Enterotoxigenic Escherichia Coli Vaccine Research Articles

F4+ ETEC infection and oral immunization with F4 fimbriae elicits an IL-17-dominated immune response.

Enterotoxigenic Escherichia coli (ETEC) are an important cause of post-weaning diarrhea (PWD) in piglets. Porcine-specific ETEC strains possess different fimbrial subtypes of which F4 fimbriae are the most frequently associated with ETEC-induced diarrhea in piglets. These F4 fimbriae are potent oral immunogens that induce protective F4-specific IgA antibody secreting cells at intestinal tissues. Recently, T-helper 17 (Th17) cells have been implicated in the protection of the host against extracellular pathogens. However, it remains unknown if Th17 effector responses are needed to clear ETEC infections. In the present study, we aimed to elucidate if ETEC elicits a Th17 response in piglets and if F4 fimbriae trigger a similar response. F4+ ETEC infection upregulated IL-17A, IL-17F, IL-21 and IL-23p19, but not IL-12 and IFN-γ mRNA expression in the systemic and mucosal immune system. Similarly, oral immunization with F4 fimbriae triggered a Th17 signature evidenced by an upregulated mRNA expression of IL-17F, RORγt, IL-23p19 and IL-21 in the peripheral blood mononuclear cells (PBMCs). Intriguingly, IL-17A mRNA levels were unaltered. To further evaluate this difference between systemic and mucosal immune responses, we assayed the cytokine mRNA profile of F4 fimbriae stimulated PBMCs. F4 fimbriae induced IL-17A, IL-17F, IL-22 and IL-23p19, but downregulated IL-17B mRNA expression. Altogether, these data indicate a Th17 dominated response upon oral immunization with F4 fimbriae and F4+ ETEC infection. Our work also highlights that IL-17B and IL-17F participate in the immune response to protect the host against F4+ ETEC infection and could aid in the design of future ETEC vaccines.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0264-2) contains supplementary material, which is available to authorized users.

Antibody-mediated disruption of the mechanics of CS20 fimbriae of enterotoxigenic Escherichia coli.

Preventive vaccines against enterotoxigenic Escherichia coli (ETEC) are being developed, many of which target common fimbrial colonization factors as the major constituent, based on empirical evidence that these function as protective antigens. Particularly, passive oral administration of ETEC anti-fimbrial antibodies prevent ETEC diarrhea. Little is, however, known regarding the specific mechanisms by which intestinal antibodies against ETEC fimbriae function to prevent disease. Using coli surface antigen 20 (CS20) fimbriae as a model ETEC colonization factor, we show using force spectroscopy that anti-fimbrial antibodies diminish fimbrial elasticity by inhibiting their natural capacity to unwind and rewind. In the presence of anti-CS20 antibodies the force required to unwind a single fimbria was increased several-fold and the extension length was shortened several-fold. Similar measurements in the presence of anti-CS20 Fab fragments did not show any effect, indicating that bivalent antibody binding is required to reduce fimbrial elasticity. Based on these findings, we propose a model for an in-vivo mechanism whereby antibody-mediated disruption of the biomechanical properties of CS20 fimbriae impedes sustained adhesion of ETEC to the intestinal mucosal surface. Further elucidation of the role played by intestinal antibodies in mechanical disruption of fimbrial function may provide insights relevant to ETEC vaccine development.

Evaluating the A-Subunit of the Heat-Labile Toxin (LT) As an Immunogen and a Protective Antigen Against Enterotoxigenic Escherichia coli (ETEC).

Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit’s potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines against ETEC.

A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles.

