Mucosal Vaccination Against SARS-CoV-2 Using Human Probiotic Bacillus subtilis Spores as an Adjuvant Induces Potent Systemic and Mucosal Immunity

<b>Background/Objectives</b>: 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. <b>Methods</b>: Here, we evaluated an intranasal (IN) vaccine formulation consisting of recombinant receptor-binding domain (RBD) adsorbed onto human probiotic <i>Bacillus subtilis</i> DG101 spores. <b>Results</b>: 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. <b>Conclusions</b>: All in all, these findings highlight the potential of intranasal vaccines adjuvanted with probiotic <i>B. subtilis</i> spores in inducing sterilizing immunity and limiting SARS-CoV-2 transmission.