Preclinical development of a novel vaccine targeting Clostridioides difficile using an attenuated Salmonella Typhimurium vector

Clostridioides difficile infection (CDI) is one of the most important nosocomial infections in the world. In 2012, approximately 40,000 CDI cases were reported in Canada with a cost of ~$300 million. In the last decade, the rate of hospital-acquired CDI cases has decreased, however community-acquired CDI rates are increasing. CDI is usually initiated by antibiotic use that disrupts the normal gastrointestinal microflora leading to C. difficile overgrowth and the production of two major toxins A and B (TcdA and TcdB) that cause CDI-associated pathology. Reducing morbidity and mortality from CDI requires the development of new tools, such as vaccines. While a small number of candidate vaccines targeting C. difficile have entered clinical trials, none has targeted the gut mucosa. Among the many potential advantages of live Salmonella-based vaccines is their ability to deliver antigens to mucosal surfaces in the context of regional bacterial ‘invasion’ – a scenario likely to elicit strong humoral and cellular immune responses at the site of C. difficile-induced pathology. We used an attenuated strain of Salmonella Typhimurium, YS1646 as a vector to develop a candidate vaccine targeting the highly immunogenic C-terminal receptor binding domains (RBD) of TcdA and TcdB of C. difficile. Anti-RBD antibodies have been shown to neutralize the corresponding toxins and protect against C. difficile challenge in animal models. To address our first aim of making a vaccine that would stimulate both mucosal and humoral immunity, we generated candidate YS1646 strains bearing plasmids that express and secrete the RBDs of TcdA or TcdB. Our best candidates elicited both systemic and mucosal antibody responses in C57BL/6 mice when given in a multimodality schedule: ie: one dose of recombinant protein intramuscularly (IM) plus 3 doses of the YS1646 candidates orally (PO) over one week. Two of our constructs were tested in a C. difficile challenge mouse model and achieved 100% protection (versus 30% survival in the control group). The PO vaccines alone gave ~80% protection. For our second aim, we examined the longevity of the responses elicited by our vaccine candidates. IgG (serum) and IgA (gut) titers elicited by multimodal vaccination were maintained up to 6 months after vaccination. Multimodal vaccination significantly protected mice that were challenged 6 months after vaccination (83-100% survival versus 33% in PBS controls). PO vaccines alone gave ~90% protection. Our third aim was to develop stable vaccine candidates without a mobile genetic element. To do this, we established 6 YS1646 strains with stable chromosomal expression of the targeted RBD antigens. After in vitro screening, we selected two candidates to move forward. When delivered in a multimodal vaccination schedule, these candidates generated IgG titers, with a skewing towards the IgG1 subtype and an increase in antigen specific IL-5 production in the mesenteric lymph node 32 days after vaccination. Oral delivery alone elicited a bias towards IgG2c antibodies and increased antigen-specific GM-CSF production in the Peyer’s patches. Upon challenge with a clinical C. difficile isolate, mice that received multimodal vaccination against both toxins had 94% survival (versus 38% survival in the PBS control group). Oral vaccination against TcdA at a higher dose elicited 100% survival (versus 30% in the control group). In summary, we have developed an orally-delivered vaccine candidates that elicit both systemic and mucosal immune responses in mice and are highly protective against C. difficile infection. Through this project, we have gained considerable insight into the immunological interactions between the host and S. Typhimurium YS1646-based vaccines and valuable information for the rational design of the first in human study of these novel vaccine candidates

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