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Mapping the neutralizing epitopes of F18 fimbrial adhesin subunit FedF of enterotoxigenic Escherichia coli (ETEC).

ABSTRACT: K88 and F18 fimbrial enterotoxigenic Escherichia coli (ETEC) are the major causes of post-weaning diarrhea (PWD) in pigs. A vaccine that induces broad immunity to prevent K88 and F18 fimbrial ETEC bacterial attachment and colonization in pig small intestines and to neutralize enterotoxin enterotoxicity would be effective for PWD. Structure-based multiepitope-fusion-antigen (MEFA) technology using a backbone immunogen to present neutralizing epitopes of representing virulence factors capacitates development of broadly protective ETEC vaccines. Neutralizing epitopes have been identified from K88 fimbrial adhesin (FaeG) and enterotoxins but not F18 fimbrial adhesin. In this study, we in silico identified immunodominant epitopes from F18ac fimbrial subunit FedF which plays a critical role in F18 fimbrial adherence, genetically fused each epitope to a carrier, examined immunogenicity of each epitope fusion, and determined epitope-derived antibodies neutralizing activities against F18 fimbrial adherence. Data showed that seven immune-dominant epitopes were identified from FedF subunit. Fused to heterologous human ETEC adhesin subunit CfaB, epitope fusions induced anti-F18 antibodies in subcutaneously immunized mice. Moreover, antibodies derived from each fusion significantly blocked adherence of a F18-fimbrial E. coli bacteria to pig intestinal cell line IPEC-J2. While all seven epitopes exhibited neutralizing activity, results from this study identified FedF epitopes #3 (IPSSSGTLTCQAGT) and #7 (QPDATGSWYD) the most effective for antibodies against F18 fimbrial adherence, and suggested their future application in PWD vaccine development.

SUBMITTER: Lu T

PROVIDER: S-EPMC7173344 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Mapping the neutralizing epitopes of F18 fimbrial adhesin subunit FedF of enterotoxigenic Escherichia coli (ETEC).

Lu Ti T Seo Hyesuk H Moxley Rodney A RA Zhang Weiping W

Veterinary microbiology 20190206

K88 and F18 fimbrial enterotoxigenic Escherichia coli (ETEC) are the major causes of post-weaning diarrhea (PWD) in pigs. A vaccine that induces broad immunity to prevent K88 and F18 fimbrial ETEC bacterial attachment and colonization in pig small intestines and to neutralize enterotoxin enterotoxicity would be effective for PWD. Structure-based multiepitope-fusion-antigen (MEFA) technology using a backbone immunogen to present neutralizing epitopes of representing virulence factors capacitates ...[more]

PMID: 30827385

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Project description:Enterotoxigenic Escherichia 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 various ETEC virulence determinants has proven 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 identified B-cell immunodominant epitopes in silico from 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 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 line 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.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing 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 conventional vaccinology approaches encounter difficulties 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 various 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.

Project description:Fimbriae mediate the initial adherence of enterotoxigenic Escherichia coli (ETEC) to the piglet small intestine and play an important role in development of ETEC-driven postweaning diarrhea (PWD). PWD inflicts huge economic losses on the swine industry each year, making development of alternative treatment and prevention measures for PWD essential. Vaccine candidates that induce antifimbria antibodies that block the initial attachment and colonization of ETEC pathogens with fimbriae are one approach that could help prevent PWD. In this study, we constructed two multiepitope fusion antigens (MEFAs) that carried, expressed, and displayed representative epitopes of F4, F5, F6, F18, and F41 ETEC fimbriae. These MEFAs used either the F4 major subunit FaeG or the F18 adhesive subunit FedF as a backbone. To assess the potential of these MEFAs as antifimbria vaccine candidates that could help prevent PWD, we generated computational models of the MEFAs, constructed them, and then tested their immunogenicity by using them to immunize mice. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface. We found that coadministration of our MEFAs in mice successfully induced five fimbria-specific antibodies in accordance with the epitopes included in the MEFA constructs. Furthermore, the induced antibodies can significantly inhibit the ability of ETEC strains that express F4, F5, F6, F18, and F41 fimbriae to adhere to piglet small intestinal IPEC-1 and IPEC-J2 cells. Our findings indicate that the antifimbria antibodies induced by our FaeG-Fim41a-FanC-FasA and FedF-FasA-Fim41a-FanC fimbria MEFAs blocked adherence of five ETEC fimbriae, suggesting these multivalent fimbria MEFAs may be useful for developing broadly protective antifimbria vaccines against PWD caused by ETEC infections.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC)-associated postweaning diarrhea (PWD) is still a leading disease in recently weaned piglets. Vaccination is considered to be the most ideal and efficacious strategy for preventing PWD. Recently, a commercialized live monovalent F4 oral vaccine and a bivalent F4/F18 oral vaccine have been demonstrated to effectively protect piglets in the F4-positive (F4+) and F18+ ETEC challenge models. However, they will not provide cross-protection against F5+, F6+, or F41+ ETEC-associated PWD cases, as they lack all five fimbria antigens. Thus, a multivalent vaccine containing all five ETEC fimbriae would be more effective in preventing ETEC-driven PWD. In this study, we designed two fimbria-targeted MEFAs using the MEFA technology, and further study demonstrated that these coadministered MEFAs in mice can induce protective antibodies against the five fimbriae expressed by ETEC. These MEFAs could be used as an efficient PWD vaccine candidate; furthermore, MEFA-based structural technology provides an alternative and promising strategy for the development of vaccines against pathogens with heterogeneous virulence factors.

Project description:F18-fimbriated Escherichia coli are associated with porcine postweaning diarrhea and edema disease. Adhesion of F18-fimbriated bacteria to the small intestine of susceptible pigs is mediated by the minor fimbrial subunit FedF. However, the target cell receptor for FedF has remained unidentified. Here we report that F18-fimbriated E. coli selectively interact with glycosphingolipids having blood group ABH determinants on type 1 core, and blood group A type 4 heptaglycosylceramide. The minimal binding epitope was identified as the blood group H type 1 determinant (Fucalpha2Galbeta3GlcNAc), while an optimal binding epitope was created by addition of the terminal alpha3-linked galactose or N-acetylgalactosamine of the blood group B type 1 determinant (Galalpha3(Fucalpha2)Galbeta3GlcNAc) and the blood group A type 1 determinant (GalNAcalpha3(Fucalpha2)-Galbeta3GlcNAc). To assess the role of glycosphingolipid recognition by F18-fimbriated E. coli in target tissue adherence, F18-binding glycosphingolipids were isolated from the small intestinal epithelium of blood group O and A pigs and characterized by mass spectrometry and proton NMR. The only glycosphingolipid with F18-binding activity of the blood group O pig was an H type 1 pentaglycosylceramide (Fucalpha2Galbeta3GlcNAc-beta3Galbeta4Glcbeta1Cer). In contrast, the blood group A pig had a number of F18-binding glycosphingolipids, characterized as A type 1 hexaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer), A type 4 heptaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GalNAcbeta3Galalpha4Galbeta4Glcbeta1Cer), A type 1 octaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer), and repetitive A type 1 nonaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GalNAcalpha3-(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer). No blood group antigen-carrying glycosphingolipids were recognized by a mutant E. coli strain with deletion of the FedF adhesin, demonstrating that FedF is the structural element mediating binding of F18-fimbriated bacteria to blood group ABH determinants.

Project description:Enterotoxigenic Escherichia coli (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 GM1-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 LTA subunit in association with LT enterotoxicity and immunogenicity has not been characterized. In this study, we identified B-cell epitopes in silico from LTA subunit and examined epitopes for immunogenicity and association with LT enterotoxicity. Epitopes identified from LTA 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 LTA epitopes 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 toxic A1 domain of LTA subunit with a 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 immunodominant LTA epitopes 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.IMPORTANCE No vaccine is licensed for enterotoxigenic Escherichia 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 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 GM1-binding activity, suggesting LT is potentially a versatile MEFA platform to present heterogeneous epitopes for multivalent vaccines against ETEC and other pathogens.

Dataset Information

Mapping the neutralizing epitopes of F18 fimbrial adhesin subunit FedF of enterotoxigenic Escherichia coli (ETEC).

Publications

Mapping the neutralizing epitopes of F18 fimbrial adhesin subunit FedF of enterotoxigenic Escherichia coli (ETEC).

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