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Escherichia coli is one of the most common bacterial pathogens in cattle. Prophylactic vaccines are considered promising strategies with the potential to reduce the incidence of colibacillosis. Some studies suggested that bacterial ghosts may serve as a novel approach for preventing bacterial infections. However, the roles of administration route on vaccine immunogenicity and efficacy have not been investigated. In this study, the efficacy of vaccination via different immune routes in generating humoral and cellular immune response was compared through subcutaneous (SC), intramuscular (IM), and oral (O) administration in female BALB/c mice with bacterial ghosts prepared using wild type Escherichia coli isolates CE9, while phosphate buffer saline (PBS) and inactivated vaccines containing aluminum adjuvants (Killed) were used as control. Our results showed that the plasmid pBV220-E-aa-SNA containing E. coli was efficiently cleaved at 42°C with 94.8% positive ratio as assessed by colony counts. Transmission electron microscopy (TEM) confirmed bacteria retained intact surface structure while devoid of cytoplasmic component. We found that total IgG titers in killed, IM and SC groups showed significant increase on 7, 14, 21 and 28 days post-immunization. The IgA level of the IM group was higher than that of all other groups on the 28th day. Meanwhile, four experimental groups showed a significant difference in IgA levels compared with PBS control. In the IM group, an increase in the relative percentages of CD3+CD4+ T cells was accompanied by an increase in the relative percentages of splenic CD3+CD8+ T cells. In comparison with the inactivated vaccine, intramuscular CE9 ghosts immunization elicited higher levels of IL-1β, IL-2, IL-6 and IL-12. Subcutaneous and intramuscular immunizations were significantly associated with improved survival in comparison with oral route, traditional vaccine and the control. Pathologic assessment revealed that less severe tissue damage and inflammation were found in lung, kidney, and intestine of IM group compared with other groups. The results above demonstrate that immunization of Escherichia coli CE9 ghosts via intramuscular injection elicits a more robust antigen-specific immune response in mice to prevent the Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of mortality and morbidity in children in low-income countries. We have tested an oral ETEC vaccine, ETVAX, consisting of inactivated E coli overexpressing the most prevalent colonization factors and a toxoid, LCTBA, administered together with a mucosal adjuvant, double-mutant heat-labile toxin (dmLT), for capacity to induce mucosal immune responses and immunological memory against the primary vaccine antigens, ie, colonization factors, heat-labile toxin B-subunit and O antigen. The studies show that ETVAX could induce strong intestine-derived and/or fecal immune responses in a majority of vaccinated Swedish adults and in different age groups, including infants, in Bangladesh.

Enterotoxigenic Escherichia coli (ETEC) are ubiquitous diarrheal pathogens that thrive in areas lacking basic human needs of clean water and sanitation. These genetically plastic organisms cause tremendous morbidity among disadvantaged young children, in the form of both acute diarrheal illness and sequelae of malnutrition and growth impairment. The recent discovery of additional plasmid-encoded virulence factors and elucidation of their critical role in the molecular pathogenesis of ETEC may inform new approaches to the development of broadly protective vaccines. Although the pathogens have been closely linked epidemiologically with nondiarrheal sequelae, these conditions remain very poorly understood. Similarly, while canonical effects of ETEC toxins on cellular signaling promoting diarrhea are clear, emerging data suggest that these toxins may also drive changes in intestinal architecture and associated sequelae. Elucidation of molecular events underlying these changes could inform optimal approaches to vaccines that prevent acute diarrhea and ETEC-associated sequelae.

Diarrhoeal disease attributable to enterotoxigenic Escherichia coli (ETEC) causes substantial morbidity and mortality predominantly in paediatric populations in low- and middle-income countries. In addition to acute illness, there is an increasing appreciation of the long-term consequences of enteric infections, including ETEC, on childhood growth and development. Provision of potable water and sanitation and appropriate clinical care for acute illness are critical to reduce the ETEC burden. However, these interventions are not always practical and may not achieve equitable and sustainable coverage. Vaccination may be the most cost-effective and equitable means of primary prevention; however, additional data are needed to accelerate the investment and guide the decision-making process for ETEC vaccines. First, to understand and quantify the ETEC disease burden, additional data are needed on the association between ETEC infection and physical and cognitive stunting as well as delayed educational attainment. Furthermore, the role of inappropriate or inadequate antibiotic treatment of ETEC-attributable diarrhoea may contribute to the development of antimicrobial resistance (AMR) and needs further elucidation. An ETEC vaccine that mitigates acute diarrhoeal illness and minimizes the longer-term disease manifestations could have significant public health impact and be a cost-effective countermeasure. Herein we review the ETEC vaccine pipeline, led by candidates compatible with the general parameters of the Preferred Product Characteristics (PPC) recently developed by the World Health Organization. Additionally, we have developed an ETEC Vaccine Development Strategy to provide a framework to underpin priority activities for researchers, funders and vaccine manufacturers, with the goal of addressing globally unmet data needs in the areas of research, product development, and policy, as well as commercialization and delivery. The strategy also aims to guide prioritization and co-ordination of the priority activities needed to minimize the timeline to licensure and use of ETEC vaccines, especially in in low- and middle-income countries, where they are most urgently needed.