Enteric infections induced by pathogens like Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) remain a massive burden in developing countries with increasing morbidity and mortality rates. Previously, we showed that the immunization with genetically detoxified outer membrane vesicles (OMVs) derived from V. cholerae elicits a protective immune response based on the generation of O antigen antibodies, which effectively block the motility by binding to the sheathed flagellum. In this study, we investigated the potential of lipopolysaccharide (LPS)-modified and toxin negative OMVs isolated from V. cholerae and ETEC as a combined OMV vaccine candidate. Our results indicate that the immunization with V. cholerae or ETEC OMVs induced a species-specific immune response, whereas the combination of both OMV species resulted in a high-titer, protective immune response against both pathogens. Interestingly, the immunization with V. cholerae OMVs alone resulted in a so far uncharacterized and cholera toxin B-subunit (CTB) independent protection mechanism against an ETEC colonization. Furthermore, we investigated the potential use of V. cholerae OMVs as delivery vehicles for the heterologously expression of the ETEC surface antigens, CFA/I, and FliC. Although we induced a detectable immune response against both heterologously expressed antigens, none of these approaches resulted in an improved protection compared to a simple combination of V. cholerae and ETEC OMVs. Finally, we expanded the current protection model from V. cholerae to ETEC by demonstrating that the inhibition of motility via anti-FliC antibodies represents a relevant protection mechanism of an OMV-based ETEC vaccine candidate in vivo.

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.

Conservation and immunogenicity of novel antigens in diverse isolates of enterotoxigenic Escherichia coli.

BackgroundEnterotoxigenic Escherichia coli (ETEC) are common causes of diarrheal morbidity and mortality in developing countries for which there is currently no vaccine. Heterogeneity in classical ETEC antigens known as colonization factors (CFs) and poor efficacy of toxoid-based approaches to date have impeded development of a broadly protective ETEC vaccine, prompting searches for novel molecular targets.MethodologyUsing a variety of molecular methods, we examined a large collection of ETEC isolates for production of two secreted plasmid-encoded pathotype-specific antigens, the EtpA extracellular adhesin, and EatA, a mucin-degrading serine protease; and two chromosomally-encoded molecules, the YghJ metalloprotease and the EaeH adhesin, that are not specific to the ETEC pathovar, but which have been implicated in ETEC pathogenesis. ELISA assays were also performed on control and convalescent sera to characterize the immune response to these antigens. Finally, mice were immunized with recombinant EtpA (rEtpA), and a protease deficient version of the secreted EatA passenger domain (rEatApH134R) to examine the feasibility of combining these molecules in a subunit vaccine approach.Principal FindingsEtpA and EatA were secreted by more than half of all ETEC, distributed over diverse phylogenetic lineages belonging to multiple CF groups, and exhibited surprisingly little sequence variation. Both chromosomally-encoded molecules were also identified in a wide variety of ETEC strains and YghJ was secreted by 89% of isolates. Antibodies against both the ETEC pathovar-specific and conserved E. coli antigens were present in significantly higher titers in convalescent samples from subjects with ETEC infection than controls suggesting that each of these antigens is produced and recognized during infection. Finally, co-immunization of mice with rEtpA and rEatApH134R offered significant protection against ETEC infection.ConclusionsCollectively, these data suggest that novel antigens could significantly complement current approaches and foster improved strategies for development of broadly protective ETEC vaccines.

Shift in phenotypic characteristics of enterotoxigenic Escherichia coli (ETEC) isolated from diarrheal patients in Bangladesh.

BackgroundEnterotoxigenic Escherichia coli (ETEC) is one of the most common causes of bacterial diarrhea. Over the last decade, from 1996 to 2012, changes in the virulence antigen properties of ETEC such as heat labile (LT) and heat stable (ST) toxins, colonization factors (CFs), and ‘O’-serogroups have been observed. The aim of this prospective study was to compare changes in antigenic profiles of ETEC strains isolated from a 2% surveillance system at the icddr,b hospital in Dhaka, Bangladesh between 2007–2012 and an earlier time period of 1996–1998 conducted at the same surveillance site.MethodologyIn the surveillance system every 50th patient attending the hospital was screened for major enteric pathogens including ETEC, Vibrio cholerae, Shigella spp. and Salmonella spp. from January 2007 to December 2012.Principal FindingsOf the 15,152 diarrheal specimens tested between 2007–2012, the overall rate of ETEC isolation was 11%; of these, 43% were LT/ST, 27% LT and 30% ST positive. Isolation rate of ST-ETEC (p<0.009) and LT/ST ETEC (p<0.011) during 2007–2012 period differed significantly compared to those seen between 1996–1998. In comparison to the 1996–1998 period, difference in CF profile of ETEC isolates during 2007–2012 was observed particularly for strains expressing CS7 (12.4%), CS14 (9.5%) and CS17 (10.0%). The predominant CF types were CS5+CS6, CFA/I, CS7, CS17, CS1+CS3, CS6 and CS14. The most common serogroups among the CF positive ETEC isolates were O115, O114, O6, O25 and O8. A strong association was found between CFs and ‘O’ serogroups i.e. between CS5+CS6 and (O115 and O126); CS7 and (O114), CFA/I and (O78 and O126), CS17 and (O8 and O167) and CS1/CS2+CS3 and (O6).ConclusionThe analyses show a shift in prevalence of antigenic types of ETEC over the study period; the information is important in designing effective ETEC vaccines with broad protective coverage.

