However, if LT is also used as an antigen for ETEC vaccine development, epitopes 7, 9, 11, and/or 3 need to be retained to induce LTA1-specific antibodies to neutralize LT enterotoxicity. without altering GM1-binding activity, suggesting LT is usually potentially a versatile MEFA platform to present heterogeneous epitopes for multivalent vaccines against ETEC and other pathogens. KEYWORDS:ETEC (enterotoxigenicEscherichia coli), LT (heat-labile toxin), epitope, vaccine platform == ABSTRACT == EnterotoxigenicEscherichia coli(ETEC) strains generating heat-labile toxin (LT) and/or heat-stable toxin (STa) are a top cause of children’s diarrhea and travelers’ diarrhea. Holotoxin-structured GM1-binding LT is usually a strong immunogen KB-R7943 mesylate 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 LTAsubunit in association with LT enterotoxicity and immunogenicity has not been characterized. In this study, we recognized B-cell epitopesin silicofrom LTAsubunit and examined epitopes for immunogenicity and association with LT enterotoxicity. Epitopes recognized from LTAsubunit were individually fused to a altered poultry ovalbumin carrier protein, and each epitope-ovalbumin fusion was used to immunize mice. Data showed all 11 LTAepitopes were immunogenic; epitope 7 (105SPHPYEQEVSA115) 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 harmful A1 domain name of LTAsubunit with a KB-R7943 mesylate foreign epitope and examined LT mutants for enterotoxicity and GM1-binding activity. Data showed that LT mutants exhibited no enterotoxicity but retained GM1-binding activity. The results from this study indicated that while not all KB-R7943 mesylate immunodominant LTAepitopes were neutralizing, LT mutants with an individual epitope substituted lost enterotoxicity but retained GM1-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. IMPORTANCENo vaccine is usually licensed for enterotoxigenicEscherichia coli(ETEC) strains, which remain a leading cause of diarrhea in children from developing countries and international travelers. GM1-binding heat-labile toxin (LT) which is a key virulence factor of ETEC diarrhea is usually a KB-R7943 mesylate 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 SFN study recognized neutralizing LT epitopes and exhibited that substitution of LT epitopes eliminated LT enterotoxicity without altering GM1-binding activity, suggesting LT is usually potentially a versatile MEFA platform to present heterogeneous epitopes for multivalent vaccines against ETEC and other pathogens. == INTRODUCTION == EnterotoxigenicEscherichia coli(ETEC) strains continue to be a leading cause of children’s diarrhea and travelers’ diarrhea (14). Currently, there KB-R7943 mesylate is no vaccine licensed for ETEC diarrhea (47). ETEC bacteria produce colonization factor antigen (CFA) or coli surface antigen (CS) adhesins and enterotoxins. CFA and CS adhesins facilitate ETEC bacteria to attach to host cell receptors and to colonize small intestines; enterotoxins, including heat-labile toxin (LT) and heat-stable type 1b toxin (STa), elevate intracellular cyclic AMP or cGMP levels in host epithelial cells to cause water and fluid hypersecretion and watery diarrhea (810). LT (LT-I) produced by ETEC is usually a typical AB5holotoxin and binds GM1receptor (9,1113). The enzymatic A subunit (LTA) consists of an A1 domain name and an A2 domain name. The A1 peptide is usually ADP-ribosylating; the A2 peptide noncovalently associates the A subunit to the GM1-binding B subunit (LTB) pentamer to form the AB5holotoxin structure. LT, detoxified LT mutants, and the nontoxic B subunit are strong immunogens to induce anti-LT antibodies; thus, they are often targeted as antigens for ETEC vaccine development (1416). Interestingly, LT and LT mutants are also effective mucosal and parenteral adjuvants (1723). In addition, LT, LTB, and derivatives can serve carrier proteins to facilitate or to up-immunoregulate immunogenicity of transporting proteins and peptides (2430). Recently, MEFA (multiepitope fusion antigen) technology has been developed to design structure-based multivalent vaccines (3135). Aided with protein computational modeling and molecular dynamics activation (35), this MEFA technology applies a special backbone protein to present multiple foreign epitopes and to mimic epitope native antigenicity, thus having a single.