Over-expression of major colonization factors of enterotoxigenic Escherichia coli, alone or together,on non-toxigenic E. coli bacteria

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveller's diarrhea. Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. We have recently described the development of recombinant strains over-expressing CFA/I; the most prevalent CF among human clinical ETEC isolates. Here, non-toxigenic recombinant E. coli strains over-expressing Coli surface antigen 2 (CS2), CS4, CS5, and CS6, either alone, or each in combination with CFA/I were constructed by cloning the genes required for expression and assembly of each CF into expression vectors harboring a strong promoter. Immunological assays showed that recombinant strains expressing single CFs produced those in significantly larger amounts than did corresponding naturally high producing reference strains. Recombinant strains co-expressing CFA/I together with another CF also expressed significantly larger amounts of both CFs compared with the corresponding references strains. Further, when tested in mice, oral immunization with formalin-killed recombinant bacteria co-expressing one such double-expression CF pair, CFA/I + CS2, induced specific serum IgG + IgM and fecal IgA antibody responses against both CFs exceeding the responses induced by immunizations with natural reference strains expressing CFA/I and CS2, respectively. We conclude that the described type of recombinant bacteria over-expressing major CFs of ETEC, alone or in combination, may be useful as candidate strains for use in an oral whole-cell CF-ETEC vaccine.     

Expression of enterotoxigenic Escherichia coli colonization factors in Vibrio cholerae

As a first step towards a vaccine against diarrhoeal disease caused by enterotoxigenic Escherichia coli (ETEC), we have studied the expression of several ETEC antigens in the live attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Colonization factors (CF) CFA/I, CS3, and CS6 were expressed at the surface of V. cholerae CVD 103-HgR. Both CFA/I and CS3 required the co-expression of a positive regulator for expression, while CS6 was expressed without regulation. Up-regulation of CF expression in V. cholerae was very efficient, so that high amounts of CFA/I and CS3 similar to those in wild-type ETEC were synthesized from chromosomally integrated CF and positive regulator loci. Increasing either the operon and/or the positive regulator gene dosage resulted in only a small increase in CFA/I and CS3 expression. In contrast, the level of expression of the non-regulated CS6 fimbriae appeared to be more dependent on gene dosage. While CF expression in wild-type ETEC is known to be tightly thermoregulated and medium dependent, it seems to be less stringent in V. cholerae. Finally, co-expression of two or three CFs in the same strain was efficient even under the control of one single regulator gene.     

Construction of non-toxic Escherichia coli and Vibrio cholerae strains expressing high and immunogenic levels of enterotoxigenic E. coli colonization factor I fimbriae

To express high quantities of colonization factor antigen I (CFA/I) derived from enterotoxigenic Escherichia coli (ETEC) for use in ETEC vaccines, the entire CFA/I operon consisting of four genes (cfa-A, -B, -C, -E) was cloned into plasmid expression vectors that could be maintained either with or without antibiotic selection. Expression from the powerful tac promoter was under the control of the lacIq repressor present on the plasmids. Fimbriae were expressed on the surface of both a non-toxigenic E. coli K12 strain and a non-toxigenic strain of Vibrio cholerae following induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). It was found that the recombinant E. coli strains expressed up to 16-fold higher levels of CFA/I fimbriae compared to a reference strain which had previously been shown to be among the highest natural producers of the CFA/I fimbriae among tested wild type ETEC strains. Oral immunization with formalin-killed recombinant E. coli bacteria over-expressing CFA/I induced significantly higher serum IgA and IgG+M antibodies responses compared to the reference strain. Oral immunization with formalin-killed recombinant V. cholerae bacteria also induce strong CFA/I-specific serum IgA and IgG+M responses. We conclude that our constructs may be useful as candidate strains in an oral killed CF-ETEC vaccine.     

Mucosal immunization of BALB/c mice using enterotoxigenic Escherichia coli colonization factors CFA/I and CS6 administered with and without a mutant heat-labile enterotoxin

Mice (BALB/c) were intranasally (IN) and intragastrically (IG) administered the ETEC colonization factors (CF), CFA/I and CS6, with and without the R192G mutant heat-labile enterotoxin (mLT), and immunogenicity and efficacy measured. The IN administration of CFA/I to mice induced strong serum and fecal IgG and IgA responses. The IG administration of CFA/I to mice induced serum IgG and fecal IgA responses, but only when mLT was co-administered with CFA/I were serum IgA titers detected. The IN administration of CS6 to mice induced serum IgG antibodies, and mLT, when co-administered with CS6, enhanced the serum IgG response. Only when the mLT was co-administered with CS6, were serum and fecal IgA responses detected. The IG administration of CS6 plus mLT induced serum IgG and fecal IgA responses. Partial protection against lethal challenge with ETEC strain H10407 was seen in the mice IN administered the CFA/I plus mLT (P<0.01), and H10407 was cleared from the lungs of CFA/I plus mLT-immunized mice at a significantly greater rate than from the control mice (P<0.05). CFA/I and CS6 administered IN and IG induced mixed Th1/Th2 immune responses with the Th2 type being predominant as evidenced by IgG1>IgG2a. The administration of colonization factors to mice, particularly by the IN route, potentially serves as a useful way to measure the serum and mucosal immune responses to these antigens prior to their use in volunteers.     

Construction of a non-toxigenic Escherichia coli oral vaccine strain expressing large amounts of CS6 and inducing strong intestinal and serum anti-CS6 antibody responses in mice

Coli surface antigen 6 (CS6) is one of the most prevalent non-fimbrial colonization factors (CFs) of enterotoxigenic Escherichia coli (ETEC) bacteria, which are the most common cause of diarrhea among infants and children in developing countries. Since immune protection against ETEC is mainly mediated by locally produced IgA antibodies in the gut, much effort is focused on the development of an oral CF-based vaccine. Previous work has described the preparation of candidate E. coli vaccine strains expressing immunogenic amounts of fimbrial CF antigens such as CFA/I and CS2, which are retained after formalin treatment. However, attempts to generate E. coli expressing immunogenic amounts of CS6 and to preserve the immunological activity of the CS6 protein in a killed whole-cell vaccine have failed until now. Here we describe the construction of a recombinant non-toxigenic E. coli strain, with thyA as a non-antibiotic-based selection, which expresses large amounts of CS6 antigen on the bacterial surface, and show that phenol inactivation of the bacteria does not destroy the CS6 antigen properties. Oral immunization of mice with such phenol-killed CS6 over-expressing E. coli bacteria induced strong fecal and intestinal IgA and serum IgG + IgM antibody responses to CS6 that exceeded the responses induced by an ETEC reference strain naturally expressing CS6 and previously used as a vaccine strain. Our data indicate that the described phenol-inactivated non-toxigenic and CS6 over-expressing E. coli strain may be a useful component in an oral ETEC vaccine.     

DNA immunisation against the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC)

The CFA/I fimbria promotes the attachment of enterotoxigenic Escherichia coli (ETEC) to the surface of human enterocytes. The generation of a protective immune response requires the induction of antibodies able to block the CFA/I-mediated binding of ETEC to receptors located on the small intestine epithelium or on the surface of human red blood cells, in hemagglutination tests. An eukaryotic expression plasmid, pBLCFA, encoding the CFA/I gene under the control of the human cytomegalovirus major immediate-early promoter was constructed as a prototype DNA vaccine against ETEC. pBLCFA-tranfected BHK-21 cells secreted a peptide cross-reacting with a monoclonal antibody raised against CFA/I subunits. BALB/c mice immunized intramuscularly with one or two doses of purified pBLCFA developed CFA/I-specific serum antibodies for at least 52 weeks, composed predominantly of the IgG1 subclass. pBLCFA-induced antibodies bind mainly to epitopes exposed on the surface of intact CFA/I fimbriae and do not react with immune recessive epitopes found in other ETEC fimbra sharing amino acid homologies with CFA/I. Furthermore, pBLCFA-induced antibodies were able to block the adhesive properties of the CFA/I fimbriae, as evaluated by the ability to inhibit the hemagglutination promoted by CFA/I-expressing ETEC cells. These results suggest that secretion of CFA/I encoded by pBLCFA preserves important conformational epitopes required for the generation of protective antibodies against the adhesive properties of the CFA/I fimbriae and open new perspectives for the development of DNA vaccines against enteric bacterial pathogens.     

