Clostridioides difficile is the main cause of health-care-associated infectious diarrhoea. Toxins, TcdA and TcdB, secreted by this bacterium damage colonic epithelial cells and in severe cases this culminates in pseudomembranous colitis, toxic megacolon and death. Vaccines in human trials have focused exclusively on the parenteral administration of toxin-based formulations. These vaccines promote toxin-neutralising serum antibodies but fail to confer protection from infection in the gut. An effective route to immunise against gut pathogens and stimulate a protective mucosal antibody response (secretory immunoglobulin A, IgA) at the infection site is the oral route. Additionally, oral immunisation generates systemic antibodies (IgG). Using this route, two different antigens were tested in the hamster model: The colonisation factor CD0873 and a TcdB fragment. Animals immunised with CD0873 generated a significantly higher titre of sIgA in intestinal fluid and IgG in serum compared to naive animals, which significantly inhibited the adherence of C. difficile to Caco-2 cells. Following challenge with a hypervirulent isolate, the CD0873-immunised group showed a mean increase of 80% in time to experimental endpoint compared to naïve animals. Survival and body condition correlated with bacterial clearance and reduced pathology in the cecum. Our findings advocate CD0873 as a promising oral vaccine candidate against C. difficile.

Mucosal vaccination aims to prevent infection mainly by inducing secretory IgA (sIgA) antibody, which neutralises pathogens and enterotoxins by blocking their attachment to epithelial cells. We previously demonstrated that encapsulated protein antigen CD0873 given orally to hamsters induces neutralising antibodies locally as well as systemically, affording partial protection against Clostridioides difficile infection. The aim of this study was to determine whether displaying CD0873 on liposomes, mimicking native presentation, would drive a stronger antibody response. The recombinant form we previously tested resembles the naturally cleaved lipoprotein commencing with a cysteine but lacking lipid modification. A synthetic lipid (DHPPA-Mal) was designed for conjugation of this protein via its N-terminal cysteine to the maleimide headgroup. DHPPA-Mal was first formulated with liposomes to produce MalLipo; then, CD0873 was conjugated to headgroups protruding from the outer envelope to generate CD0873-MalLipo. The immunogenicity of CD0873-MalLipo was compared to CD0873 in hamsters. Intestinal sIgA and CD0873-specific serum IgG were induced in all vaccinated animals; however, neutralising activity was greatest for the CD0873-MalLipo group. Our data hold great promise for development of a novel oral vaccine platform driving intestinal and systemic immune responses.