Designing vaccines to neutralize effective toxin delivery by enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) are a leading cause of diarrheal illness in developing countries. Despite the discovery of these pathogens as a cause of cholera-like diarrhea over 40 years ago, and decades of vaccine development effort, there remains no broadly protective ETEC vaccine. The discovery of new virulence proteins and an improved appreciation of the complexity of the molecular events required for effective toxin delivery may provide additional avenues to pursue in development of an effective vaccine to prevent severe diarrhea caused by these important pathogens.

Mo1949 Rapamycin Protects Intestinal Ischemia/Reperfusion-Induced Remote Lung Injury in Mice

Background: Enterotoxigenic Escherichia coli (ETEC), defined by their production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of infantile diarrhea in developing countries and of travelers' diarrhea in visitors to the developing world. Despite our understanding of ETEC pathogenesis, no broadly protective ETEC vaccine is available. This is largely a result of the difficulty in inducing immunity to the small ST molecule. Our goal is to develop a broadly protective live attenuated vaccine against ETEC by taking advantage of the recent demonstration (Zhang et al.,2013,PLoSONe 8 (10):e77386) that genetically produced fusions of mutant ST with LT components can induce effective immunity against both toxins. Methods: To achieve this aim, we engineered an attenuated strain of E. coli, which we have developed as a vaccine vector for mucosal antigen delivery, to express immunogenic fusion constructs of LT and mutant ST. Our vaccine vector strain ZCR533, is a derivative of O157:H7 enterohemorrhagic E. coli (EHEC) strain 86-24 attenuated by deleting its genes for Shiga toxin production and truncating its major adhesin, intimin, such that it can no longer adhere intimately, but can still induce anti-intimin antibody. To express LT-ST fusions in immunogenic form in our vector strain, we used restriction-free cloning technology to incorporate them into the passenger domain of an autotransporter protein (EspP) expressed on a medium copy number plasmid. This system is designed to express the toxin antigens in either surface-bound or secreted forms, and to provide effective delivery to the mucosal immune system as we have done for other toxin antigens. Results: The incorporation of the LT-ST fusions into the passenger domain of the EspP autotransporter plasmids was confirmed by PCR and DNA sequencing. Protein expression by ZCR533 containing the autotransporter plasmids was confirmed by SDS-PAGE, and Western blot of whole cell lysates and culture supernates, using poly-clonal antisera to the LT component. Moreover, appropriate expression of the LT-ST fusions on the bacterial surface of ZCR533 was confirmed by immuno-fluorescent staining with the specific antisera. Conclusions: These studies show that immunogenic fusions of LT and mutant ST can be expressed on the surface, and in the culture supernates, of our attenuated E. coli vaccine vector ZCR533 via the EspP autotransporter. Since our recent studies using the similarly-sized Shiga toxin B subunit antigen have indicated the validity of this approach to mucosal immunization against toxin antigens, these studies could lead to the development of broadly protective vaccines directed against both LTand ST-producing ETEC, as well as against attaching/ effacing E. coli strains expressing intimin.