Immune responses elicited against multiple enterotoxigenic Escherichia coli fimbriae and mutant LT expressed in attenuated Shigella vaccine strains

Shigella and enterotoxigenic Escherichia coli (ETEC) continue to be important causes of diarrheal disease in infants and young children in developing countries and are major etiologic agents of traveler's diarrhea. Since attenuated strains of Shigella have been developed as live oral vaccines against shigellosis, we have adapted these attenuated Shigella strains to serve as carriers of ETEC antigens, thereby constituting a hybrid vaccine. Since protective immunity against ETEC is largely directed against fimbrial antigens (of which there are multiple antigenic types), we have individually expressed four different ETEC fimbriae, including CFA/I, CS2, CS3, and CS4, using deltaguaBA attenuated Shigella vaccine strain CVD 1204 as a prototype live vector. Following mucosal (intranasal) immunization of guinea pigs, serum IgG and mucosal IgA responses were elicited against each fimbrial type. An additional strain was constructed expressing a detoxified version of the human ETEC variant of heat labile toxin (LThK63). Following mucosal immunization of guinea pigs with a mixed inoculum containing five Shigella strains each expressing a different ETEC antigen, immune responses were observed against each ETEC antigen plus the Shigella vector.     

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.     

Prime-boost vaccine regimen confers protective immunity to human-derived enterotoxigenic Escherichia coli

Development of effective vaccines against diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains is still a priority for those living at or traveling to endemic regions. In this work, we evaluated the protective role of an anti-ETEC vaccine regimen based on parenteral priming with a DNA vaccine, pRECFA, followed by oral boosting with a recombinant attenuated Salmonella Typhimurium vaccine strain, HG3, both encoding the same antigen, the structural subunit (CfaB) of the ETEC CFA/I fimbriae. The DNA-priming Salmonella-boosting protocol enhanced both murine anti-CfaB serum IgG and fecal IgA antibody responses and increased the ability of serum antibodies to inhibit the adhesive properties of the CFA/I fimbriae expressed by live bacteria, as compared to mice immunized with only one vaccine type. Addition of a mucosal adjuvant (LTR192G) to the Salmonella vaccine strain further enhanced the synergic effects of the vaccine regimen on the induced CfaB-specific antibody responses. DBA/2 dams submitted to the prime-boost regimen transferred complete passive protection to suckling neonates challenged with a virulent ETEC strain. Detection of milk anti-CfaB IgA antibodies and protection conferred by vaccinated dams to neonates born from non-vaccinated dams indicated that secretion of antigen-specific IgA is the immune response induced by the protective vaccine regimen. These results demonstrate that priming with a DNA vaccine and boosting with a Salmonella strain enhances both quantitatively and qualitatively the antibody responses to the CfaB antigen and represents an alternative for either active or passive immunization approach to ETEC-associated diarrhea.     

A systematic review of ETEC epidemiology focusing on colonization factor and toxin expression

Introduction

Vaccine development for enterotoxigenic Escherichia coli (ETEC) is dependent on in-depth understanding of toxin and colonization factor (CF) distribution. We sought to describe ETEC epidemiology across regions and populations, focusing on CF and toxin prevalence.

Methods

We conducted a systematic review of the published literature, including studies reporting data on ETEC CF and toxin distributions among those with ETEC infection. Point estimates and confidence intervals were calculated using random effects models.

Results

Data on 17,205 ETEC isolates were abstracted from 136 included studies. Approximately half of the studies (49%) involved endemic populations, and an additional 17% involved only travel populations. Globally, 60% of isolates expressed LT either alone (27%) or in combination with ST (33%). CFA/I-expressing strains were common in all regions (17%), as were ETEC expressing CFA/II (9%) and IV (18%). Marked variation in toxins and CFs across regions and populations was observed.

Discussion/conclusions

These results demonstrate the relative importance of specific CFs in achieving target product profiles for a future ETEC vaccine. However, heterogeneity across time, population, and region, confounded by variability in CF and toxin detection methodologies, obfuscates rational estimates for valency requirements.     

Induction of long term mucosal immunological memory in humans by an oral inactivated multivalent enterotoxigenic Escherichia coli vaccine

We have evaluated the capacity of an oral multivalent enterotoxigenic Escherichia coli (ETEC) vaccine (MEV) to induce mucosal immunological memory. MEV consists of four inactivated E. coli strains over-expressing the major colonization factors (CFs) CFA/I, CS3, CS5 and CS6 and the LTB-related toxoid LCTBA. Memory responses were analyzed by comparing the magnitudes and kinetics of intestine-derived antibody-secreting cell responses to a single dose of MEV in three groups of adult Swedish volunteers (n = 16–19 subjects per group) in a Phase I trial: non-immunized controls (I) and subjects who in a previous Phase I trial 13–23 months earlier had received two biweekly doses of MEV (II) or MEV + double mutant LT (dmLT) adjuvant (III). Responses against CFs and LTB were analyzed in antibodies in lymphocyte secretions (ALS) of blood mononuclear cells collected before (day 0) and 4/5 and 7 days after immunization. Specific circulating memory B cells present at the time of the single dose vaccination were also studied to determine if such cells may reflect mucosal memory.Considerably higher and significantly more frequent IgA ALS responses against all CFs and LTB were induced by the single vaccine dose in the previously immunized than in non-immunized volunteers. Furthermore, peak IgA ALS responses against all antigens were observed on days 4/5 in most of the previously immunized subjects whereas only a few previously non-vaccinated individuals responded before day 7. Priming with adjuvant did not influence memory responses. Circulating vaccine specific IgA memory B cells were not detected, whereas anti-toxin IgG memory B cells were identified 13–23 months after priming vaccination.We conclude that MEV induces functional mucosal immunological memory which remains at least 1–2 years. Furthermore, our results support that analysis of antibody-secreting cell responses after booster vaccination may be a useful approach to evaluate longstanding mucosal immunological memory in humans.Clinical trials registration: ISRCTN27096290.     

Cross-reactivity and avidity of antibody responses induced in humans by the oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX

We investigated whether the oral inactivated, multivalent enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, consisting of four E. coli strains over-expressing the colonisation factors (CFs) CFA/I, CS3, CS5 and CS6, combined with the toxoid LCTBA, could induce cross-reactive antibodies to CFs related to the CFA/I and CS5 families. We also evaluated the avidity of vaccine induced antibodies against the toxoid and CFs.Cross-reactivity was analysed in mucosal (faecal and antibodies in lymphocyte supernatants, ALS) samples, and antibody avidity in serum and ALS samples, from two phase I trials: a primary vaccination study, where two oral doses of ETVAX were given ± the double mutant heat labile toxin (dmLT) adjuvant at a 2-week interval, and a booster vaccination study, where a single booster dose of ETVAX was given 13–23 months after primary vaccinations.We found that 65–90% of subjects who had responded to CFA/I in ALS or faecal specimens also developed cross-reactive antibodies to the related CFs tested, i.e. CS1, CS14 and CS17, and that approximately 80% of those responding to CS5 also responded to the closely related CS7. For subjects who had developed cross-reactive antibodies, the magnitudes of responses against vaccine CFs and related non-vaccine CFs were comparable. Using both a simple method of antibody avidity determination based on limiting antigen dilution, as well as a chaotropic ELISA method, we found that the avidity of serum and ALS antibodies to key vaccine antigens increased after a late booster dose compared to after primary vaccination.Our results suggest that the cross-reactive antibody responses against multiple CFs may result in expanded ETEC strain coverage of ETVAX and that repeated vaccinations induce vaccine-specific antibodies with increased binding capacity.     

Surveys of human enterotoxigenic Escherichia coli from three different geographical areas for possible colonization factors.

Enterotoxigenic Escherichia coli (ETEC) from Burma, central Africa (Rwanda and Zaire) and Peru, were screened by enzyme-linked immunoassays for the colonization factor antigens (CFAs) and putative colonization factors (PCFs): CFA/I, CFA/II, which consists of three coli surface-associated (CS) antigens, CS1, CS2 and CS3, CFA/III, CFA/IV (CS4, CS5, CS6), CS7, PCFO9, PCFO159. H4, PCFO166, and CS17. The highest proportion of ETEC with identifiable colonization factors (71%) were found in the strains from Burma, which were mainly positive for CFA/I (38%), but strains producing CFA/II (4%), CFA/IV (11%), CS7 (10%), CS17 (4%), PCFO159, H4 (2%) and PCFO166 (2%) were also found. Sixty-nine percent of the ETEC from central Africa were positive for known colonization factors. While CFA/I positive strains were important (12%), a higher number of ETEC producing CFA/IV (33%) and CS17 (24%) were found. Fifty-two percent of the Peruvian strains produced identifiable colonization factors. The largest group of strains produced antigens of the CFA/IV complex (17%), while ETEC producing CFA/II (6%), CFA/III and CS6 (2%), CS7 (6%), PCFO9 (6%), PCFO166 (8%) and CS17 (7%) were also found. These surveys show that there is a considerable variation in the proportions and types of colonization factor found in different geographical areas. From 29 to 48% of the ETEC did not possess an identifiable colonization factor. These were particularly of the LT only producing type. These results have important implications for vaccine formulation.     

