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1). == FIG 1. antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide. KEYWORDS:enterotoxigenicEscherichia coli, FaeG, K88 (F4), neutralizing epitope, postweaning diarrhea, vaccine == ABSTRACT == EnterotoxigenicEscherichia coli(ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against numerous ETEC virulence determinants has confirmed challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we recognized B-cell immunodominant epitopesin silicofrom the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived JAK2-IN-4 from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell collection IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development. IMPORTANCEEnterotoxigenicEscherichia coli(ETEC) strains generating K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While standard vaccinology methods encounter troubles at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from numerous heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide. == INTRODUCTION == EnterotoxigenicEscherichia coli(ETEC) bacteria that express K88 (F4) or F18 fimbria and enterotoxins, including heat-labile toxin (LT), heat-stable toxin type I (STa), heat-stable toxin type II (STb), and Shiga toxin type 2e (Stx2e), are the primary cause of diarrhea in pigs (16). Porcine neonatal diarrhea is largely prevented by passive protection of maternal antibodies through immunization of pregnant sows. Postweaning diarrhea, however, is yet to be effectively controlled (4). Various preventive approaches, including feeding ETEC-specific JAK2-IN-4 antibody-containing materials and treatment with prebiotics, probiotics, or dietary supplements, were attempted but found either inconsistent or unviable commercially (4,7). JAK2-IN-4 Developing effective vaccines for postweaning diarrhea has encountered challenges JAK2-IN-4 as well. Difficulties include a very narrow windows for immunization, the cost of the vaccine and vaccination, and more importantly the need of cross protection against heterogeneous ETEC strains. Ideally, piglets JAK2-IN-4 are vaccinated when their maternal antibodies (against neonatal diarrhea) drop to certain levels. These antibody levels can still sufficiently protect against neonatal diarrhea but do not significantly interfere with a vaccine product from activation of antigen-specific active immunity. Pig neonatal diarrhea and postweaning diarrhea can be caused by the same ETEC pathogens or strains sharing virulence factors. To be guarded against postweaning diarrhea, piglets need to develop active immunity against ETEC contamination by the time of weaning and after they are weaned. Vaccines need to Rabbit polyclonal to ZNF484 be low cost and easy to administer. Furthermore, since.