Phenotypic and functional evaluation of T cell responses after intradermal immunization with LTR192G, an attenuated form of the enterotoxigenic E. coli (ETEC) labile toxin (LT), as an adjuvant for an ETEC vaccine (VAC8P.1004)

Abstract The skin is an appealing route for delivery of vaccines. It is easily accessible, is rich in antigen presenting cells (i.e. dendritic cells), and several reports have shown that intradermal (ID) vaccination can use less antigen (dose sparing). However, few new adjuvants have been developed and tested for skin immunization. Here, we report our findings on the evaluation of the T cell response after immunization of volunteers with two prototype vaccines against ETEC given ID with LTR192G. Volunteers were immunized on days 0, 21 and 42 with the tip adhesin of the class 5a CFA/I fimbriae, CfaE, or an adhesin-toxoid chimera protein, CfaE-sCTA2/LTB5, admixed with the adjuvant. We found marked activation of LTB- and CfaE-specific CD4+ T cells post-immunization as measured by CD69 upregulation. Within the LTB-specific CD4+ population, we found a greater frequency of skin-homing CCR4+ cells, which is consistent with the route of immunization and a strong response to LTR192G. In addition, there was a higher frequency of LTB-specific CD4+ T cells with an effector memory phenotype. The frequency of CfaE-specific IFNγ+ CD4+ T cells was significantly higher post-immunization and was validated by high levels of IFNγ in CfaE-stimulated supernatants. Cytokine analysis also revealed higher levels of CfaE-specific IL-6 secretion compared to LTB. Taken together, the data demonstrate a strong CD4+ T cell response was generated to both antigen and adjuvant after ID immunization.

Mo1948 Toward a Broadly Protective Vaccine Against Enterotoxigenic E.coli (ETEC) Infections

Background: Enterotoxigenic Escherichia coli (ETEC), defined by their production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of infantile diarrhea in developing countries and of travelers' diarrhea in visitors to the developing world. Despite our understanding of ETEC pathogenesis, no broadly protective ETEC vaccine is available. This is largely a result of the difficulty in inducing immunity to the small ST molecule. Our goal is to develop a broadly protective live attenuated vaccine against ETEC by taking advantage of the recent demonstration (Zhang et al.,2013,PLoSONe 8 (10):e77386) that genetically produced fusions of mutant ST with LT components can induce effective immunity against both toxins. Methods: To achieve this aim, we engineered an attenuated strain of E. coli, which we have developed as a vaccine vector for mucosal antigen delivery, to express immunogenic fusion constructs of LT and mutant ST. Our vaccine vector strain ZCR533, is a derivative of O157:H7 enterohemorrhagic E. coli (EHEC) strain 86-24 attenuated by deleting its genes for Shiga toxin production and truncating its major adhesin, intimin, such that it can no longer adhere intimately, but can still induce anti-intimin antibody. To express LT-ST fusions in immunogenic form in our vector strain, we used restriction-free cloning technology to incorporate them into the passenger domain of an autotransporter protein (EspP) expressed on a medium copy number plasmid. This system is designed to express the toxin antigens in either surface-bound or secreted forms, and to provide effective delivery to the mucosal immune system as we have done for other toxin antigens. Results: The incorporation of the LT-ST fusions into the passenger domain of the EspP autotransporter plasmids was confirmed by PCR and DNA sequencing. Protein expression by ZCR533 containing the autotransporter plasmids was confirmed by SDS-PAGE, and Western blot of whole cell lysates and culture supernates, using poly-clonal antisera to the LT component. Moreover, appropriate expression of the LT-ST fusions on the bacterial surface of ZCR533 was confirmed by immuno-fluorescent staining with the specific antisera. Conclusions: These studies show that immunogenic fusions of LT and mutant ST can be expressed on the surface, and in the culture supernates, of our attenuated E. coli vaccine vector ZCR533 via the EspP autotransporter. Since our recent studies using the similarly-sized Shiga toxin B subunit antigen have indicated the validity of this approach to mucosal immunization against toxin antigens, these studies could lead to the development of broadly protective vaccines directed against both LTand ST-producing ETEC, as well as against attaching/ effacing E. coli strains expressing intimin.