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.

The broiler industry in the Middle East (ME) faces many challenges related to bacterial infections, including M. gallisepticum, M. synoviae, E. coli, and other gram-negative bacteria, exacerbated by various errors in the brooding process. Antibiotics use in the first three days of life, such as Linco-Spectin 100 SP, tilmicosin, enrofloxacin, tylosin, colistin, and doxycycline, is the trend in the market to control such challenges. This study aimed to evaluate the efficacy of the newly introduced aroA E. coli vaccine (Poulvac E. coli) and its ability to reduce over-reliance on the heavy use of antibiotics in the ME. The study was conducted on 160 broiler chicks, divided into eight even groups. Each group was treated differently in terms of antibiotic therapy and ages at the time of Poulvac E. coli administration and the challenge of virulent avian pathogenic E. coli (APEC), serotype O78. Spray application of Poulvac E. coli at seven days of age plus Linco-Spectin 100 SP during the first three days provided the best results for zero mortality after challenge with APEC, while Poulvac E. coli at seven days with enrofloxacin during the early three days resulted in 10% mortality. Poulvac E. coli hatchery vaccination protected birds against mortality but reduced body weight gain compared to the 7-day group vaccinated with Linco-Spectin 100 SP during the first three days. Poulvac E. coli given on day one or day seven did not affect the immune response to concurrent respiratory viral vaccines and, in some cases, improved response. This study shows that Poulvac E. coli at seven days of age, together with Linco-Spectin 100 during the first three days, has produced the best results in terms of protection and performance in the ME high presence of avian pathogenic E. coli field challenge.

Currently, there are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea in developing countries and the most common cause of travelers' diarrhea. A vaccine preventing ETEC bacteria from colonization at small intestines and neutralizing enterotoxin toxicity is expected to be effective against ETEC diarrhea. Protein-based multivalent vaccine candidate MecVax was demonstrated recently to induce antibodies neutralizing heat-labile toxin (LT) and heat-stable toxin (STa) enterotoxicity and inhibiting adherence of seven ETEC adhesins (CFA/I, CS1 to CS6) but also to protect against ETEC toxin-mediated clinical diarrhea in a pig challenge model. To further evaluate MecVax preclinical efficacy against ETEC colonization at small intestines, in this study, we intramuscularly immunized adult rabbits with MecVax, challenged rabbits with ETEC strain H10407 (CFA/I, LT, STa), and examined prevention of bacteria intestinal colonization. Data showed that rabbits immunized with MecVax developed antibodies to both ETEC toxins (LT, STa) and seven adhesins (CFA/I, CS1 to CS6) and had over 99.9% reduction of H10407 intestinal colonization, indicating that the broadly immunogenic ETEC vaccine candidate MecVax is protective against ETEC H10407 intestinal colonization. This study also confirmed that parenteral administration of a protein-based vaccine can prevent bacteria intestinal colonization. Protection against ETEC intestinal colonization demonstrated by this rabbit study, in conjugation with protection against ETEC enterotoxin-mediated clinical diarrhea from a previous pig challenge study, suggested that MecVax can potentially be an effective ETEC vaccine and a combined pig and rabbit challenge model can evaluate ETEC vaccine preclinical efficacy.

Avian pathogenic

The study assessed long-term immunopersistence and safety of the

Antibiotic resistance among gram-negative bacteria continues to rise globally at an alarming rate. New vaccines that prevent bacterial infections and reduce antibiotic use could provide a potential solution to these problems. This study focused on development of an investigational vaccine to prevent recurrent urinary traction infections (UTI) caused by gram-negative bacteria that use type 1 pili to adhere to, invade, and colonize human bladders. The vaccine antigen is FimH, an adhesin protein on the tip of type 1 pili with a lectin binding domain that enables attachment to glycoproteins on mammalian bladders. This was a phase 1, open-label, dose escalation study evaluating the vaccine in 67 healthy women with and without histories of recurrent UTI. The objectives of the study were to evaluate the safety, tolerability, and immunogenicity of different dosages of the antigen and adjuvant of the vaccine. All dosages were well-tolerated and a low incidence of systemic reactions occurred. No serious adverse events related to the vaccine were reported. The vaccine induced both binding and functional antibodies. The women with histories of recurrent UTI demonstrated greater than 150-fold increases in antibodies against the N-terminal region of FimH. Based on the results of this phase 1 study, this vaccine is proceeding to a double-blind, randomized, placebo-controlled phase 2 study. If this vaccine is successful in future studies, it could potentially prevent millions of recurrent UTI globally and reduce the development of antibiotic resistance.

