Dual-adjuvant mucosal vaccine leveraging mast cell and TLR9 agonists for protection against poxvirus infection.

Mast cells (MC) are innate immune cells that are predominantly localized under the skin and at mucosal surfaces, and play a role in numerous physiological processes, including host response to pathogens. Recently, mast cell activators (MCA) have been identified as mucosal vaccine adjuvants that are able to promote a strong and antigen-specific immune response. We performed an extensive structure-activity relationship (SAR) analysis on the previously identified small molecule MCA, ST101036, to further optimize its adjuvanticity. This led to the development of the derivative, VAP-1185, which demonstrated improved mast cell degranulation activity in vitro, and a Th2-biased in vivo immune response. While mucosal vaccines with a single adjuvant have shown effectiveness, combining two adjuvants can activate multiple immune pathways, leading to a stronger and more comprehensive immune response. Additionally, dual adjuvant vaccines can elicit a balanced Th1/Th2 response, leading to equally effective cellular and humoral responses. We combined VAP-1185 with the FDA-approved adjuvant, cytosine phosphoguanine (CpG), which activates toll-like receptor 9 (TLR9) and promotes a Th1-biased immune response. This dual adjuvant formulation promotes inflammatory cytokine production in vitro, additive humoral effects and an active cellular response in vivo, as well as a favorable safety profile when intranasally administered to C57BL/6 mice. Subsequently, this adjuvant formulation was also able to confer protection against a lethal challenge of vaccinia virus in BALB/c mice. Thus, we report a novel mucosal vaccine formulation that produces an effective Th1/Th2 balanced immune response.