Abstract

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. There is currently no vaccine licensed to prevent ETEC. Development of prophylaxis against ETEC is challenging due to the vast heterogeneity of the ETEC strains. The discovery of nanobodies has emerged as a successful new biologics in treating mucosal infectious disease as nanobodies can recognize conserved epitopes on hypervariable pathogens. In this study, we performed large screens using immunized llamas and a naive nanobody yeast display library against adhesins of colonization factors. Cross-protective nanobodies were selected with in vitro activities inhibiting mannose-resistant hemagglutination (MRHA) against all eleven major pathogenic ETEC strains. Oral administration of nanobodies led to significant reduction of bacterial colonization in animals challenged with multiple ETEC strains. Structural analysis revealed novel conserved epitopes as critical structural features for pan-ETEC vaccine design. Two of the lead nanobodies, 2R215 and 1D7, were further engineered as trimer or fused with human IgA Fc-fragments as fusionbodies. Oral administration of the trimers or fusionbodies protected mice from infection at a much lower dose compared to the monomeric format. Importantly, fusionbodies prevented infection as a pre-treatment when administrated 2 hours before ETEC challenge to the animals. Together, our study provides the first proof of concept that oral administration of a single nanobody could confer broad protection against major pathogenic ETEC strains. Technological advances in large-scale manufacturing of biological proteins in plants and microorganisms will make nanobody-based immunotherapy a potent and cost-effective prophylaxis or treatment for ETEC.