Characterization of enterotoxigenic Escherichia coli strains isolated from Nicaraguan children in hospital, primary care and community settings

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhoea among young children in developing countries. ETEC vaccines offer promise in reducing the burden of ETEC disease, but the development of these vaccines relies on the characterization of ETEC isolates from a variety of settings. To best reflect the full spectrum of ETEC disease in León, Nicaragua, the aim of this study was to characterize ETEC strains isolated from children with diarrhoea attending different settings (hospital, primary care clinics and in the community) and children from different age groups. We characterized ETEC isolates in terms of their colonization factors (CFs) and enterotoxins, and determined whether these factors varied with setting and age group. Diarrhoeal stool samples were obtained from children under the age of 60 months from: (1) the regional public hospital, (2) four public primary care clinics, and (3) a population-based cohort. In total, 58 ETEC-positive isolates were analysed by multiplex-PCR assays for the identification of CFs (CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS12, CS13, CS14, CS15, CS17, CS18, CS19, CS20, CS21, CS22 and CFA/I), and enterotoxins [heat-labile toxin (LT) and heat-stable variants STh and STp]. The frequency of CFs and enterotoxins was compared among the three settings and for different age groups, using Fisher's exact test or a χ(2) test. At least one CF was detected among one-half of samples; CS19 was detected among all strains in which a CF was identified, either alone or in combination with another CF. Among all CFs detected, 91.7 % were identified as members of the class 5 fimbrial family. CFs were detected more commonly among samples from infants captured in the health facility setting compared with the community setting. Overall, LT was detected among 67.2 % of samples, STh was detected among 20.7 % and both enterotoxins were detected among 12.1 %. The enterotoxin STh was detected more commonly among cases in the community, whilst a combination of STh and LT was detected more commonly among cases treated in health facilities. Our results suggest that, to protect against diarrhoeal cases associated with this E. coli pathotype in León, Nicaragua, an ETEC vaccine that effectively targets the archeotype CFA/I of the class 5 fimbrial family would be the most effective in this setting.

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.

Toxicity and Immunogenicity of Enterotoxigenic Escherichia coli Heat-Labile and Heat-Stable Toxoid Fusion 3xSTaA14Q-LTS63K/R192G/L211A in a Murine Model

Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT) and heat-stable type Ib toxin (STa) disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STaP13F) fused at the N- or C-terminus, or inside the A subunit of LTR192G elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011). In this study, we generated a different STa toxoid (STaA14Q) and a triple-mutant LT toxoid (LTS63K/R192G/L211A, tmLT), constructed a toxoid fusion (3xSTaA14Q-tmLT) that carried 3 copies of STaA14Q for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTaA14Q-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.

Vaccines for the prevention of diarrhea due to cholera, shigella, ETEC and rotavirus

BackgroundDiarrhea is a leading cause of mortality in children under 5 years along with its long-term impact on growth and cognitive development. Despite advances in the understanding of diarrheal disorders and management strategies, globally nearly 750,000 children die annually as a consequence of diarrhea.MethodsWe conducted a systematic review of the efficacy and effectiveness studies. We used a standardized abstraction and grading format and performed meta-analyses for all outcomes. The estimated effect of cholera, shigella, Enterotoxigenic Escherichia coli (ETEC) and rotavirus vaccines was determined by applying the standard Child Health Epidemiology Reference Group (CHERG) rules.ResultsA total of 24 papers were selected and analyzed for all the four vaccines. Based on the evidence, we propose a 74% mortality reduction in rotavirus specific mortality, 52% reduction in cholera incidence due to their respective vaccines. We did not find sufficient evidence and a suitable outcome to project mortality reductions for cholera, ETEC and shigella in children under 5 years.ConclusionVaccines for rotavirus and cholera have the potential to reduce diarrhea morbidity and mortality burden. But there is no substantial evidence of efficacy for ETEC and shigella vaccines, although several promising vaccine concepts are moving from the development and testing pipeline towards efficacy and Phase 3 trials.

