A Polyvalent Adhesin–Toxoid Multiepitope-Fusion-Antigen-Induced Functional Antibodies against Five Enterotoxigenic Escherichia coli Adhesins (CS7, CS12, CS14, CS17, and CS21) but Not Enterotoxins (LT and STa)

Abstract

The increasing prevalence and association with moderate-to-severe diarrhea make enterotoxigenic Escherichia coli (ETEC) adhesins CS7, CS12, CS14, CS17, and CS21 potential targets of ETEC vaccines. Currently, there are no vaccines licensed to protect against ETEC, a top cause of children’s diarrhea and travelers’ diarrhea. Recently, a polyvalent adhesin protein (adhesin MEFA-II) was demonstrated to induce antibodies that inhibited adherence from these five ETEC adhesins and reduced the enterotoxicity of ETEC heat-stable toxin (STa), which plays a key role in causing ETEC-associated diarrhea. To improve adhesin MEFA-II for functional antibodies against STa toxin and the other ETEC toxin, heat-labile toxin (LT), we modified adhesin MEFA-II by adding another STa toxoid and an LT epitope; we examined the new antigen immunogenicity (to five adhesins and two toxins) and more importantly antibody functions against ETEC adherence and STa and LT enterotoxicity. Data show that mice intramuscularly immunized with the new antigen (adhesin MEFA-IIb) developed robust IgG responses to the targeted adhesins (CS7, CS12, CS14, CS17, and CS21) and toxins (STa and LT). Mouse antibodies inhibited the adherence of ETEC strains expressing any of these five adhesins but failed to neutralize STa or LT enterotoxicity. In further studies, rabbits intramuscularly immunized with adhesin MEFA-IIb developed robust antigen-specific antibodies; when challenged with an ETEC isolate expressing CS21 adhesin (JF2101, CS21, and STa), the immunized rabbits showed a significant reduction in intestinal colonization by ETEC bacteria. These data indicate that adhesin MEFA-IIb is broadly immunogenic and induces functional antibodies against the targeted ETEC adhesins but not the toxins.

A Novel Multiepitope Fusion Antigen as a Vaccine Candidate for the Prevention of Enterotoxigenic E. coli-Induced Calf Diarrhea

“…Therefore, the level of cGMP can serve as a direct indicator of the extent of STa stimulation on cells. Notably, the stimulation of HCT-8 cells was significantly decreased when STa toxin was neutralized by anti-MEFA serum, which is in accordance with a previous study on STa demonstrating that toxoid epitopes elicited modest antibody level and substantial neutralizing activity against STa toxin [18]. Moreover, the inhibitory effect of anti-MEFA serum on the stimulation of the STa + ETEC strain (Figure 5) was significantly weaker than that of STa toxin stimulation.…”

“…Adhesion and adhesion inhibition experiments were performed using F17 + , F5 + , and F41 + ETEC strains. Following the method described previously [18], HCT-8 cells (1 × 10 5 /well) were inoculated into 12-well tissue culture plates containing Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. F17 + , F5 + , and F41 + ETEC strains were cultured overnight at 37 • C, and then the bacterial solution was counted using a plate and added to each well after treatment.…”

“…A previous study has reported that a vaccine utilizing a recombinant adenoviral vector expressing the STa toxin with ETEC F5 fimbrial adhesin could effectively stimulate the production of antibodies targeting both the F5 fimbriae and STa [17]. Additionally, it has been observed that the fusion of STa toxoid epitopes with immunogenic protein subunits, such as the FanC subunit of F5 fimbriae or CfaB subunit of CFA adhesin, can also elicit the production of antibodies against the STa toxin [18,19]. However, no research has been reported on the potential of fusing F17 fimbriae with the STa enterotoxin as a potential vaccine candidate.…”

Efficacy Evaluation of an Intradermally Delivered Enterotoxigenic Escherichia coli CF Antigen I Fimbrial Tip Adhesin Vaccine Coadministered with Heat-Labile Enterotoxin with LT(R192G) against Experimental Challenge with Enterotoxigenic E. coli H10407 in Healthy Adult Volunteers

“…While there was not a significant reduction in the rates of moderate-to-severe diarrhea in vaccinees, the consistent and significant observed reduction in disease severity and loose stool output is encouraging and supports the continued development of a subunit, parenterally administered ETEC vaccine. Additional research studies utilizing other potential components of a multivalent vaccine have already been described [ 35 , 36 , 37 , 38 , 39 ], one of which has advanced to a Phase 1 clinical trial [ 40 ]. Presuming that the CS6-based component, CssBA, reduces the ETEC-attributable disease upon challenge with a CS6-expressing strain, a conceivable pathway towards the development of a multivalent subunit becomes more apparent.…”