Establishment and Validation of Pathogenic CS17+ and CS19+ Enterotoxigenic Escherichia coli Challenge Models in the New World Primate Aotus nancymaae.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrheal illness in the military, travelers, and children living in low- to middle-income countries. Increased antibiotic resistance, the absence of a licensed vaccine, and the lack of broadly practical therapeutics perpetuate the significant health and financial burden resulting from ETEC infection. A critical step in the evaluation of vaccines and therapeutics is preclinical screening in a relevant animal disease model that closely replicates human disease. We previously developed a diarrheal model of class 5a colonization factor (CF) CFA/I-expressing ETEC in the New World owl monkey species Aotus nancymaae using ETEC strain H10407. In order to broaden the use of the model, we report here on the development of A. nancymaae models of ETEC expressing the class 5b CFs CS17 and CS19 with strains LSN03-016011/A and WS0115A, respectively. For both models, we observed diarrheal attack rates of ≥80% after oral inoculation with 5 × 1011 CFU of bacteria. These models will aid in assessing the efficacy of future ETEC vaccine candidates and therapeutics.

Evaluation of the Immunogenicity and Protective Efficacy of an Enterotoxigenic Escherichia coli CFA/I Adhesin-Heat-Labile Toxin Chimera.

Recent efforts to develop an enterotoxigenic Escherichia coli (ETEC) vaccine have focused on the antigenically conserved tip adhesins of colonization factors. We showed previously that intranasal immunization with dsc19CfaE, a soluble variant of the in cis donor strand-complemented tip adhesin of a colonization factor of the class 5 family (CFA/I) fimbria, is highly immunogenic and protects against oral challenge with CFA/I-positive (CFA/I+) ETEC strain H10407 in the Aotus nancymaae nonhuman primate. We also reported a cholera toxin (CT)-like chimera (called dsc19CfaE-CTA2/CTB) in which the CTA1 domain of CT was replaced by dsc19CfaE that was strongly immunogenic when administered intranasally or orogastrically in mice. Here, we evaluate the immunogenicity and protective efficacy (PE) of a refined and more stable chimera comprised of a pentameric B subunit of ETEC heat-labile toxin (LTB) in lieu of the CTB pentamer and a donor strand truncation (dsc14) of CfaE. The refined chimera, dsc14CfaE-sCTA2/LTB, was highly immunogenic in mice when administered intranasally or intradermally, eliciting serum and fecal antibody responses against CfaE and LTB, as well as strong hemagglutination inhibition titers, a surrogate for neutralization of intestinal adhesion mediated by CfaE. Moreover, the chimera was safe and highly immunogenic when administered intradermally to guinea pigs. In A. nancymaae, intradermal (i.d.) immunization with chimera plus single-mutant heat-labile toxin [LT(R192G)] elicited strong serum anti-CfaE and anti-LTB antibody responses and conferred significant reduction of diarrhea compared to phosphate-buffered saline (PBS) controls (PE = 84.1%; P < 0.02). These data support the further evaluation of dsc14CfaE-sCTA2/LTB as an ETEC vaccine in humans.

Hyperimmune Bovine Colostral Anti-CS17 Antibodies Protect Against Enterotoxigenic Escherichia coli Diarrhea in a Randomized, Doubled-Blind, Placebo-Controlled Human Infection Model.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) commonly cause diarrhea in children living in developing countries and in travelers to those regions. ETEC are characterized by colonization factors (CFs) that mediate intestinal adherence. We assessed if bovine colostral IgG (bIgG) antibodies against a CF, CS17, or antibodies against CsbD, the minor tip subunit of CS17, would protect subjects against diarrhea following challenge with a CS17-expressing ETEC strain. METHODS: Adult subjects were randomized (1:1:1) to receive oral bIgG against CS17, CsbD, or placebo. Two days prior to challenge, subjects began dosing 3 times daily with the bIgG products (or placebo). On day 3, subjects ingested 5 × 109 cfu ETEC strain LSN03-016011/A in buffer. Subjects were assessed for diarrhea for 120 hours postchallenge. RESULTS: A total of 36 subjects began oral prophylaxis and 35 were challenged with ETEC. While 50.0% of the placebo recipients had watery diarrhea, none of the subjects receiving anti-CS17 had diarrhea (P = .01). In contrast, diarrhea rates between placebo and anti-CsbD recipients (41.7%) were comparable (P = 1.0). CONCLUSIONS: This is the first study to demonstrate anti-CS17 antibodies provide significant protection against ETEC expressing CS17. More research is needed to better understand why anti-CsbD was not comparably efficacious. Clinical Trials Registration. NCT00524004.

