The forgotten pandemic: single-domain antibodies for equitable protection against diarrheal diseases

Diarrheal diseases claim the lives of millions of people every year, many of them children in low and middle income countries. During rainy season in these resource-limited settings, the water- and foodborne bacteria Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) can cause up to half of all diarrheal episodes. The pathogenic mechanism of these two pathogens is highly similar, due to the secretion of the genetically related and functionally comparable cholera toxin (CTX) of V. cholerae and heat-labile enterotoxin (LT) of ETEC. Oral cholera vaccines are available, but the success of mass vaccination campaigns is hindered by a global production shortage, their relative short-term protection, and challenges associated with distribution and administration. Interestingly, because of their similarity, the oral cholera vaccine containing the receptor-binding subunit of CTX remains the only solution with a documented cross-protective effect against LT⁺ ETEC. The purpose of the studies presented in this PhD thesis was to determine the potential role of VHHs, commonly referred to as single-domain antibodies, against CTX and LT in mitigating V. cholerae and ETEC pathogenicity.

In this PhD thesis, we demonstrate that an orally delivered bivalent V_HH construct able to specifically abrogate CTX–GM1 receptor interaction can significantly reduce cholera- associated diarrhea and V. cholerae colonization of the small intestine in an infant mouse model. Moreover, a previously characterized bivalent V_HH against LT is shown to hinder intestinal cell colonization of the prototypical human ETEC strain (H10407) in a flow chamber model simulating the human intestine. These findings provide insights into the molecular pathogenesis of V. cholerae and ETEC and indicates that precision blocking of a single virulence factor is sufficient to attenuate bacterial virulence. Their high target affinity, robust format, and efficient microbial expression could position V_HHs against CTX and LT as complementary tools against enteric disease available to the global at-risk population. In addition, these anti-toxin V_HH constructs could be utilized to elucidate the specific contribution of LT to long-term disorders, such as growth-stunting, observed among children suffering from recurrent ETEC infections.

Future studies should aim to determine the role of toxin-neutralizing V_HH constructs in reducing household transmission of V. cholerae and ETEC, as well as their potential ability to curtail antimicrobial resistance development in areas endemic to diarrheal disease. For V_HHs to significantly reduce the global risk of bacterial enteric infections, the development of standardized in vivo performance indicators, such as in situ target-neutralization efficiency, should be prioritized together with cost-effective antibody production.