Biosynthesis of a nanoscale Vibrio cholerae conjugate vaccine elicits potent immunity through size and receptor effects

Cholera, caused by Vibrio cholerae , remains a significant global health threat, with the O1 serotype being the predominant cause of recent global outbreaks. Although conjugate vaccines against V. cholerae have been developed via chemical methods, the feasibility of applying novel biosynthetic approaches remains uncertain due to the special structure of its O-polysaccharide, which should be recognized by oligosaccharyltransferases. Here, we successfully established an oligosaccharyltransferase PglL-based glycosylation system in an engineered V. cholerae strain to biosynthesize a nanoscale conjugate vaccine, NP-OPS Vc(I) . The carrier NP was designed by fusing the cholera toxin B subunit (CTB) with a trimer-forming protein (Tri), which could self‑assemble into a nanoparticle during expression. The resulting glycoprotein exhibited high purity, stability, and retained GM1-binding capacity. We found that the size of NP-OPS Vc(I) and its GM1 receptor targeting both contribute to a potent humoral immune response, while its efficient delivery is additionally associated with the induction of intestinal mucosal immunity. In mouse models, the NP-OPS Vc(I) demonstrated a favorable safety profile and elicited substantially stronger systemic and mucosal immune responses than conventional conjugates, providing great protection against lethal V. cholerae challenge even at a 10‑fold lower dose without requiring an adjuvant. Furthermore, the immune responses were confirmed in non-human primates, supporting its potential for clinical translation. This work not only developed a novel highly effective candidate vaccine against V. cholerae but also significantly expanded the application scope of the PglL-based platform technology, establishing it as a versatile and broadly applicable enzymatic toolkit for vaccine development.