Targeting the Toxin B glucosyltransferase domain of NAP1/B1/027 Clostridioides difficile using an attenuated Salmonella enterica Typhimurium vaccine vector

Thesis

Upadhyaya, P;

Clostridioides difficile (C. difficile) is the most common cause of nosocomial antibiotic-associated diarrhea in developed countries and is classified as an urgent threat by the Centers of Disease Control and Prevention (CDC). Most strains of C. difficile release toxins A (TcdA) and B (TcdB) that mediate disease. Annually, there are approximately 450,000 new cases and 29,000 deaths in the USA alone resulting in an economic burden of $5.4 billion dollar. The emergence of hypervirulent strains, notably NAP1/B1/027, in the 2000s and the relative inefficiency of current treatments against these strains highlight the need for a vaccine. Therefore, we proposed a vaccine consisting of attenuated Salmonella enterica serovar Typhimurium (S. Tm) strains YS1646 that express the immunogenic portions of TcdB (i.e. the glucosyltransferase domain (GTD) and the receptor binding domain (RBD)). Since GTD is highly conserved across many C. difficile strains, we hypothesized that this vaccine would protect mice from lethal challenge with NAP1/B1/027 strains and possibly contribute to protection against all strains. We aimed to modify a murine model for NAP1/B1/027 challenge and to evaluate the protective efficacy against NAP1/B1/027 challenge of a GTD-expressing YS1646 vaccine compared to a more traditional vaccine targeting the RBD of C. difficile TcdB (rbdB). The optimal dose for infection with a NAP1/B1/027 clinical isolate was demonstrated to range between 4.07×10^4 and 9.83×10^4 colony forming units (CFU)/ mouse. The GTD was confirmed to be immunogenic in C57BL/6 mice when three intramuscular (i.m.) doses of rGTD (10µg alum-adjuvanted) were administered on days 0, 21 and 35 (serum anti-TcdB IgG titers >1.3×10^4 ng/mL). Female C57BL/6 mice that were vaccinated with the traditional rbdB multimodal vaccine (a single i.m. dose of alum-adjuvanted recombinant rbdB (rrbdB) on D0 and three p.o. doses of YS1646-vectored rbdB on D0, D2, D4) yielded an efficacy of 80% against lethal challenge with a NAP1/B1/027 strain. The ‘solo’ GTD multimodal (three i.m. doses of alum-adjuvanted rGTD on D0, D21, D35; and three p.o. doses of YS1646-vectored GTD on D35, D37, D39) and the combined GTD + rbdB multimodal (three i.m. doses of rGTD on D0, D21, D35; a single i.m. dose of rrbdB on D35; and three p.o. doses of each antigen in a YS1646 vector on D35, D37, D39) vaccines yielded an efficacy of 100%. Interestingly, TcdB-specific IgG titers were higher in the combined GTD + rbdB vaccine group compared to the ‘solo’ GTD vaccine group (3.15 x 10^4 ng/mL and 7.3 x 10^3 ng/mL, respectively). While the combined GTD + rbdB multimodal vaccine seemed to be the most promising construct, an evaluation of the mucosal response is required

Citation

Targeting the Toxin B glucosyltransferase domain of NAP1/B1/027 Clostridioides difficile using an attenuated Salmonella enterica Typhimurium vaccine vector