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Introduction: Adjuvants added to subunit vaccines augment antigen-specific immune responses. One mechanism of adjuvant action is activation of pattern recognition receptors (PRRs) on innate immune cells. Bordetella colonization factor A (BcfA); an outer membrane protein with adjuvant function, activates T H 1/T H 17-polarized immune responses to protein antigens from Bordetella pertussis and SARS CoV-2. Unlike other adjuvants, BcfA does not elicit a T H 2 response., Methods: To understand the mechanism of BcfA-driven T H 1/T H 17 vs. T H 2 activation, we screened PRRs to identify pathways activated by BcfA. We then tested the role of this receptor in the BcfA-mediated activation of bone marrow-derived dendritic cells (BMDCs) using mice with germline deletion of TLR4 to quantify upregulation of costimulatory molecule expression and cytokine production in vitro and in vivo. Activity was also tested on human PBMCs., Results: PRR screening showed that BcfA activates antigen presenting cells through murine TLR4. BcfA-treated WT BMDCs upregulated expression of the costimulatory molecules CD40, CD80, and CD86 and produced IL-6, IL-12/23 p40, and TNF-α while TLR4 KO BMDCs were not activated. Furthermore, human PBMCs stimulated with BcfA produced IL-6. BcfA-stimulated murine BMDCs also exhibited increased uptake of the antigen DQ-OVA, supporting a role for BcfA in improving antigen presentation to T cells. BcfA further activated APCs in murine lungs. Using an in vitro T H cell polarization system, we found that BcfA-stimulated BMDC supernatant supported T FH and T H 1 while suppressing T H 2 gene programming., Conclusions: Overall, these data provide mechanistic understanding of how this novel adjuvant activates immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Shamseldin, Read, Hall, Tuazon, Brown, Guo, Gupta, Deora, Oestreich and Dubey.)

Introduction: Resurgence of pertussis, caused by Bordetella pertussis, necessitates novel vaccines and vaccination strategies to combat this disease. Alum-adjuvanted acellular pertussis vaccines (aPV) delivered intramuscularly reduce bacterial numbers in the lungs of immunized animals and humans, but do not reduce nasal colonization. Thus, aPV-immunized individuals are sources of community transmission. We showed previously that modification of a commercial aPV (Boostrix) by addition of the Th1/17 polarizing adjuvant Bordetella Colonization Factor A (BcfA) attenuated Th2 responses elicited by alum and accelerated clearance of B. pertussis from mouse lungs. Here we tested whether a heterologous immunization strategy with systemic priming and mucosal booster (prime-pull) would reduce nasal colonization., Methods: Adult male and female mice were immunized intramuscularly (i.m.) with aPV or aPV/BcfA and boosted either i.m. or intranasally (i.n.) with the same formulation. Tissue-resident memory (TRM) responses in the respiratory tract were quantified by flow cytometry, and mucosal and systemic antibodies were quantified by ELISA. Immunized and naïve mice were challenged i.n. with Bordetella pertussis and bacterial load in the nose and lungs enumerated at days 1-14 post-challenge., Results: We show that prime-pull immunization with Boostrix plus BcfA (aPV/BcfA) generated IFNγ+ and IL-17+ CD4+ lung resident memory T cells (TRM), and CD4+IL-17+ TRM in the nose. In contrast, aPV alone delivered by the same route generated IL-5+ CD4+ resident memory T cells in the lungs and nose. Importantly, nasal colonization was only reduced in mice immunized with aPV/BcfA by the prime-pull regimen., Conclusions: These results suggest that TH17 polarized TRM generated by aPV/BcfA may reduce nasal colonization thereby preventing pertussis transmission and subsequent resurgence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yount, Hall, Caution, Shamseldin, Guo, Marion, Fullen, Huang, Maynard, Quataert, Deora and Dubey.)