Development of a fast-dissolving tablet formulation of a live attenuated enterotoxigenic E. coli vaccine candidate

Vaccination is considered the most cost-effective approach to preventing infectious diseases, yet better formulations and delivery methods for efficient distribution and administration of vaccines are needed, especially for low-resource settings. A fast-dissolving tablet (FDT) that could be packaged in a compact stackable blister sheet is a potentially attractive option for formulating oral vaccines, since it would minimally impact the cold chain and could potentially be administered directly to patients without reconstitution. This study focused on using one component of a live attenuated trivalent vaccine under development to produce a FDT for the prevention of diarrhea induced by enterotoxigenic Escherichia coli (ETEC). Ten formulations were prepared and freeze dried to produce FDTs. Three freezing conditions were explored, along with different drying and package sealing methods. Physical properties examined included structural integrity, dissolution time, moisture content, and glass transition temperature. Bacterial viability was tested by assaying for colony-forming units. The formulation compositions and freeze-drying parameters were adjusted in an iterative process to arrive at a promising formulation for the ETEC vaccine tablet. This formulation included sucrose and trehalose as cryoprotectants; phosphate and glutamate salts as buffers and stabilizers; and Natrosol®, polyvinylpyrrolidone, and mannitol as binders. The process loss in viability during freeze drying was less than 0.3log10 (50% recovery) for the optimized vaccine tablet formulation. The final tablets were robust, disintegrated in less than 10s, and preserved the bacteria at 2–8°C for at least 12 months with less than 0.4log10 loss (40% recovery) in viability during storage. This study indicates that the FDT produced by freeze drying directly in a blister sheet could be a practical option for formulating ETEC vaccines for oral immunization and help to facilitate delivery of lifesaving vaccines, particularly in low-resource settings.

Lactobacillus acidophilussupplementation in human subjects and their resistance to enterotoxigenicEscherichia coliinfection

To assess the effect of Lactobacillus acidophilus (American Type Culture Collection (ATCC) 700396) on enterotoxigenic Escherichia coli (ETEC) infection, in the present study, a parallel, double-blind, placebo-controlled 4-week intervention was performed in healthy males. The subjects largely consumed their habitual diet, but had to abstain from consuming dairy foods generally high in Ca. The subjects were randomised into the L. acidophilus (dose 10⁹ colony-forming units twice daily; n 20) or the placebo (n 19) group. After an adaptation period of 2 weeks, the subjects were orally infected with a live, but attenuated, ETEC vaccine, able to induce mild, short-lived symptoms. Before and after the challenge, the subjects recorded stool consistency, bowel habits, and frequency and severity of gastrointestinal complaints. The ETEC challenge led to a significant increase in faecal output on the 2nd day and a concomitant increase in Bristol stool scale scores. Likewise, abdominal pain, bloating, flatulence, fever, headache and nausea peaked 1 d after the oral challenge. The concentrations of faecal calprotectin and IgA peaked 2 d after and that of serum IgM peaked 9 and 15 d after the oral challenge. The concentrations of serum IgA and IgG were unaffected. The ETEC challenge led to a reduction in the number of Bacteroides-Prevotella, Bifidobacterium, Clostridium cluster XIVab and total faecal bacteria. Probiotic treatment was associated with a larger increase in Bristol stool scale scores and more fever, headache and nausea after the ETEC challenge compared with the placebo treatment. These differences were, however, small and with substantial variation within the groups. Oral application of an attenuated live ETEC vaccine provides a useful model for food-borne infections. Supplementation with L. acidophilus ATCC 700396, however, was ineffective in reducing ETEC infection symptoms in healthy men.

