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Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic  Escherichia coli  (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTa A14Q -dmLT or 3xSTa N12S -dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STa A14Q -dmLT and CFA/I/II/IV-STa N12S -dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa -toxoid -dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTa N12S -dmLT. Moreover, induced antibodies showed  in vitro  adherence inhibition activities against ETEC or  E .  coli  strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa -toxoid -dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTa N12S -dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent vaccine development.

plos one

Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity