Significance of Enterotoxigenic Escherichia coli (ETEC) Heat-Labile Toxin (LT) Enzymatic Subunit Epitopes in LT Enterotoxicity and Immunogenicity.

Enterotoxigenic (ETEC) strains producing heat-labile toxin (LT) and/or heat-stable toxin (STa) are a top cause of children's diarrhea and travelers' diarrhea. Holotoxin-structured GM-binding LT is a strong immunogen and an effective adjuvant, and can serve a carrier or a platform for multivalent vaccine development. However, the significance of peptide domains or epitopes of LT particularly enzymatic LT subunit in association with LT enterotoxicity and immunogenicity has not been characterized. In this study, we identified B-cell epitopes from LT subunit and examined epitopes for immunogenicity and association with LT enterotoxicity. Epitopes identified from LT subunit were individually fused to a modified chicken ovalbumin carrier protein, and each epitope-ovalbumin fusion was used to immunize mice. Data showed all 11 LT epitopes were immunogenic; epitope 7 (SPHPYEQEVSA) induced greater titers of anti-LT antibodies which neutralized LT enterotoxicity more effectively. To examine these epitopes for the significance in LT enterotoxicity, we constructed LT mutants by substituting each of 10 epitopes at the toxic A1 domain of LT subunit with a foreign epitope and examined LT mutants for enterotoxicity and GM-binding activity. Data showed that LT mutants exhibited no enterotoxicity but retained GM-binding activity. The results from this study indicated that while not all immunodominant LT epitopes were neutralizing, LT mutants with an individual epitope substituted lost enterotoxicity but retained GM-binding activity. These results provided additional information to understand LT immunogenicity and enterotoxicity and suggested the potential application of LT platform for multivalent vaccines against ETEC diarrhea and other diseases. No vaccine is licensed for enterotoxigenic (ETEC) strains, which remain a leading cause of diarrhea in children from developing countries and international travelers. GM-binding heat-labile toxin (LT) which is a key virulence factor of ETEC diarrhea is a strong vaccine antigen and a self-adjuvant. LT can also serve a backbone or platform for MEFA (multiepitope fusion antigen), a newly developed structural vaccinology technology, to present heterogeneous epitopes (by replacing LT epitopes) and to mimic epitope antigenicity for development of broadly protective vaccines. Data from this study identified neutralizing LT epitopes and demonstrated that substitution of LT epitopes eliminated LT enterotoxicity without altering GM-binding activity, suggesting LT is potentially a versatile MEFA platform to present heterogeneous epitopes for multivalent vaccines against ETEC and other pathogens.