Sr. Research Scientist UMass Chan Med. Sch., Massachusetts, United States
Disclosure(s):
Gaurav Chauhan, PhD: No financial relationships to disclose
Introduction/Rationale: Enterotoxigenic Escherichia coli (ETEC) remains a major cause of diarrheal illness in low- and middle-income countries and among travelers to endemic regions. The antigenic diversity of ETEC colonization factors (CFs) has hindered the development of broadly protective vaccines or therapeutics. Passive immunization with antibodies offers a promising strategy for rapid protection; however, conventional IgG antibodies lack stability in the gastrointestinal tract. To address this limitation, we designed a bispecific single-domain antibody (VHH) fused to an IgA backbone to enhance mucosal stability and broaden strain coverage.
Methods: Llamas were immunized with a panel of ETEC CFs, and two lead VHHs (V1 and V2) were identified based on cross-reactivity and binding kinetics using ELISA and BLI. V1 and V2 were fused to a human IgA backbone to generate a bispecific VHH-IgA antibody. Functional binding to purified CFs and native ETEC cells was evaluated by ELISA and flow cytometry. The ability of the bispecific antibody to block bacterial adhesion to intestinal epithelial cells was assessed using a Caco-2 cell adhesion assay.
Results: V1 and V2 collectively showed strong binding to ten CFs by ELISA, with partial gaps in coverage for two CFs by each antibody alone. The bispecific VHH-IgA antibody retained high-affinity binding to all ten CFs and to ETEC strains expressing them, as confirmed by ELISA and flow cytometry respectively. It effectively inhibited ETEC adhesion to Caco-2 cells by 70–90%, demonstrating enhanced breadth and functional potency.
Conclusion: We report the design and functional characterization of a bispecific VHH-IgA fusion antibody that provides broad binding and potent inhibition of ETEC adherence across multiple CFs. By combining the cross-reactivity of nanobodies with the mucosal resilience of IgA, this platform represents a promising prophylactic strategy for the prevention of ETEC infection and may be adaptable to other enteric pathogens.