Establishment, Validation, and Application of a New World Primate Model of Enterotoxigenic Escherichia coli Disease for Vaccine Development.

The establishment of an animal model that closely approximates enterotoxigenic Escherichia coli (ETEC) disease in humans is critical for the development and evaluation of vaccines against this enteropathogen. Here, we evaluated the susceptibility of Aotus nancymaae, a New World monkey species, to ETEC infection. Animals were challenged orogastrically with 109 to 1011 CFU of the human pathogenic CFA/I+ ETEC strain H10407 and examined for evidence of diarrhea and fecal shedding of bacteria. A clear dose-range effect was obtained, with diarrheal attack rates of 40% to 80%, validated in a follow-on study demonstrating an attack rate of 80% with 1011 CFU of H10407 ETEC. To determine whether this model is an effective approach for assessing ETEC vaccine candidates, we used it to evaluate the ability of the donor strand-complemented CFA/I adhesin CfaE (dscCfaE) to protect against H10407 challenge. In a series of experiments, animals were intranasally vaccinated with dscCfaE alone, dscCfaE with either cholera toxin B-subunit (CTB) or heat-labile toxin (LTB), or phosphate-buffered saline (PBS) alone and then challenged with 1011 CFU of H10407. Control animals vaccinated with PBS had attack rates of 70 to 90% on challenge. Vaccination with dscCfaE, or dscCfaE admixed with CTB or LTB, resulted in a reduction of attack rates, with vaccine efficacies of 66.7% (P = 0.02), 77.7% (P = 0.006), and 42.9% (P = 0.370) to 83.3% (P = 0.041), respectively. In conclusion, we have shown the H10407 ETEC challenge of A. nancymaae to be an effective, reproducible model of ETEC disease, and importantly, we have demonstrated that in this model, vaccination with the prototype vaccine candidate dscCfaE is protective against CF-homologous disease.

Prophylactic Efficacy of Hyperimmune Bovine Colostral Antiadhesin Antibodies Against Enterotoxigenic Escherichia coli Diarrhea: A Randomized, Double-Blind, Placebo-Controlled, Phase 1 Trial.

Background: Tip-localized adhesive proteins of bacterial fimbriae from diverse pathogens confer protection in animal models, but efficacy in humans has not been reported. Enterotoxigenic Escherichia coli (ETEC) commonly elaborate colonization factors comprising a minor tip adhesin and major stalk-forming subunit. We assessed the efficacy of antiadhesin bovine colostral IgG (bIgG) antibodies against ETEC challenge in volunteers. Methods: Adults were randomly assigned (1:1:1) to take oral hyperimmune bIgG raised against CFA/I minor pilin subunit (CfaE) tip adhesin or colonization factor I (CFA/I) fimbraie (positive control) or placebo. Two days before challenge, volunteers began a thrice-daily, 7-day course of investigational product administered in sodium bicarbonate 15 minutes after each meal. On day 3, subjects drank 1 × 109 colony-forming units of colonization factor I (CFA/I)-ETEC strain H10407 with buffer. The primary efficacy endpoint was diarrhea within 120 hours of challenge. Results: After enrollment and randomization, 31 volunteers received product, underwent ETEC challenge, and were included in the per protocol efficacy analysis. Nine of 11 placebos developed diarrhea, 7 experiencing moderate to severe disease. Protective efficacy of 63% (P = .03) and 88% (P = .002) was observed in the antiadhesin bIgG and positive control groups, respectively. Conclusions: Oral administration of anti-CFA/I minor pilin subunit (CfaE) antibodies conferred significant protection against ETEC, providing the first clinical evidence that fimbrial tip adhesins function as protective antigens.

Volunteer challenge with enterotoxigenic Escherichia coli that express intestinal colonization factor fimbriae CS17 and CS19.

Human challenges with enterotoxigenic Escherichia coli (ETEC) have broadened our understanding of this important enteropathogen. We report findings from the first challenge studies using ETEC-expressing colonization factor fimbria CS17 and CS19. LSN03-016011/A (LT, CS17) elicited a dose-dependent effect, with the upper dose (6 × 10(9) organisms) causing diarrhea in 88% of recipients. WS0115A (LTSTp, CS19) also showed a dose response, with a 44% diarrhea rate at 9 × 10(9) organisms. Both strains elicited homologous antifimbrial and anti-LT antibody seroconversion. These studies establish the relative pathogenicity of ETEC expressing newer class 5 fimbriae and suggest suitability of the LT|CS17-ETEC challenge model for interventional trials.

