Preclinical Characterization of Immunogenicity and Efficacy against Diarrhea from MecVax, a Multivalent Enterotoxigenic E. coli Vaccine Candidate.

There are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading cause of diarrhea for children in developing countries and international travelers. Virulence heterogeneity among strains and difficulties identifying safe antigens for protective antibodies against STa, a potent but poorly immunogenic heat-stable toxin which plays a key role in ETEC diarrhea, are challenges in ETEC vaccine development. To overcome these challenges, we applied a toxoid fusion strategy and a novel epitope- and structure-based multiepitope fusion antigen (MEFA) vaccinology platform to construct two chimeric multivalent proteins, toxoid fusion 3xSTaN12S-mnLTR192G/L211A and adhesin CFA/I/II/IV MEFA, and demonstrated that the proteins induced protective antibodies against STa and heat-labile toxin (LT) produced by all ETEC strains or the seven most important ETEC adhesins (CFA/I and CS1 to CS6) expressed by the ETEC strains causing 60 to 70% of diarrheal cases and moderate to severe cases. Combining two proteins, we prepared a protein-based multivalent ETEC vaccine, MecVax. MecVax was broadly immunogenic; mice and pigs intramuscularly immunized with MecVax developed no apparent adverse effects but had robust antibody responses to the target toxins and adhesins. Importantly, MecVax-induced antibodies were broadly protective, demonstrated by significant adherence inhibition against E. coli bacteria producing any of the seven adhesins and neutralization of STa and cholera toxin (CT) enterotoxicity. Moreover, MecVax protected against watery diarrhea and provided over 70% and 90% protection against any diarrhea from an STa-positive or an LT-positive ETEC strain in a pig challenge model. These results indicated that MecVax induces broadly protective antibodies and prevents diarrhea preclinically, signifying that MecVax is potentially an effective injectable vaccine for ETEC. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) bacteria are a top cause of children's diarrhea and travelers' diarrhea and are responsible for over 220 million diarrheal cases and more than 100,000 deaths annually. A safe and effective ETEC vaccine can significantly improve public health, particularly in developing countries. Data from this preclinical study showed that MecVax induces broadly protective antiadhesin and antitoxin antibodies, becoming the first ETEC vaccine candidate to induce protective antibodies inhibiting adherence of the seven most important ETEC adhesins and neutralizing the enterotoxicity of not only LT but also STa toxin. More importantly, MecVax is shown to protect against clinical diarrhea from STa-positive or LT-positive ETEC infection in a pig challenge model, recording protection from antibodies induced by the protein-based, injectable, subunit vaccine MecVax against ETEC diarrhea and perhaps the possibility of intramuscularly administered protein vaccines for protection against intestinal mucosal infection.

Enterotoxigenic Escherichia coli Adhesin-Toxoid Multiepitope Fusion Antigen CFA/I/II/IV-3xSTaN12S-mnLTG192G/L211A-Derived Antibodies Inhibit Adherence of Seven Adhesins, Neutralize Enterotoxicity of LT and STa Toxins, and Protect Piglets against Diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Vaccines inducing antibodies to broadly inhibit bacterial adherence and to neutralize toxin enterotoxicity are expected to be effective against ETEC-associated diarrhea. 6×His-tagged adhesin-toxoid fusion proteins were shown to induce neutralizing antibodies to several adhesins and LT and STa toxins (X. Ruan, D. A. Sack, W. Zhang, PLoS One 10:e0121623, 2015, https://doi.org/10.1371/journal.pone.0121623). However, antibodies derived from His-tagged CFA/I/II/IV-2xSTaA14Q-dmLT or CFA/I/II/IV-2xSTaN12S-dmLT protein were less effective in neutralizing STa enterotoxicity and were not evaluated in vivo for efficacy against ETEC diarrhea. Additionally, His-tagged proteins are considered less desirable for human vaccines. In this study, we produced a tagless adhesin-toxoid MEFA (multiepitope fusion antigen) protein, enhanced anti-STa immunogenicity by including a third copy of STa toxoid STaN12S, and examined antigen immunogenicity in a murine model. Moreover, we immunized pregnant pigs with the tagless adhesin-toxoid MEFA protein and evaluated passive antibody protection against STa+ or LT+ ETEC infection in a pig challenge model. Results showed that tagless adhesin-toxoid MEFA CFA/I/II/IV-3xSTaN12S-mnLTR192G/L211A induced broad antiadhesin and antitoxin antibody responses in the intraperitoneally immunized mice and the intramuscularly immunized pigs. Mouse and pig serum antibodies significantly inhibited adherence of seven colonization factor antigen (CFA) adhesins (CFA/I and CS1 to CS6) and effectively neutralized both toxins. More importantly, suckling piglets born to the immunized mothers acquired antibodies and were protected against STa+ ETEC and LT+ ETEC diarrhea. These results indicated that tagless CFA/I/II/IV-3xSTaN12S-mnLTR192G/L211A induced broadly protective antiadhesin and antitoxin antibodies and demonstrate that this adhesin-toxoid MEFA is a potential antigen for developing broadly protective ETEC vaccines.

Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) strains are the main cause of diarrhea in pigs. Pig diarrhea especially post-weaning diarrhea remains one of the most important swine diseases. ETEC bacterial fimbriae including K88, F18, 987P, K99 and F41 promote bacterial attachment to intestinal epithelial cells and facilitate ETEC colonization in pig small intestine. ETEC enterotoxins including heat-labile toxin (LT) and heat-stable toxins type Ia (porcine-type STa) and type II (STb) stimulate fluid hyper-secretion, leading to watery diarrhea. Blocking bacteria colonization and/or neutralizing enterotoxicity of ETEC toxins are considered effective prevention against ETEC diarrhea. In this study, we applied the MEFA (multiepitope fusion antigen) strategy to create toxoid MEFAs that carried antigenic elements of ETEC toxins, and examined for broad antitoxin immunogenicity in a murine model. By embedding STa toxoid STaP12F (NTFYCCELCCNFACAGCY), a STb epitope (KKDLCEHY), and an epitope of Stx2e A subunit (QSYVSSLN) into the A1 peptide of a monomeric LT toxoid (LTR192G), two toxoid MEFAs, 'LTR192G-STb-Stx2e-STaP12F' and 'LTR192G-STb-Stx2e-3xSTaP12F' which carried three copies of STaP12F, were constructed. Mice intraperitoneally immunized with each toxoid MEFA developed IgG antibodies to all four toxins. Induced antibodies showed in vitro neutralizing activities against LT, STa, STb and Stx2e toxins. Moreover, suckling piglets born by a gilt immunized with 'LTR192G-STb-Stx2e-3xSTaP12F' were protected when challenged with ETEC strains, whereas piglets born by a control gilt developed diarrhea. Results from this study showed that the toxoid MEFA induced broadly antitoxin antibodies, and suggested potential application of the toxoid MEFA for developing a broad-spectrum vaccine against ETEC diarrhea in pigs.

Enterotoxigenic Escherichia coli heat-stable toxin and heat-labile toxin toxoid fusion 3xSTaN12S-dmLT induces neutralizing anti-STa antibodies in subcutaneously immunized mice.

Enterotoxigenic Escherichia coli (ETEC) bacteria producing heat-stable toxin (STa) and/or heat-labile toxin (LT) are among top causes of children's diarrhea and travelers' diarrhea. Currently no vaccines are available for ETEC associated diarrhea. A major challenge in developing ETEC vaccines is the inability to stimulate protective antibodies against the key STa toxin that is potently toxic and also poorly immunogenic. A recent study suggested toxoid fusion 3xSTaN12S-dmLT, which consists of a monomer LT toxoid (LTR192G/L211A) and three copies of STa toxoid STaN12S, may represent an optimal immunogen inducing neutralizing antibodies against STa toxin [IAI 2014, 82(5):1823-32]. In this study, we immunized mice with this fusion protein following a different parenteral route and using different adjuvants to further characterize immunogenicity of this toxoid fusion. Data from this study showed that 3xSTaN12S-dmLT toxoid fusion induced neutralizing anti-STa antibodies in the mice following subcutaneous immunization, as effectively as in the mice under intraperitoneal route. Data also indicated that double mutant LT (dmLT) can be an effective adjuvant for this toxoid fusion in mice subcutaneous immunization. Results from this study affirmed that toxoid fusion 3xSTaN12S-dmLT induces neutralizing antibodies against STa toxin, suggesting this toxoid fusion is potentially a promising immunogen for ETEC vaccine development.

