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Monitoring of Vaccine-Specific Gamma Interferon Induction in Genital Mucosa of Mice by Real-Time Reverse Transcription-PCR

Véronique Revaz, Anne Debonneville, Martine Bobst, and Denise Nardelli-Haefliger^*

Institute of Microbiology, Centre Hospitalier Universitaire Vaudois and University of Lausanne, CH-1011 Lausanne, and NCCR Molecular Oncology, 1066 Epalinges, Switzerland

Received 27 September 2007/ Returned for modification 6 November 2007/ Accepted 12 March 2008

Monitoring of T-cell responses in genital mucosa has remained a major challenge because of the absence of lymphoid aggregates and the low abundance of T cells. Here we have adapted to genital tissue a sensitive real-time reverse transcription-PCR (TaqMan) method to measure induction of gamma interferon (IFN-) mRNA transcription after 3 h of antigen-specific activation of CD8 T cells. For this purpose, we vaccinated C57BL/6 mice subcutaneously with human papillomavirus type 16 L1 virus-like particles and monitored the induction of CD8 T cells specific to the L1_165-173 H-2D^b-restricted epitope. Comparison of the responses induced in peripheral blood mononuclear cells and lymph nodes (LN) by L1-specific IFN- enzyme-linked immunospot assay and TaqMan determination of the relative increase in L1-specific IFN- mRNA induction normalized to the content of CD8b mRNA showed a significant correlation, despite the difference in the readouts. Most of the cervicovaginal tissues could be analyzed by the TaqMan method if normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA was used and a significant L1-specific IFN- induction was found in one-third of the immunized mice. This local response did not correlate with the immune responses measured in the periphery, with the exception of the sacral LN, an LN draining the genital mucosa, where a significant correlation was found. Our data show that the TaqMan method is sensitive enough to detect antigen-specific CD8 T-cell responses in the genital mucosa of individual mice, and this may contribute to elaborate effective vaccines against genital pathogens.

Published ahead of print on 26 March 2008.

Present address: Department of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland.