Mapping the Neutralizing Epitopes of Enterotoxigenic Escherichia coli K88 (F4) Fimbrial Adhesin and Major Subunit FaeG

Abstract

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

Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea

“…Wild-type porcine E. coli strains 3030-2 (K88ac/LT/STb) [27] and 8516 (F18ac) [5] were used as the K88 + and F18 + bacteria in antibody adherence inhibition assays. STb recombinant E. coli strain 8020 [27] and wild-type Stx2e + strain 9168 (F18/Stx2e) [5] Serum samples collected from each mouse before the immunization, and two weeks after the final booster, were examined for antibodies specific to K88 and F18 fimbriae, and toxins (LT, STa, STb and Stx2e) in ELISAs as described previously [20,23,24]. Briefly, 100 ng K88 or F18 fimbria, CT (Sigma), recombinant protein MBP-STx2e or MBP-STb, or 10 ng STaovalbumin conjugate was coated onto each well of 96-well Immulon 2HB plates (Thermo Fisher Scientific) at 37°C for 1 h, followed by overnight incubation at 4°C.…”

“…Plates were washed with PBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000; Sigma). OD650 values were read after exposure to TMB (3,3',5,5'tetranethylbenzidine) Microwell peroxidase substrate (KPL, Gaithersburg, MD) and converted into antibody titers (in log10) as previously described [20,23,24].…”

“…We also showed that antigenic domains of K88 major subunit FaeG and F18 adhesin subunit FedF carried by a LT toxoid induced antibodies inhibiting adherence K88-fimbria or F18-fimbrial ETEC and protecting pigs against K88-fimbrial ETEC diarrhea [21,22]. More recently, we mapped and identified the neutralizing epitopes of K88 fimbrial subunit FaeG [23], F18 fimbrial FedF subunit [24], and LT A subunit [17].…”

“…toxins and expressed recombinant MEFA proteins. Four neutralizing fimbrial epitopes, two from F18 minor subunit adhesin FedF (QPDATGSWYD, IPSSSGTLTCQAGT) [24] and two from K88 major subunit adhesin FaeG (GRTKEAFATP, PMKNAGGTKVGSVKVN) [23] representing K88 and F18 antigens, and four toxin epitopes, KKDLCEHY of STb toxin, QSYVSSLN of Stx2e A subunit, and two copies of STa toxoid (STaN11S) epitope domain CCELCCSPACAGCY were used to replace eight surface-exposed but less antigenic epitopes at the A1 domain of LT A subunit to construct PWD fimbria-toxin MEFA (PWD MEFA) gene.…”

“…porcine intestinal cell line. Pig intestinal cell line IPEC-J2, K88 + strain 3030-2, and F18 + strain 8516 were used to assess antibody adherence inhibition activities as previously described [21,23,24]. Briefly, overnight cultures of 3030-2 and 8516 bacteria were harvested by centrifugation (3000 X g for 5 min) and resuspended in 5 mL PBS.…”