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. Mucosal vaccination offers the possibility of establishing immune protection directly at portals of pathogen entry, but its development remains constrained by biological, technological, and implementation barriers. Objective. To integrate the major challenges limiting mucosal vaccination within a common framework and examine emerging strategies capable of addressing several of these barriers simultaneously. Results and Discussion. We consider six interconnected challenges: incomplete recognition of the determinant role of adjuvants; scarcity of safe and accessible mucosal adjuvants; the need to overcome mucosal tolerance while preserving tissue homeostasis; dependence on coldchain logistics; reliance on multidose immunization schedules to achieve protective immunity; and limited breadth of vaccine-induced cross-reactive protection. To better capture the complexity of modern vaccine platforms, we discuss expand adjuvant activity as exogenous, self-adjuvanting, or endogenous according to the origin of the immunostimulatory signal. Particular emphasis is placed on proteoliposome- and cochleate-based systems and probiotic Bacillus subtilis spores as multifunctional platforms combining antigen delivery with intrinsic immunostimulatory properties. Preclinical studies support their capacity to induce systemic IgG, mucosal secretory IgA, cellular responses, and memory response, while their heat resistance may facilitate the development of more thermostable formulations. We also discuss the Single Time Vaccination Strategy as an approach to combined-route immunization and consider glycans and other microbial structures as potential contributors to antigen-specific and cross-reactive mucosal responses. Conclusions. Future mucosal vaccine development should consider adjuvants as tools for coordinated antigen delivery, innate sensing, immune programming with polarization activity, and mucosal memory rather than solely as immune potentiators and delivery system. Probiotic spores, cochleates, and combined-route strategies provide promising examples of this integrated approach, although their translational value will require rigorous demonstration of formulation stability, mechanisms of action, safety, protective breadth, and clinical efficacy.