Evaluation of the safety and immunogenicity of the oral inactivated multivalent enterotoxigenic Escherichia coli vaccine ETVAX in Bangladeshi adults in a double-blind,randomized, placebo-controlled Phase I trial using electrochemiluminescence and ELISA assays for immunogenicity analyses

The safety and immunogenicity of the second generation oral enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, consisting of inactivated recombinant E. coli strains over-expressing the colonization factors (CFs) CFA/I, CS3, CS5 and CS6 and the heat labile toxoid LCTBA, were evaluated in Bangladeshi volunteers. To enable analysis of antibody responses against multiple vaccine antigens for subsequent use in small sample volumes from children, a sensitive electrochemiluminescence (ECL) assay for analysis of intestine-derived antibody-secreting cell responses using the antibodies in lymphocyte secretions (ALS) assay was established using Meso Scale Discovery technology.Three groups of Bangladeshi adults (n = 15 per group) received two oral doses of ETVAX with or without double mutant LT (dmLT) adjuvant or placebo in the initial part of a randomized, double-blind, placebo-controlled, age-descending, dose-escalation trial. CF- and LTB-specific ALS and plasma IgA responses were analyzed by ECL and/or ELISA.ETVAX was safe and well tolerated in the adults. Magnitudes of IgA ALS responses determined by ECL and ELISA correlated well (r = 0.85 to 0.98 for the five primary antigens, P < 0.001) and ECL was selected as the ALS readout method. ALS IgA responses against each of the primary antigens were detected in 87–100% of vaccinees after the first and in 100% after the second vaccine dose. Plasma IgA responses against different CFs and LTB were observed in 62–93% and 100% of vaccinees, respectively. No statistically significant adjuvant effect of dmLT on antibody responses to any antigen was detected, but the overall antigenic breadth of the plasma IgA response tended to favor the adjuvanted vaccine when responses to 4 or more or 5 vaccine antigens were considered. Responses in placebo recipients were infrequent and mainly detected against single antigens.The promising results in adults supported testing ETVAX in descending age groups of children.ClinicalTrials.gov Identifier: NCT02531802.     

Safety and immunogenicity of an oral, inactivated enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Bangladeshi adults and children

We have compared the B cell responses evoked in Bangladeshi, adults (n=11, median age 25 years) and children (n=21, median age 4.5 years), 7 days after intake of each of two doses of an oral, inactivated enterotoxigenic Escherichia coli (ETEC) vaccine composed of formalin-killed ETEC strains expressing the colonization factors, CFA/I, CFA/II and CFA/IV together with 1 mg of recombinant cholera toxin B-subunit (rCTB). The vaccine was well tolerated and only gave rise to negligible side effects. Peak antibody-secreting cell (ASC) response of the IgA isotype were seen 7 days after the first dose of the vaccine. The ASC responses to the different colonization factors (CFs) increased from a 29- to 46-fold (responder frequency 90-100%) in the adults and 13- to 24-fold (responder frequency 67-90%) in the children. The IgA-ASC response to rCTB also peaked after the first dose in the adults (426-fold, responder frequency 100%) and the children (46-fold, responder frequency 95%). Increased IgA antibody levels against CFA/I as well as IgA and IgG antibody levels to rCTB were seen in plasma after immunisation. About 86% of the children and 80% of the adults responded with faecal antibodies to rCTB, whereas about 67% of both groups responded to CFA/I.These results show that a single dose of the ETEC vaccine may elicit significant mucosal immune responses in both children and adults residing in an ETEC-endemic country such as Bangladesh.     

