The adjuvant BcfA activates antigen presenting cells through TLR4 and supports T and T1 while attenuating T2 gene programming.

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. colonization factor A (BcfA); an outer membrane protein with adjuvant function, activates T1/T17-polarized immune responses to protein antigens from and SARS CoV-2. Unlike other adjuvants, BcfA does not elicit a T2 response.

Methods: To understand the mechanism of BcfA-driven T1/T17 vs. T2 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 T cell polarization system, we found that BcfA-stimulated BMDC supernatant supported T and T1 while suppressing T2 gene programming.

Conclusions: Overall, these data provide mechanistic understanding of how this novel adjuvant activates immune responses.