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•We developed and tested a new oral ETEC vaccine in mice, alone and with an adjuvant.•The vaccine contains E. coli overexpressing CFA/I, CS3, CS5 and CS6 and B-subunit.•Oral immunization induced intestinal and serum antibody responses to all antigens.•These responses were further enhanced by dmLT, a nontoxic E. coli adjuvant.•The vaccine, both with and without dmLT, was stable and well tolerated. 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.

Enterotoxigenic Escherichia coli causes diarrhoea, leading to substantial mortality and morbidity in children, but no specific vaccine exists. This trial tested an oral, inactivated, enterotoxigenic E coli vaccine (ETVAX), which has been previously shown to be safe and highly immuongenic in Swedish and Bangladeshi adults. We tested the safety and immunogenicity of ETVAX, consisting of four E coli strains overexpressing the most prevalent colonisation factors (CFA/I, CS3, CS5, and CS6) and a toxoid (LCTBA) administered with or without a double-mutant heat-labile enterotoxin (dmLT) as an adjuvant, in Bangladeshi children. We did a randomised, double-blind, placebo-controlled, dose-escalation, age-descending, phase 1/2 trial in Dhaka, Bangladesh. Healthy children in one of three age groups (24–59 months, 12–23 months, and 6–11 months) were eligible. Children were randomly assigned with block randomisation to receive either ETVAX, with or without dmLT, or placebo. ETVAX (half [5·5 × 1010 cells], quarter [2·5 × 1010 cells], or eighth [1·25 × 1010 cells] adult dose), with or without dmLT adjuvant (2·5 μg, 5·0 μg, or 10·0 μg), or placebo were administered orally in two doses 2 weeks apart. Investigators and participants were masked to treatment allocation. The primary endpoint was safety and tolerability, assessed in all children who received at least one dose of vaccine. Antibody responses to vaccine antigens, defined as at least a two-times increase in antibody levels between baseline and post-immunisation, were assessed as secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02531802. Between Dec 7, 2015, and Jan 10, 2017, we screened 1500 children across the three age groups, of whom 430 were enrolled and randomly assigned to the different treatment groups (130 aged 24–59 months, 100 aged 12–23 months, and 200 aged 6–11 months). All participants received at least one dose of vaccine. No solicited adverse events occurred that were greater than moderate in severity, and most were mild. The most common solicited event was vomiting (ten [8%] of 130 patients aged 24–59 months, 13 [13%] of 100 aged 12–23 months, and 29 [15%] of 200 aged 6–11 months; mostly of mild severity), which appeared related to dose and age. The addition of dmLT did not modify the safety profile. Three serious adverse events occurred but they were not considered related to the study drug. Mucosal IgA antibody responses in lymphocyte secretions were detected against all primary vaccine antigens (CFA/I, CS3, CS5, CS6, and the LCTBA toxoid) in most participants in the two older age groups, whereas such responses to four of the five antigens were less frequent and of lower magnitude in infants aged 6–11 months than in older children. Faecal secretory IgA immune responses were recorded against all vaccine antigens in infants aged 6–11 months. 78 (56%) of 139 infants aged 6–11 months who were vaccinated developed mucosal responses against at least three of the vaccine antigens versus 14 (29%) of 49 of the infants given placebo. Addition of the adjuvant dmLT enhanced the magnitude, breadth, and kinetics (based on number of responders after the first dose of vaccine) of immune responses in infants. The encouraging safety and immunogenicity of ETVAX and benefit of dmLT adjuvant in young children support its further assessment for protective efficacy in children in enterotoxigenic E coli-endemic areas. PATH (Bill & Melinda Gates Foundation and the UK's Department for International Development), the Swedish Research Council, and The Swedish Foundation for Strategic Research.

Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens causing severe human infections including hemorrhagic colitis and hemolytic uremic syndrome, particularly in children. Ruminants are the main reservoir of EHEC which colonize their intestinal tract through a mechanism involving the bacterial adhesin intimin. Vaccination of cattle has shown efficacy in reducing EHEC O157:H7 shedding in feces. However, most of these vaccines rely on purified proteins and/or adjuvants, making them expensive and not used by breeders. This study introduced the development of a new type of vaccine based on Outer Membrane Vesicles (OMVs) carrying the C-terminal domain of intimin (Int280). A vaccine which combines OMVs carrying luminal Int280 and OMVs displaying surface-exposed Int280 was produced using two addressing systems based on PelB peptide signal and Lpp-OmpA hybrid protein, respectively. Dot blot experiments on OMVs combined with FAS assay with bacteria confirmed the correct localization of the fusion proteins and the functionality of Lpp-OmpA-Int280, respectively. As a proof of concept, the efficiency of the mixed vaccine was tested in a mouse model using the pathogen Citrobacter rodentium which shares a similar intimin-based adhesion mechanism with EHEC. Intraperitoneal vaccination of mice, at two-week intervals with 1 μg of the mixture of OMV-Int280, elicited a strong anti-intimin IgG response. Interestingly, we observed a shortened C. rodentium fecal shedding duration in immunized mice compared to the control unvaccinated group, with significant reduction of C. rodentium colonization from day 14 (q < 0.0001) to day 18 (q = 0.0068). This OMV-Int280 vaccine therefore represents a promising candidate for the control of EHEC intestinal carriage and fecal shedding in ruminants. •Outer membrane vesicles (OMVs) enriched with intimin C-terminal domain were used as vaccine.•Two addressing systems successfully exported Int280 to the lumen or surface of OMVs.•Int280 was addressed to the outer membrane surface of E. coli and was functional.•The OMV-Int280 based vaccine induced a seroconversion against intimin in mice.•OMV-Int280 immunized mice had a shortened duration of C. rodentium fecal shedding.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of moderate to severe diarrhoea in children for which there is no licensed vaccine. We evaluated ETVAX®, an oral, inactivated ETEC vaccine containing four E. coli strains over-expressing the major colonization factors CFA/I, CS3, CS5, and CS6, a toxoid (LCTBA) and double mutant heat-labile enterotoxin (dmLT) adjuvant for safety, tolerability, and immunogenicity. A double-blind, placebo-controlled, age-descending, dose-finding trial was undertaken in 40 adults, 60 children aged 10–23 months, and 146 aged 6–9 months. Adults received one full dose of ETVAX® and children received 3 doses of either 1/4 or 1/8 dose. Safety was evaluated as solicited and unsolicited events for 7 days following vaccination. Immunogenicity was assessed by evaluation of plasma IgA antibody responses to CFA/I, CS3, CS5, CS6, and LTB, and IgG responses to LTB. Solicited adverse events were mostly mild or moderate with only 2 severe fever reports which were unrelated to the vaccine. The most common events were abdominal pain in adults (26.7 % in vaccinees vs 20 % in placebos), and fever in children aged 6–9 months (44 % vs 54  %). Dosage, number of vaccinations and decreasing age had no influence on severity or frequency of adverse events. The vaccine induced plasma IgA and IgG responses against LTB in 100 % of the adults and 80–90 % of the children. In the 6–23 months cohort, IgA responses to more than 3 vaccine antigens after 3 doses determined as ≥2-fold rise was significantly higher for 1/4 dose compared to placebo (56.7 % vs 27.2 %, p = 0.01). In the 6–9 months cohort, responses to the 1/4 dose were significantly higher than 1/8 dose after 3 rather than 2 doses. ETVAX® was safe, tolerable, and immunogenic in Zambian adults and children. The 1/4 dose induced significantly stronger IgA responses and is recommended for evaluation of protection in children. Clinical trials registration: The trial is registered with the Pan African Clinical Trials Registry (PACTR Ref. 201905764389804) and a description of this clinical trial is available on: https://pactr.samrc.ac.za/Trial Design.

Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis, which can progress to hemolytic uremic syndrome. These complications represent a serious global public health problem that requires laborious public health interventions and safety control measures to combat recurrent outbreaks worldwide. Today, there are no effective interventions for the control of EHEC infections, and, in fact, the use of antibiotics is counterindicated for EHEC disease. Therefore, a viable alternative for the prevention of human infections is the development of vaccines; however, no such vaccines are approved for human use. In this study, we developed a novel gold nanoparticle platform which acts as a scaffold for the delivery of various antigens, representing a nanovaccine technology which can be applied to several disease models. Here we exploit the natural properties of a synthetic nanoparticle (NP) scaffold as a subunit vaccine against enterohemorrhagic Escherichia coli (EHEC). Two EHEC-specific immunogenic antigens, namely, LomW and EscC, either alone or in combination, were covalently linked on the surface of gold nanoparticles (AuNPs) and used to immunize mice prior to challenge with EHEC O157:H7 strain 86-24. LomW is a putative outer membrane protein encoded in bacteriophage BP-933W, while EscC is a structural type III secretion system protein which forms a ring in the outer membrane. The resulting AuNP preparations, AuNP-LomW and AuNP-EscC, showed that the nanoparticles were able to incorporate the antigens, forming stable formulations that retained robust immunogenicity in vivo after subcutaneous immunization. When administered subcutaneously, AuNP-LomW or AuNP-EscC or a combination containing equivalent amounts of both candidates resulted in higher IgG titers in serum and secretory IgA titers in feces. The serum IgG titers correlated with a significant reduction in EHEC intestinal colonization after 3 days postinoculation. In addition, we showed that serum from antigen-coated AuNP-immunized mice resulted in a reduction of adherence to human intestinal epithelial cells for EHEC, as well as for two other E. coli pathotypes (enteropathogenic E. coli [EPEC], encoding EscC, and enteroaggregative E. coli [EAEC], encoding LomW). Further, the serum had antigen-specific bactericidal properties, engaging the classical complement pathway. Overall, our results demonstrate the immunogenicity and stability of a novel nanovaccine against EHEC. These results also strengthen the prospect of development of a synthetic nanoparticle vaccine conjugated to E. coli antigens as a promising platform against other enteric pathogens. IMPORTANCE Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis, which can progress to hemolytic uremic syndrome. These complications represent a serious global public health problem that requires laborious public health interventions and safety control measures to combat recurrent outbreaks worldwide. Today, there are no effective interventions for the control of EHEC infections, and, in fact, the use of antibiotics is counterindicated for EHEC disease. Therefore, a viable alternative for the prevention of human infections is the development of vaccines; however, no such vaccines are approved for human use. In this study, we developed a novel gold nanoparticle platform which acts as a scaffold for the delivery of various antigens, representing a nanovaccine technology which can be applied to several disease models.