Antibodies derived from an enterotoxigenic Escherichia coli (ETEC) adhesin tip MEFA (multiepitope fusion antigen) against adherence of nine ETEC adhesins: CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA.

Diarrhea continues to be a leading cause of death in children younger than 5 years in developing countries. Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of children's diarrhea and travelers' diarrhea. ETEC bacteria initiate diarrheal disease by attaching to host receptors at epithelial cells and colonizing in small intestine. Therefore, preventing ETEC attachment has been considered the first line of defense against ETEC diarrhea. However, developing vaccines effectively against ETEC bacterial attachment encounters challenge because ETEC strains produce over 23 immunologically heterogeneous adhesins. In this study, we applied MEFA (multiepitope fusion antigen) approach to integrate epitopes from adhesin tips or adhesive subunits of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA adhesins and to construct an adhesin tip MEFA peptide. We then examined immunogenicity of this tip MEFA in mouse immunization, and assessed potential application of this tip MEFA for ETEC vaccine development. Data showed that mice intraperitoneally immunized with this adhesin tip MEFA developed IgG antibody responses to all nine ETEC adhesins. Moreover, ETEC and E. coli bacteria expressing these nine adhesins, after incubation with serum of the immunized mice, exhibited significant reduction in attachment to Caco-2 cells. These results indicated that anti-adhesin antibodies induced by this adhesin tip MEFA blocked adherence of the most important ETEC adhesins, suggesting this multivalent tip MEFA may be useful for developing a broadly protective anti-adhesin vaccine against ETEC diarrhea.

Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen) and a toxoid fusion of heat-stable toxin (STa) and heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC) retain broad anti-CFA and antitoxin antigenicity.

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent vaccine development.

Characterization of heat-stable (STa) toxoids of enterotoxigenic Escherichia coli fused to double mutant heat-labile toxin peptide in inducing neutralizing Anti-STa antibodies.

A long-standing challenge in developing vaccines against enterotoxigenic Escherichia coli (ETEC), the most common bacteria causing diarrhea in children of developing countries and travelers to these countries, is to protect against heat-stable toxin type Ib (STa or hSTa). STa and heat-labile toxin (LT) are virulence determinants in ETEC diarrhea. LT antigens are often used in vaccine development, but STa has not been included because of its poor immunogenicity and potent toxicity. Toxic STa is not safe for vaccines, but only STa possessing toxicity is believed to be able to induce neutralizing antibodies. However, recent studies demonstrated that nontoxic STa derivatives (toxoids), after being fused to an LT protein, induced neutralizing antibodies and suggested that different STa toxoids fused to an LT protein might exhibit different STa antigenic propensity. In this study, we selected 14 STa toxoids from a mini-STa toxoid library based on toxicity reduction and reactivity to anti-native STa antibodies, and genetically fused each toxoid to a monomeric double mutant LT (dmLT) peptide for 14 STa-toxoid-dmLT toxoid fusions. These toxoid fusions were used to immunize mice and were characterized for induction of anti-STa antibody response. The results showed that different STa toxoids (in fusions) varied greatly in anti-STa antigenicity. Among them, STaN12S, STaN12T, and STaA14H were the top toxoids in inducing anti-STa antibodies. In vitro neutralization assays indicated that antibodies induced by the 3×STaN12S-dmLT fusion antigen exhibited the greatest neutralizing activity against STa toxin. These results suggested 3×STaN12S-dmLT is a preferred fusion antigen to induce an anti-STa antibody response and provided long-awaited information for effective ETEC vaccine development.

Multiepitope fusion antigen induces broadly protective antibodies that prevent adherence of Escherichia coli strains expressing colonization factor antigen I (CFA/I), CFA/II, and CFA/IV.

Diarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens.

A multiepitope fusion antigen elicits neutralizing antibodies against enterotoxigenic Escherichia coli and homologous bovine viral diarrhea virus in vitro.

Diarrhea is one of the most important bovine diseases. Enterotoxigenic Escherichia coli (ETEC) and bovine viral diarrhea virus (BVDV) are the major causes of diarrhea in calves and cattle. ETEC expressing K99 (F5) fimbriae and heat-stable type Ia (STa) toxin are the leading bacteria causing calf diarrhea, and BVDV causes diarrhea and other clinical illnesses in cattle of all ages. It is reported that maternal immunization with K99 fimbrial antigens provides passive protection to calves against K99 fimbrial ETEC and that BVDV major structural protein E2 elicits antibodies neutralizing against BVDV viral infection. Vaccines inducing anti-K99 and anti-STa immunity would protect calves more effectively against ETEC diarrhea, and those also inducing anti-E2 neutralizing antibodies would protect calves and cattle against diarrhea caused by both ETEC and BVDV. In this study, we used the ETEC K99 major subunit FanC as a backbone, genetically embedded the STa toxoid STaP12F and the most-antigenic B-cell epitope and T-cell epitope predicted from the BVDV E2 glycoprotein into FanC for the multivalent antigen FanC-STa-E2, and examined immunogenicity of this multivalent antigen to assess vaccine potential against bovine diarrhea. Mice intraperitoneally (i.p.) immunized with this multivalent antigen developed anti-K99, anti-STa, and anti-BVDV antibodies. Moreover, elicited antibodies showed neutralization activities, as they inhibited adherence of K99 fimbrial E. coli, neutralized STa toxin, and prevented homologous BVDV viral infection in vitro. Results from this study suggest that this multiepitope fusion antigen can potentially be developed as a vaccine for broad protection against bovine diarrhea and that the multiepitope fusion strategy may be generally applied for multivalent vaccine development against heterogeneous pathogens.

Oral immunization of a live attenuated Escherichia coli strain expressing a holotoxin-structured adhesin-toxoid fusion (1FaeG-FedF-LTA2:5LTB) protected young pigs against enterotoxigenic E. coli (ETEC) infection.

ETEC strains expressing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of porcine post-weaning diarrhea (PWD). PWD continues causing significant economic losses to swine producers worldwide. Vaccines effectively protecting against PWD are needed. Our recent study revealed that a tripartite adhesin-toxin monomer (FaeG-FedF-LT(A2-B)) elicited protective antibodies. In this study, we constructed a new adhesin-toxoid fusion, expressed it as a 1A:5B holotoxin-structured antigen (1FaeG-FedF-LT(192A2):5LT(B)) in an avirulent Escherichia coli strain, and evaluated its vaccine potential in pig challenge studies. Piglets orally inoculated with this live strain showed no adverse effects but developed systemic and mucosal antibodies that neutralized cholera toxin and inhibited adherence of K88 and F18 fimbriae in vitro. Moreover, the immunized piglets, when were challenged with ETEC strain 3030-2 (K88ac/LT/STb), had significant fewer bacteria colonized at small intestines and did not develop diarrhea; whereas the control piglets developed severe diarrhea and died. These results indicated the 1FaeG-FedF-LT(192A2):5LT(B) fusion antigen induced protective antiadhesin and antitoxin immunity in pigs, and suggested a live attenuated vaccine can be potentially developed against porcine ETEC diarrhea. Additionally, presenting antigens in a holotoxin structure to target host local mucosal immunity can be used in vaccine development against other enteric diseases.

Flagella from F18+Escherichia coli play a role in adhesion to pig epithelial cell lines.

