PROJECT SUMMARY Despite high vaccination coverage, pertussis outbreaks caused by the gram-negative obligate human pathogen Bordetella pertussis (Bp) are observed in many countries. Pertussis resurgence correlates with the switch in the 1990s from whole cell vaccines (wPV) which elicit long-lived Th1/17 immune responses, to acellular vaccines (aPV) which elicit Th1/2 skewed immune responses. Furthermore, aPV do not prevent nasal colonization or transmission of Bp. Current aPV are comprised of 1-5 bacterial proteins that were selected for their roles in pathogenesis and ability to elicit antibodies. In contrast, wPV present an undefined large number of antigens. The combination of limited antigenic diversity and Th2 skewed immune profile is a likely explanation for the incomplete protection provided by aPV. Recent studies including that from our laboratory also show that circulating Bp strains (CBp) from globally diverse countries have absent/reduced expression of current aPV antigens, suggesting that aPV may be significantly less effective against CBp strains. There is increasing recognition that CD4+ T cell responses are critical for long-lived protective immune responses that clear the entire respiratory tract. However, it is not clear that current aPV antigens are optimal CD4+ T cell targets. We will use state-of-the-art mass spectrometry, bioinformatics, and phenotypic and functional assays to identify proteins expressed by CBp that are processed and presented on Class II histocompatibility antigens of humans and mice, and that stimulate CD4+ T cell responses. This foundational data set will be coupled with a prime-pull vaccination strategy and a Th1/17 skewing adjuvant developed in our laboratory, to determine the immunogenicity and protective efficacy of newly defined antigens to create a next-gen aPV. Specific Aim 1: Define the set of naturally derived Bp peptides presented on MHC II and recognized by CD4+ T cells. We will identify the Bp antigens from circulating Bp strains (CBp) that are expressed on human and murine Class II, and use proliferation and flow cytometry assays to determine which antigens are recognized by CD4+ T cells of wPV-immunized individuals and convalescent mice. Specific Aim 2: To test the immunogenicity and protective efficacy of novel antigens against circulating Bp strains and their role in pathogenesis. We will test the immunogenicity and protective efficacy of the novel proteins using a murine model of Bp infection. We will create deletion mutants of the novel proteins in CBp to determine their role in pathogenesis and colonization of the respiratory tract. IMPACT: Our integrated approach to identify, test, and leverage novel Bp antigens will permit the rational design of next-gen aPVs that elicit long-lasting protection in the respiratory tract, prevent nasopharyngeal carriage and thereby reduce the spread of the disease pertussis.