ENGINEERING AND PRECLINICAL EVALUATION OF DRIED FOOD-FORMULATED YEAST-SECRETED ANTIBODIES AS PERI-EXPOSURE PROPHYLAXIS AGAINST CLOSTRIDIOIDES DIFFICILE DISEASE

Clostridioides difficile gastrointestinal infection (CDI) causes significant disease burden, often following gut dysbiosis induced by antibiotic treatment in at-risk individuals. A non-invasive, easy to use targeted prophylaxis in such cases of acute elevated CDI risk, especially in those with a history of recurrent CDI, would prevent much suffering. In this study, we envisioned a simple prophylactic intervention for CDI enabled by the oral administration of yeast-secreted dried food-formulated C. difficile toxin A (TcdA)-and B (TcdB)-neutralizing VHH-based antibodies. We engineered two antibody versions: a previously reported tetravalent VHH (VHH ₄ ) comprising dual specificities for TcdA and TcdB, and its murine IgA Fc fusion counterpart (VHH ₄ -mIgA Fc). We explored Generally Recognized as Safe (GRAS) Komagataella phaffii yeast as a low-cost, highly scalable production platform. In a first experiment, we produced the antibodies at small scale in shake flask yeast cultivations and formulated the secreted antibody-containing growth medium into standard-composition dried mouse feed pellets. The antibody food pellet-administration intervention was tested in a C. difficile spore-challenged mouse model in which CDI leads to 50% mortality of untreated animals. We started the antibody-feed prophylaxis dosed at 10 nmol/g pellet one day prior to C. difficile challenge and continued until the infection was cleared with ad libitum feeding. We found that the VHH ₄ feed almost completely prevented mortality and reduced disease-related weight loss. Despite its higher in vitro potency against TcdA and equipotent potency against TcdB, the VHH ₄ -mIgA Fc fusion did not confer additional in vivo benefit, and further work focused on the simpler VHH ₄ format. Scalable high-cell-density bioreactor production of VHH ₄ yielded equally effective prophylactic formulations at the same dose of 10 nmol/g pellet, achieving complete survival and reduced disease burden. Our findings suggest that, with further antibody- and process development, oral prophylaxis with scalable, cost-effective, yeast-produced, dried food-formulated anti-toxin VHHs could offer a simple non-invasive, non-antibiotic prophylaxis option for the high-risk (r)CDI patient population.