 – 2 month after 2^nd and 3^rd dose. A cytometric ex-vivo stimulation assay and the new fluorogenic ELISpot assay were performed. PBMC were stimulated with HPV16L1 or HPV18L1 synthetic 30mer peptides. Intracellular IFN-γ/IL-2 expression was assessed (IC-FACS) in antigen-specific CD154+/CD4+ T cells. PBMC (2 × 10⁵) were stimulation with L1-synthetic peptides in microwells. Secreted cytokines were visualized by fluorochrome-labeled mABs and spot numbers read out using iDOT Elispot reader (AID, Strassberg, Germany).

Results:

After Cervarix™ vaccination, IFN-γ and IL-2 expressing T cells were significantly increased in all samples after 2^nd and 3^rd dose by FACS measurement (3 – 41fold for IFN-γ, 7 – 60fold for IL-2). Similar results were found by IFN-γ/IL-2 double-stained FluoroSpot assay (4 – 60fold by IFN-γ, 3 – 43fold by IL-2). The results assessed by FluoSpot assay and IC-FACS measurement correlated excellently.

Conclusion:

Results from FluoroSpot assay demonstrated good analytical performance and showed excellent concordance with the ex vivo IC-FACS. The L1 FluoroSpot is much more easy and robust to perform and may be an alternative option to monitor the cellular immune responses after HPV vaccination.