Background/Objectives: The ongoing evolution of SARS-CoV-2 has highlighted the limitations of parenteral vaccines in preventing viral transmission, largely due to their failure to elicit robust mucosal immunity. Methods: Here, we evaluated an intranasal (IN) vaccine formulation consisting of recombinant receptor-binding domain (RBD) adsorbed onto human probiotic Bacillus subtilis DG101 spores. Results: In BALB/c mice, IN spore-RBD immunization induced strong systemic and mucosal humoral responses, including elevated specific IgG, IgM, and IgA levels in serum, bronchoalveolar lavage fluid (BALF), nasal-associated lymphoid tissue (NALT), and saliva. It further promoted mucosal B cell and T cell memory, along with a Th1/Tc1-skewed T cell response, characterized by increased IFN-γ-expressing CD4⁺ and CD8⁺ T cells in the lungs. Conclusions: All in all, these findings highlight the potential of intranasal vaccines adjuvanted with probiotic B. subtilis spores in inducing sterilizing immunity and limiting SARS-CoV-2 transmission.

Background. Most vaccinologists have looking for specific antigens and the regulators have been pushing them to purified products eliminating the immunopotentiators agents coming from self-adjuvants. Nevertheless, adjuvants are determinant vaccine component, and undoubtedly the future of vaccine formulations; particularly for mucosal applications. Mucosa is challenged by the huge majority of infectious including commensal which do not produce disease but inducing immune response. In addition, mucosa is the only route capable to protect them self and to induce systemic immune response as parenteral route did. Nevertheless, few mucosal vaccines exist mainly due to the insufficient cognizance of mucosal immune functions and the absence of mucosal adjuvants. Aim. To update adjuvant Finlay strategy and to show promising results combining mucosal and parenteral simultaneously priming. Results. Adjuvant Finlay (AF), specifically the AFPL® and the AFCo were design for parenteral and mucosal application, respectively. AFPL®1 is derived from Neisseria meningitidis serogroup B proteoliposome (PL) and adsorbed onto Al(OH)3 in order to increase stability and safety. AFPL1 has been widely used in humans in a prophylactic meningococcal vaccine as an essential adjuvant. A comparable PL has been used in the 4CMenB Novartis vaccine. AFPL®1 has been also evaluated as an adjuvant for a therapeutic/prophylactic allergen vaccine shifting the allergen-specific Th2 pattern to Th1/Tr1 in mice and the Phase I clinical trial was recently concluded. AFPL®1 was used at preclinical stage with several other antigens. AFCo1, a cochleate structure derived from AFPL®1, is a mucosal adjuvant. AFCo3 non-derived from AFPL®1, has been tested as immunopotentiator in fish decreasing their mortality. As mucosal route required more doses than parenteral one two simultaneous primes by parenteral and mucosal routes were performed and surprising this strategy overcome the three doses required, inducing systemic and also mucosal immune response. Lastly, as covalent conjugation is the hallmark of thymus-independence bacterial polysaccharides vaccines we explored the coadministration of AFCo1 with plain polysaccharide and this combination overcome the thymus-independence inducing a Th1 polarization and memory immune response in mice. Conclusion. AF are very promising adjuvants for unsolved-vaccine diseases including mucosal approaches and therapeutic vaccines.