Clostridioides difficile infection (CDI) is primarily driven by two protein toxins, toxin A (TcdA) and toxin B (TcdB). Additionally, about 5–30% of C. difficile strains produce a third toxin, binary toxin (CDT), which is linked to higher morbidity and mortality rates in CDI. Vaccination is a cost effective approcah to prevent CDI. Current;y, no vaccine has been licenced against CDI. Major effects have been devloted to developing vaccines against TcdA and TcdB. How a fully ecctive vaccine should target all 3 toxins. CDT consists of an enzymatic component (CDTa) and a binding/translocation component (CDTb), the latter facilitating CDTa entry into host cells. CDTb contains two receptor-binding domains (RBD1 and RBD2), with RBD2 playing a critical role in host cell toxicity, making it a promising target for intervention.

In this study, we assessed the homology and immunogenicity of RBD2. Our in-silico analyses revealed that RBD2 is highly conserved across diverse toxinotypes and ribotypes. Immunization of mice and hamsters with RBD2 elicited robust IgG/A antibody responses against CDT. Notably, vaccinated mice were fully protected against lethal systemic CDT challenge, and hamsters were completely protected against a lethal oral spore challenge with the CDT-only C. difficile strain DSM101085. Furthermore, we demonstrated for the first time that CDT is lethal in both animal models, causing significant damage to the colon, cecum, and spleen—a previously unreported finding. Collectively, our data highlight RBD2 as a promising vaccine component for CDI prevention.