Antimicrobial-resistant bacteria originating from broilers pose an ongoing challenge as they can spread in the environment and food chain. One approach to lower colonization is to administer live bacteria to broilers. A live Escherichia coli (E. coli) vaccine, consisting of a single E. coli strain used as a competitive exclusion (CE) approach, was evaluated to decrease the colonization with ESBL-/pAmpC- producing E. coli . 168 ESBL-/pAmpC- negative Ranger Gold broilers were divided into six groups (3 x n  = 46 experimental groups; 3 x n  = 10 control groups for quality assurance). The E. coli vaccine was administered on day one through coarse spray or by drinking water on day five. Experimental groups were orally co-colonized with 10 2 cfu of one ESBL- (ST410, bla CTX−M−15 ) and one pAmpC- producing E. coli (ST10, bla CMY−2 / mcr -1) on day three. Colonization status was monitored throughout the trial and quantified at the end of the study (day 49). A transient reduction in ESBL-producing E. coli colonization ( p  < 0.001) was observed following coarse spray administration. However, this decrease was not sustained over time. It can be concluded that a single E. coli strain originating from a live vaccine cannot decrease colonization of broilers with ESBL-/pAmpC-  E. coli throughout a fattening period.

To evaluate the immunoprotective effect of bacterial biomimetic vesicles (BBVs) against avian pathogenic Escherichia coli (APEC), a ΔtolA J11 mutant strain was generated by deleting the tolA gene in the low pathogenic O78 serotype J11 strain. The total protein content of outer membrane vesicles (OMVs) derived from the ΔtolA J11 strain exhibited a sevenfold increase compared to the wild-type strain. Additionally, high-pressure homogenization technology was employed to produce BBVs, resulting in a sixfold increase in total protein content compared to spontaneously secreted OMVs from ΔtolA J11. The immunogenicity of both OMVs and BBVs was assessed through intranasal or intramuscular immunization in specific pathogen-free (SPF) chickens. Results demonstrated that intranasal immunization with OMVs or BBVs in chickens elicited specific IgY antibodies against APEC outer membrane proteins and specific sIgA antibodies in the nasal cavity and trachea, as well as a significant increase in the proliferation response of chicken peripheral blood lymphocytes. The bacterial load in the blood and various organs of the challenged chickens were significantly reduced, resulting in a 66.67% and 58.30% survival rate against a high pathogenic serotype O78 strain challenge, while the control group exhibited only a 16.67% survival rate. The intramuscular immunization with OMVs or BBVs in chickens only induced specific IgY antibodies, with a survival rate of only 33.33% for challenged chickens during the same period. Therefore, intranasal vaccination of the highly productive BBVs is capable of eliciting an immune response similar to that of OMVs and providing protection against APEC infection, thus offering innovative insights for the advancement of APEC vaccines.

