A Tripartite Fusion, FaeG-FedF-LT₁₉₂A2:B, of Enterotoxigenic Escherichia coli (ETEC) Elicits Antibodies That Neutralize Cholera Toxin, Inhibit Adherence of K88 (F4) and F18 Fimbriae, and Protect Pigs against K88ac/Heat-Labile Toxin Infection

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT)and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT 192 ) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT 192 A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT 192 A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT 192 A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrial E. coli strains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea.

A Multiepitope Fusion Antigen Elicits Neutralizing Antibodies against Enterotoxigenic Escherichia coli and Homologous Bovine Viral Diarrhea VirusIn Vitro

“…Blood and fecal pellets were collected from each mouse before the immunization and weekly after each immunization. Fecal pellets were suspended in fecal reconstitution buffer supplemented with the protease inhibitor phenylmethylsulfonyl fluoride (Sigma) at a ratio of 1:6 (1 g feces to 5 ml buffer) (37). Fecal suspensions were centrifuged, and supernatants were collected.…”

“…To titrate anti-FanC antibodies, heat-extracted K99 fimbriae were used as ELISA coating antigens. K99 fimbriae were extracted based on methods previously described (37,39). Briefly, K99 fimbrial E. coli strain I297 bacteria, grown on sheep blood agar plates for 18 h at 37°C, were harvested and suspended gently in PBS.…”

“…Purified 6ϫHis-tagged protein was refolded using a protein-refolding kit by following the manufacturer's protocol (Novagen, Madison, WI). Refolded protein was dialyzed in 20 mM Tris-HCl buffer overnight at 4°C and was concentrated (to 1 to 2 mg/ml) using Spectra/Por molecular porous membrane tubing (Spectrum Laboratories Inc., Rancho Dominquez, CA) and polyethylene glycol compound (PEG; Sigma, St. Louis, MO) as previously described (24,37). Ten microliters of the refolded protein was analyzed in 10 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot assay.…”

“…As described previously (22,24,37), splice overlapping extension (SOE) PCRs using specifically designed PCR primers (Table 2) were conducted to generate the chimeric gene. Briefly, we first overlapped two PCR products, one amplified with primers FanCNheI-F and STa12/ FanC107-R and the other amplified with primers STa12/FanC91-F and FanCEagI-R, with pK99 plasmid as a DNA template, to replace nucleotides encoding amino acid residues 91 to 107 of FanC with those of STa P12F toxoid for a FanC-STa chimeric gene.…”

Characterization of Heat-Stable (STa) Toxoids of Enterotoxigenic Escherichia coli Fused to Double Mutant Heat-Labile Toxin Peptide in Inducing Neutralizing Anti-STa Antibodies

“…Two booster injections at the same dose as the primary injection but with Freund incomplete adjuvant followed at biweekly intervals. Serum and fecal suspension samples (1 g of feces was suspended in 5 ml of fecal reconstitution buffer [10 mM Tris, 100 mM NaCl, 0.05% Tween 20, 5 mM sodium azide, pH 7.4; 1:6 dilution]) (22,27) were collected from each mouse prior to immunization and 10 to 12 days after each immunization and were then stored at Ϫ80°C until use. On day 37, mice were anesthetized with CO 2 and exsanguinated.…”