Immunogenicity of RSV Fusion Protein Adsorbed to Non-Pathogenic Bacillus subtilis Spores: Implications for Mucosal Vaccine Delivery in Nonclinical Animal Models

<b>Background/Objectives</b>: Mucosal vaccines are rare but commercially desirable because of their real and theoretical biological advantages. Spores and vegetative forms from Bacillus have been used as probiotics due to their stability under various environmental conditions, including heat, gastric acidity, and moisture. Preclinical studies have shown that <i>Bacillus subtilis</i> (<i>B. subtilis</i>) spores can serve as effective mucosal adjuvants. Our study aimed to evaluate B. subtilis spores as a mucosal adjuvant. <b>Methods and Results</b>: We demonstrate in rodents that the fusion protein (F) from respiratory syncytial virus (RSV), when combined with either heat-inactivated or live <i>B. subtilis</i> spores, elicits robust IgG binding and neutralizes antibody titers following both systemic and intranasal administration in mice. The spores facilitate TH-1 and local IgA responses, which could enhance antiviral protection. However, this vaccine failed to elicit measurable antibodies when immunized using a strict intranasal administration method in cotton rats. <b>Conclusions</b>: Our findings illustrate the differing immune responses between the two rodent species, highlighting the need for the careful consideration of validated methods when evaluating intranasal vaccines in preclinical studies.