Vaccines for preventing enterotoxigenicEscherichia coli(ETEC) diarrhoea

Infection with enterotoxigenic Escherichia coli (ETEC) bacteria is a common cause of diarrhoea in adults and children in developing countries and is a major cause of 'travellers' diarrhoea' in people visiting or returning from endemic regions. A killed whole cell vaccine (Dukoral®), primarily designed and licensed to prevent cholera, has been recommended by some groups to prevent travellers' diarrhoea in people visiting endemic regions. This vaccine contains a recombinant B subunit of the cholera toxin that is antigenically similar to the heat labile toxin of ETEC. This review aims to evaluate the clinical efficacy of this vaccine and other vaccines designed specifically to protect people against diarrhoea caused by ETEC infection. To evaluate the efficacy, safety, and immunogenicity of vaccines for preventing ETEC diarrhoea. We searched the Cochrane Infectious Disease Group Specialized Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, LILACS, and http://clinicaltrials.gov up to December 2012. Randomized controlled trials (RCTs) and quasi-RCTs comparing use of vaccines to prevent ETEC with use of no intervention, a control vaccine (either an inert vaccine or a vaccine normally given to prevent an unrelated infection), an alternative ETEC vaccine, or a different dose or schedule of the same ETEC vaccine in healthy adults and children living in endemic regions, intending to travel to endemic regions, or volunteering to receive an artificial challenge of ETEC bacteria. Two authors independently assessed each trial for eligibility and risk of bias. Two independent reviewers extracted data from the included studies and analyzed the data using Review Manager (RevMan) software. We reported outcomes as risk ratios (RR) with 95% confidence intervals (CI). We assessed the quality of the evidence using the GRADE approach. Twenty-four RCTs, including 53,247 participants, met the inclusion criteria. Four studies assessed the protective efficacy of oral cholera vaccines when used to prevent diarrhoea due to ETEC and seven studies assessed the protective efficacy of ETEC-specific vaccines. Of these 11 studies, seven studies presented efficacy data from field trials and four studies presented efficacy data from artificial challenge studies. An additional 13 trials contributed safety and immunological data only. Cholera vaccinesThe currently available, oral cholera killed whole cell vaccine (Dukoral®) was evaluated for protection of people against 'travellers' diarrhoea' in a single RCT in people arriving in Mexico from the USA. We did not identify any statistically significant effects on ETEC diarrhoea or all-cause diarrhoea (one trial, 502 participants, low quality evidence).Two earlier trials, one undertaken in an endemic population in Bangladesh and one undertaken in people travelling from Finland to Morocco, evaluated a precursor of this vaccine containing purified cholera toxin B subunit rather than the recombinant subunit in Dukoral®. Short term protective efficacy against ETEC diarrhoea was demonstrated, lasting for around three months (RR 0.43, 95% CI 0.26 to 0.71; two trials, 50,227 participants). This vaccine is no longer available. ETEC vaccinesAn ETEC-specific, killed whole cell vaccine, which also contains the recombinant cholera toxinB-subunit, was evaluated in people travelling from the USA to Mexico or Guatemala, and from Austria to Latin America, Africa, or Asia. We did not identify any statistically significant differences in ETEC-specific diarrhoea or all-cause diarrhoea (two trials, 799 participants), and the vaccine was associated with increased vomiting (RR 2.0, 95% CI 1.16 to 3.45; nine trials, 1528 participants). The other ETEC-specific vaccines in development have not yet demonstrated clinically important benefits. There is currently insufficient evidence from RCTs to support the use of the oral cholera vaccine Dukoral® for protecting travellers against ETEC diarrhoea. Further research is needed to develop safe and effective vaccines to provide both short and long-term protection against ETEC diarrhoea.

Development and preclinical evaluation of safety and immunogenicity of an oral ETEC vaccine containing inactivated E. coli bacteria overexpressing colonization factors CFA/I, CS3, CS5 and CS6 combined with a hybrid LT/CT B subunit antigen, administered alone and together with dmLT adjuvant

A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTBA), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant.Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell+LCTBA vaccine, and (2) A “complete” inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell+LCTBA vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans.

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.The prototype vaccine was found to be safe and equally well-tolerated as the reference vaccine at either dosage tested. The prototype vaccine induced mucosal IgA (fecal secretory IgA and intestine-derived IgA antibody secreting cell) responses to both LTB and CFA/I, as well as serum IgA and IgG antibody responses to LTB. Immunization with LCTBA resulted in about twofold higher mucosal and systemic IgA responses against LTB than a comparable dose of CTB. The higher dose of the prototype vaccine induced significantly higher fecal and systemic IgA responses to LTB and fecal IgA responses to CFA/I than the reference vaccine.These results demonstrate that CF over-expression and inclusion of the LCTBA hybrid protein in an oral inactivated ETEC vaccine does not change the safety profile when compared to a previous generation of such a vaccine and that the prototype vaccine induces significant dose dependent mucosal immune responses against CFA/I and LTB.