F18 fimbriae and toxins produced by F18 fimbriae-carrying Escherichia coli (E. coli) strains are known virulence factors responsible for post-weaning diarrhea (PWD) and edema disease (ED). In this study, we showed that fliC isogenic mutants constructed in two reference wild-type F18 fimbriae (F18+) E. coli were markedly impaired in adherence in vitro cell models (p < 0.05). Flagella purified from F18+E. coli could directly bind to cultured piglet epithelial cells and block adherence of F18+E. coli to cells when pre-incubated. In addition, the F18+E. coli fliC deletion mutants up-regulated the expression of type I fimbriae produced by F18+E. coli strains. These results demonstrated that expression of flagella is essential for the adherence of F18+E. coli in vitro.

Escherichia coli expressing EAST1 toxin did not cause an increase of cAMP or cGMP levels in cells, and no diarrhea in 5-day old gnotobiotic pigs.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we constructed E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation. CONCLUSION/SIGNIFICANCE: Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea.

The flagella of F18ab Escherichia coli is a virulence factor that contributes to infection in a IPEC-J2 cell model in vitro.

Bacterial flagella contribute to pathogen virulence; however, the role of flagella in the pathogenesis of F18ab E. coli-mediated swine edema disease (ED) is not currently known. We therefore evaluated the role of flagella in F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production using an in vitro cell infection model approach with gene-deletion mutant and complemented bacterial strains. We demonstrated that the flagellin-deficient fliC mutant had a marked decrease in the ability to adhere to and invade porcine epithelial IPEC-J2 cells. Surprisingly, there was no difference in adhesion between the F18 fimbriae-deficient ΔfedA mutant and its parent strain. In addition, both the ΔfedA and double ΔfliCΔfedA mutants exhibited an increased ability to invade IPEC-J2 cells compared to the wild-type strain, although this may be due to increased expression of other adhesins following the loss of F18ab fimbriae and flagella. Compared to the wild-type strain, the ΔfliC mutant showed significantly reduced ability to form biofilm, whereas the ΔfedA mutant increased biofilm formation. Although ΔfliC, ΔfedA, and ΔfliCΔfedA mutants had a reduced ability to stimulate IL-8 production from infected Caco-2 cells, the ΔfliC mutant impaired this ability to a greater extent than the ΔfedA mutant. The results from this study clearly demonstrate that flagella are required for efficient F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production in vitro.

A tripartite fusion, FaeG-FedF-LT(192)A2: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.

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.

Heat-labile- and heat-stable-toxoid fusions (LTR192G-STaP13F) of human enterotoxigenic Escherichia coli elicit neutralizing antitoxin antibodies.

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Adhesins and enterotoxins, including heat-labile (LT) and heat-stable (STa) toxins, are the key virulence factors. Antigenic adhesin and LT antigens have been used in developing vaccines against ETEC diarrhea. However, STa has not been included because of its poor immunogenicity and potent toxicity. Our recent study showed that porcine-type STa toxoids became immunogenic and elicited neutralizing anti-STa antibodies after being genetically fused to a full-length porcine-type LT toxoid, LT(R192G) (W. Zhang et al., Infect. Immun. 78:316-325, 2010). In this study, we mutated human-type LT and STa genes, which are highly homologous to porcine-type toxin genes, for a full-length LT toxoid (LT(R192)) and a full-length STa toxoid (STa(P13F)) and genetically fused them to produce LT192-STa13 toxoid fusions. Mice immunized with LT192-STa13 fusion antigens developed anti-LT and anti-STa IgG (in serum and feces) and IgA antibodies (in feces). Moreover, secretory IgA antibodies from immunized mice were shown to neutralize STa and cholera toxins in T-84 cells. In addition, we fused the STa13 toxoid at the N terminus and C terminus, between the A1 and A2 peptides, and between the A and B subunits of LT192 to obtain different fusions in order to explore strategies for enhancing STa immunogenicity. This study demonstrated that human-type LT192-STa13 fusions induce neutralizing antitoxin antibodies and provided important information for developing toxoid vaccines against human ETEC diarrhea.