Development and Comparison of a Panel of Modified CS17 Fimbrial Tip Adhesin Proteins as Components for an Adhesin-Based Vaccine against Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in travelers and children in resource-limited countries. ETEC colonization factors, fimbrial tip adhesins and enterotoxins are key virulence factors, and thus have been studied as vaccine candidates. Some prevalent colonization factors, including CFA/I and CS17, belong to the class 5 family. We previously found that passive oral administration of hyperimmune bovine colostral IgG (bIgG) raised against dscCfaE (donor strand complemented CFA/I tip adhesin) protected volunteers against CFA/I+ ETEC challenge, while anti-dscCsbD bIgG (CS17 tip adhesin) did not confer protection. These findings led us to develop and optimize a panel of alternative CsbD-based vaccine candidates based on allele matching and in silico protein engineering. Physicochemical characterizations revealed that an optimized vaccine candidate dscCsbDLSN139(P218A/G3) had the greatest thermal stability among the six tested dscCsbD adhesins, whereas the overall secondary structures and solubility of these adhesins had no obvious differences. Importantly, dscCsbDLSN139(P218A/G3) elicited significantly higher CS17+ ETEC hemagglutination inhibition titers in sera from mice intranasally immunized with the panel of dscCsbD adhesins, while no significant difference was observed among heterologous neutralizing titers. Our results strongly advocate for the incorporation of these modifications into a new generation of CsbD-based ETEC vaccine candidates.

Biochemical and immunological characterization of an ETEC CFA/I adhesin cholera toxin B subunit chimera.

Surface-expressed colonization factors and their subunits are promising candidates for inclusion into a multivalent vaccine targeting enterotoxigenic Escherichia coli (ETEC), a leading cause of acute bacterial diarrhea in developing regions. However, soluble antigens are often poorly immunogenic in the absence of an adjuvant. We show here that the serum immune response to CfaE, the adhesin of the ETEC colonization factor CFA/I, can be enhanced in BALB/c mice by immunization with a chimeric antigen containing CfaE and pentameric cholera toxin B subunit (CTB) of cholera toxin from Vibrio cholerae. We constructed this antigen by replacing the coding sequence for the A1 domain of the cholera toxin A subunit (CTA) with the sequence of donor strand complemented CfaE (dscCfaE) within the cholera toxin operon, resulting in a dscCfaE-CTA2 fusion. After expression, via non-covalent interactions between CTA2 and CTB, the fusion and CTB polypeptides assemble into a complex containing a single dscCfaE-CTA2 protein bound to pentameric CTB (dscCfaE-CTA2/CTB). This holotoxin-like chimera retained the GM1 ganglioside binding activity of CTB, as well as the ability of CfaE to mediate the agglutination of bovine red blood cells when adsorbed to polystyrene beads. When administered intranasally to mice, the presence of CTB in the chimera significantly increased the serum immune response to CfaE compared to dscCfaE alone, stimulating a response similar to that obtained with a matched admixture of dscCfaE and CTB. However, by the orogastric route, immunization with the chimera elicited a superior functional immune response compared to an equivalent admixture of dscCfaE and CTB, supporting further investigation of the chimera as an ETEC vaccine candidate.

Evaluation of transcutaneous immunization as a delivery route for an enterotoxigenic E. coli adhesin-based vaccine with CfaE, the colonization factor antigen 1 (CFA/I) tip adhesin.

dscCfaE is a recombinant form of the CFA/I tip adhesin CfaE, expressed by a large proportion of enterotoxigenic E. coli (ETEC). It is highly immunogenic by the intranasal route in mice and Aotus nancymaae, protective against challenge with CFA/I+ ETEC in an A. nancymaae challenge model, and antibodies to dscCfaE passively protect against CFA/I+ ETEC challenge in human volunteers. Here, we show that transcutaneous immunization (TCI) with dscCfaE in mice resulted in strong anti-CfaE IgG serum responses, with a clear dose-response effect. Co-administration with heat-labile enterotoxin (LT) resulted in enhanced immune responses over those elicited by dscCfaE alone and strong anti-LT antibody responses. The highest dose of dscCfaE administered transcutaneously with LT elicited strong HAI titers, a surrogate for the neutralization of intestinal adhesion. Fecal anti-adhesin IgG and IgA antibody responses were also induced. These findings support the feasibility of TCI for the application of an adhesin-toxin based ETEC vaccine.

