Background: In immunocompromised patients Aspergillus fumigatus causes most cases of fatal invasive aspergillosis. However, cell-mediated adaptive immunity may provide protection. Here we report a proteomic strategy to identify T cell epitopes as potential vaccine candidates. Method: CF1 mice were immunized with either the recombinant A. fumigatus vaccine candidate, rAsp f 3, or with crude fungal extracts. Subsequently, the animals were immunosuppressed with cortisone and intranasally challenged with conidia. T cells of the survivors were co-cultured with antigen-presenting cells and HPLC-fractionated pepsin or trypsin-derived peptides of the antigens used for immunizations. Antigenicities were measured by T cell proliferation and the peptides and their protein origins were identified by mass spectrometry (MS). Results: The proliferative response of T cells from immunized mice to stimulation with proteolytic peptides from rAsp f 3 was significantly greater than in non-immunized mice. T cells from mice immunized with protein extracts of A. fumigatus proliferated in response to several HPLC fractions of the trypsin-derived peptides of the same extracts. Several of these T cell-stimulating fractions contained partially digested peptides that overlapped a common sequence EDVAEAVK, in NAD-dependent formate dehydrogenase. The functionality of this potential T cell epitope will be evaluated using synthetic peptides. Conclusion: Although, most HPLC fractions contained more than one peptide, we demonstrate a strategy to identify T cell epitopes by MS analysis of overlapping peptide sequences that result from incomplete cleavages using various proteases with distinct proteolytic specificities. Our methodology is a valuable tool for the discovery of vaccine candidates that may protect against aspergillosis.
Full conference title:
47th Interscience Conference on Antimicrobial agents and Chemotherapy
- ICAAC 47th