MecVax is a protein-based vaccine candidate targeting the seven most important adhesins and the two toxins of enterotoxigenic Escherichia coli (ETEC), the top cause of children's diarrhea and travelers' diarrhea. In this study, we formulated MecVax protein antigens, toxoid fusion 3xSTaN12S-mnLTR192G/L211A and CFA/I/II/IV MEFA, with 5 % lactose and/or 0.1 % polysorbate 80 (Tween-80) or phosphate-buffered saline (PBS), and explored buffer formulations for this ETEC vaccine candidate. Data showed that CFA/I/II/IV MEFA protein remained stable at 37 °C for eight weeks in 0.1 % Tween-80 buffer or PBS, whereas toxoid fusion protein showed apparent physical degradation after three weeks, particularly buffered with 5 % lactose, based on visual examination with SDS-PAGE Coomassie blue staining. Mice intramuscularly immunized with MecVax, composed of the toxoid fusion protein and CFA/I/II/IV MEFA that were shelved at 37 °C for three and six weeks, respectively, developed robust antibody responses to ETEC heat-stable toxin (STa) and heat-labile toxin (LT) and seven ETEC adhesins (CFA/I, CS1-CS6). MecVax in 0.1 % Tween-80 buffer, with or without 5 % lactose, or in PBS induced significantly better anti-adhesin and antitoxin IgG responses, and the derived mouse serum antibodies had significantly better activities against adherence of the seven ETEC adhesins and STa and LT enterotoxicity in vitro. These results indicated that a 0.1 % Tween-80 buffer and PBS significantly improved MecVax's thermal stabilization and immunogenicity, providing instructive information for future buffer formulation and the development of this multivalent vaccine candidate against ETEC diarrhea.

Enterotoxigenic Escherichia coli (ETEC), Shigella, and Campylobacter have been identified as major causes of diarrheal diseases worldwide. In addition to overt disease and death, they are responsible for stunting in children with the risk of lifelong consequences on health and economic opportunities. All three of these bacterial pathogens, which collectively account for approximately 30 % of the cases of diarrheal diseases, are recognized as antimicrobial resistance (AMR) threats. In spite of the dangers these pathogens represent for both children and adults, there is as yet no licensed vaccine available for any of them. Fortunately, much has been accomplished to identify conserved antigens against each of these pathogens so that now relatively simple vaccines have the potential to be developed into multi-pathogen vaccines which could have a major impact on reduction of diarrheal diseases. Conserved antigens may be used even more efficiently if consolidated and expressed on a cellular vector or as part of a conjugate vaccine. A new mucosal adjuvant, double mutant heat-labile toxin (dmLT), has been shown to not only be among the conserved antigens against ETEC, but to also have properties which drive robust mucosal and systemic immune responses for antigens given orally or intramuscularly. Conserved antigens and the strategies for their use such as co-administration with dmLT will be presented in this review. •Vaccines against ETEC, Shigella, and Campylobacter are needed.•Conserved antigens are available for ETEC, Shigella, and Campylobacter.•Conserved antigens can facilitate multi-pathogen vaccine strategies.•dmLT benefits mucosal immunization by oral and parenteral routes.

•We have tested a new oral killed ETEC vaccine±dmLT adjuvant in Swedish adults.•The vaccine contained E. coli bacteria overexpressing common colonization factors.•The vaccine also contained a recombinant hybrid LTB/CTB protein; LCTBA.•The vaccine was safe and induced immune responses to all major vaccine antigens.•dmLT enhanced mucosal responses to antigens present in low amounts in the vaccine. We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC), which is the most common cause of bacterial diarrhea in children in developing countries and in travelers. The vaccine was tested for safety and immunogenicity alone and together with double-mutant heat-labile toxin (dmLT) adjuvant in a double-blind, placebo-controlled Phase I study in 129 Swedish adults. The vaccine consists of four inactivated recombinant E. coli strains overexpressing the major ETEC colonization factors (CFs) CFA/I, CS3, CS5, and CS6 mixed with an LT B-subunit related toxoid, LCTBA. Volunteers received two oral doses of vaccine alone, vaccine plus 10μg or 25μg dmLT or placebo. Secretory IgA antibody responses in fecal samples and IgA responses in secretions from circulating intestine-derived antibody secreting cells were assessed as primary measures of vaccine immunogenicity. The vaccine was safe and well tolerated; adverse events were few and generally mild with no significant differences between subjects receiving placebo or vaccine with or without adjuvant. As many as 74% of subjects receiving vaccine alone and 83% receiving vaccine plus 10μg dmLT showed significant mucosal IgA responses to all five primary vaccine antigens and about 90% of all vaccinees responded to at least four of the antigens. Subjects receiving vaccine plus 10μg dmLT responded with significantly increased intestine-derived anti-CS6 responses compared to subjects receiving vaccine alone. The vaccine was safe and broadly immunogenic. dmLT further enhanced mucosal immune responses to CF antigens present in low amounts in the vaccine. Based on these encouraging results, the vaccine will be tested for safety and immunogenicity in different age groups including infants in Bangladesh and for protective efficacy in travelers.