Evaluation of the immunogenicity and protective efficacy of a recombinant CS6-based ETEC vaccine in an Aotus nancymaae CS6 + ETEC challenge model

Colonization factors or Coli surface antigens (CFs or CS) are important virulence factors of Enterotoxigenic E. coli (ETEC) that mediate intestinal colonization and accordingly are targets of vaccine development efforts. CS6 is a highly prevalent CF associated with symptomatic ETEC infection both in endemic populations and amongst travelers. In this study, we used an Aotus nancymaae non-human primate ETEC challenge model with a CS6 + ETEC strain, B7A, to test the immunogenicity and protective efficacy (PE) of a recombinant CS6-based subunit vaccine. Specifically, we determined the ability of dscCssBA, the donor strand complemented recombinant stabilized fusion of the two subunits of the CS6 fimbriae, CssA and CssB, to elicit protection against CS6 + ETEC mediated diarrhea when given intradermally (ID) with the genetically attenuated double mutant heat-labile enterotoxin LT(R192G/L211A) (dmLT). ID vaccination with dscCssBA + dmLT induced strong serum antibody responses against CS6 and LT. Importantly, vaccination with dscCssBA + dmLT resulted in no observed diarrheal disease (PE = 100%, p = 0.03) following B7A challenge as compared to PBS immunized animals, with an attack rate of 62.5%. These data demonstrate the potential role that CS6 may play in ETEC infection and that recombinant dscCssBA antigen can provide protection against challenge with the homologous CS6 + ETEC strain, B7A, in the Aotus nancymaae diarrheal challenge model. Combined, these data indicate that CS6, and more specifically, a recombinant engineered derivative should be considered for further clinical development.     

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.     

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.     

Epidemiology and properties of heat-stable enterotoxin-producing Escherichia coli serotype O169:H41.

Enterotoxigenic Escherichia coli (ETEC) serotype O169:H41 organisms have become the most prevalent ETEC in Japan since the first outbreak in 1991. It was assumed that the outbreaks were due to clonal spread of this new ETEC serotype. The relationship of 32 strains isolated from 6 outbreaks were examined for biotype, antibiotic susceptibility, enterotoxigenicity, protein banding pattern, lipopolysaccharide banding pattern, plasmid analysis, and ribotyping. Further, the strains were examined by haemagglutination, surface hydrophobicity, and the ability to adhere to HEp-2 cells. The present study suggests that the outbreaks were caused by multiple clones of STp-producing O169:H41 since they showed differences in ribotype and outer membrane protein banding patterns. The strains did not agglutinate human or bovine red blood cells in a mannose-resistant manner. They adhered to HEp-2 cells in a manner resembling enteroaggregative E. coli. Five strains were examined by dot-blot tests for the colonization factor antigens CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, PCFO159, PCFO166 and CFA/III. Although four strains expressed CS6, no structure for CS6 was identified. A strain that the anti-CS6 MAbs did not react with could adhere to HEp-2 cells in mannose resistant manner; thus, it is unlikely that CS6 play an important role in the adhesion to the cells. Electron microscopy studies of the O169:H41 strains suggested that curly fimbriae, a possible new colonization factor, may be playing an important role in the adhesion of the bacteria to HEp-2 cells. In conclusion, outbreaks due to ETEC O169:H41 were caused by multiple clones, and the strains should be examined in detail for a possible new colonization factor.     

Clinical trial to evaluate safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid protein

We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea containing killed recombinant E. coli bacteria expressing increased levels of ETEC colonization factors (CFs) and a recombinant protein (LCTBA), i.e. a hybrid between the binding subunits of E. coli heat labile toxin (LTB) and cholera toxin (CTB). We describe a randomized, comparator controlled, double-blind phase I trial in 60 adult Swedish volunteers of a prototype of this vaccine. The safety and immunogenicity of the prototype vaccine, containing LCTBA and an E. coli strain overexpressing the colonization factor CFA/I, was compared to a previously developed oral ETEC vaccine, consisting of CTB and inactivated wild type ETEC bacteria expressing CFA/I (reference vaccine). Groups of volunteers were given two oral doses of either the prototype or the reference vaccine; the prototype vaccine was administered at the same or a fourfold higher dosage than the reference vaccine.