Immunogenicity of a prototype enterotoxigenic Escherichia coli adhesin vaccine in mice and nonhuman primates.

Enterotoxigenic Escherichia coli (ETEC) are the most common cause of bacterial diarrhea in young children in developing countries and in travelers. Efforts to develop an ETEC vaccine have intensified in the past decade, and intestinal colonization factors (CFs) are somatic components of most investigational vaccines. CFA/I and related Class 5 fimbrial CFs feature a major stalk-forming subunit and a minor, antigenically conserved tip adhesin. We hypothesized that the tip adhesin is critical for stimulating antibodies that specifically inhibit ETEC attachment to the small intestine. To address this, we compared the capacity of donor strand complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, and CFA/I fimbriae to elicit anti-adhesive antibodies in mice, using hemagglutination inhibition (HAI) as proxy for neutralization of intestinal adhesion. When given with genetically attenuated heat-labile enterotoxin LTR192G as adjuvant by intranasal (IN) or orogastric (OG) vaccination, dscCfaE exceeded CFA/I fimbriae in eliciting serum HAI titers and anti-CfaE antibody titers. Based on these findings, we vaccinated Aotus nancymaae nonhuman primates (NHP) with dscCfaE alone or admixed with one of two adjuvants, LTR192G and cholera toxin B-subunit, by IN and OG administration. Only IN vaccination with dscCfaE with either adjuvant elicited substantial serum HAI titers and IgA and IgG anti-adhesin responses, with the latter detectable a year after vaccination. In conclusion, we have shown that dscCfaE elicits robust HAI and anti-adhesin antibody responses in both mice and NHPs when given with adjuvant by IN vaccination, encouraging further evaluation of an ETEC adhesin-based vaccine approach.

A Salmonella enterica serovar Typhi vaccine expressing Yersinia pestis F1 antigen on its surface provides protection against plague in mice.

A recombinant strain of attenuated Salmonella enterica serovar Typhi surface-expressing Yersinia pestis F1 antigen was generated by transforming strain BRD1116 (aroA aroC htrA) with plasmid pAH34L encoding the Y. pestis caf operon. BRD1116/pAH34L was stable in vitro and in vivo. An immunisation regimen of two intranasal doses of 1 x 10(8) cfu of BRD1116/pAH34L given intranasally to mice 7 days apart induced the strongest immune response compared to other regimens and protected 13 out of 20 mice from lethal challenge with Y. pestis. Intranasal immunisation of mice constitutes a model for oral immunisation with Salmonella vaccines in humans. Thus, the results demonstrate that attenuated strains of S. enterica serovar Typhi which express Y. pestis F1 antigen may be developed to provide an oral vaccine against plague suitable for use in humans.

Attenuated Salmonella enterica serovar Typhi expressing urease effectively immunizes mice against Helicobacter pylori challenge as part of a heterologous mucosal priming-parenteral boosting vaccination regimen.

Recombinant vaccine strains of Salmonella enterica serovar Typhi capable of expressing Helicobacter pylori urease were generated by transforming strains CVD908 and CVD908-htrA with a plasmid harboring the ureAB genes under the control of an in vivo-inducible promoter. The plasmid did not interfere with the ability of either strain to replicate and persist in human monocytic cells or with their transient colonization of mouse lungs. When administered to mice intranasally, both recombinant strains elicited antiurease immune responses skewed towards a Th1 phenotype. Vaccinated mice exhibited strong immunoglobulin G2a (IgG2a)-biased antiurease antibody responses as well as splenocyte populations capable of proliferation and gamma interferon (IFNgamma) secretion in response to urease stimulation. Boosting of mice with subcutaneous injection of urease plus alum enhanced immune responses and led them to a more balanced Th1/Th2 phenotype. Following parenteral boost, IgG1 and IgG2a antiurease antibody titers were raised significantly, and strong urease-specific splenocyte proliferative responses, accompanied by IFNgamma as well as interleukin-4 (IL-4), IL-5, and IL-10 secretion, were detected. Neither immunization with urease-expressing S. enterica serovar Typhi alone nor immunization with urease plus alum alone conferred protection against challenge with a mouse-adapted strain of H. pylori; however, a vaccination protocol combining both immunization regimens was protective. This is the first report of effective vaccination against H. pylori with a combined mucosal prime-parenteral boost regimen in which serovar Typhi vaccine strains are used as antigen carriers. The significance of these findings with regard to development of a human vaccine against H. pylori and modulation of immune responses by heterologous prime-boost immunization regimens is discussed.