Poultry colibacillosis has been one of the major causes behind economic losses in the poultry production; however, no effective method for its prevention has been developed so far. Vaccination against colibacillosis is capturing increasing interest. The aim of this study was to demonstrate benefits from using a live, aroA gene–deleted vaccine against colibacillosis in broiler chickens and its potential impact on reduced use of antibiotics, the efficacy of vaccination against infectious bronchitis (IB), and the structure and properties of Escherichia coli population in broilers under commercial farm conditions. In 2 experiments, carried out on 3 farms, broiler chickens of one chicken house from each farm were vaccinated against Escherichia coli (E. coli), whereas birds of other chicken houses of each farm were not vaccinated against E. coli. In experiment 1 , which was carried out on 2 farms, for 3 consecutive production cycles, spray vaccination of day-old broilers against E. coli decreased the number of E. coli isolates from internal organs but not from the respiratory system in the sixth week of birds' life. In experiment 1 , E. coli–vaccinated broilers did not receive the antimicrobials until 14 d after the vaccination. Escherichia coli isolates from the E. coli–vaccinated birds were more susceptible to the antimicrobials. Escherichia coli vaccination had no impact on the IB vaccination efficiency; it has reduced the mean number of days of the antimicrobial treatment and improved broiler production parameters. In experiment 2 , chickens of both houses received the antimicrobials for the first 4 d of their life. Birds of chicken house 1 were vaccinated against E. coli on the ninth day of life, whereas birds of chicken house 2 were not vaccinated. In both houses, further antimicrobial usage was the same, and antimicrobials were not used until 14 d after E. coli vaccination. Similar to experiment 1 , in experiment 2 , vaccination decreased the number of E. coli isolates, and these isolates were more susceptible to the antimicrobials. Vaccination of broilers against E. coli should be considered in terms of routine immunoprophylaxis.

Uropathogenic Escherichia coli (UPEC) are common pathogens in urinary tract infections (UTIs), which show resistance to antibiotics. Therefore, there is a need for a vaccine to reduce susceptibility to the infection. In the present study, bioinformatics approaches were employed to predict the best B and T-cell epitopes of UPEC virulence proteins to develop a multiepitope vaccine candidate against UPEC. Then, the efficacy of the candidate was studied with and without Freund adjuvant. Using bioinformatics methods, 3 epitope-rich domains of IutA and FimH antigens were selected to construct the fusion. Molecular docking and Molecular dynamics (MD) simulation were employed to investigate in silico interaction between designed vaccine and Toll-like receptor 4 (TLR4). Our results showed that the levels of IgG and IgA antibodies were improved in the serum and mucosal samples of the vaccinated mice, and the IgG responses were maintained for at least 6 months. The fusion protein was also able to enhance the level of cytokines IFN.γ (Th1), IL.4 (Th2), and IL.17. In challenge experiments, all vaccine combinations showed high potency in the protection of the urinary tract even after 6 months post first injection. The present study indicates that the designed candidate is able to evoke strong protective responses which warrant further studies.

Highlights • Oral co-administration of dmLT with U-Omp19 increases dmLT immunogenicity. • U-Omp19 oral co-delivery with dmLT induces anti-LT antibody responses. • U-Omp19 co-administered with dmLT protects against oral challenge with LT. • U-Omp19 can allow antigen dose sparing by oral route. • U-Omp19 can be used as adjuvant in an oral vaccine formulation against ETEC., Acute diarrhea disease caused by bacterial infections is a major global health problem. Enterotoxigenic Escherichia coli (ETEC) is one of the top causes of diarrhea-associated morbidity and mortality in young children and travelers to low-income countries. There are currently no licensed vaccines for ETEC. Induction of immunity at the site of entry of the bacteria is key to prevent infection. Current approaches to ETEC vaccines include a less toxic mutant form of E. coli heat-labile toxin (double-mutant heat-labile enterotoxin -dmLT-) with both antigenic and immunostimulatory properties. U-Omp19 is a protease inhibitor from Brucella spp. with immunostimulatory properties that has been used as oral adjuvant. In this work, we use U-Omp19 as adjuvant in an oral vaccine formulation against ETEC containing dmLT in outbred and inbred mice. To evaluate antigen dose sparing by U-Omp19 three different immunization protocols with three different doses of dmLT were evaluated. We demonstrated that U-Omp19 co-delivery increases anti-LT IgA in feces using a mid-dose of dmLT following a prime-boost protocol (after one or two boosts). Oral immunization with U-Omp19 induced protection against LT challenge when co-formulated with dmLT in CD-1 and BALB/c mice. Indeed, there was a significant increase in anti-LT IgG and IgA avidity after a single oral administration of dmLT plus U-Omp19 in comparison with dmLT delivered alone. Interestingly, sera from dmLT plus U-Omp19 vaccinated mice significantly neutralize LT effect on intestine inflammation in vivo compared with sera from the group immunized with dmLT alone. These results demonstrate the adjuvant capacity of U-Omp19 to increase dmLT immunogenicity by the oral route and support its use in an oral subunit vaccine formulation against ETEC.

Background The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its numerous serogroups has promoted the development of biological immunostimulatory materials as new vaccine candidates in poultry farms. Bacterial outer membrane vesicles (OMVs), known as spherical nanovesicles enriched with various immunostimulants, are naturally secreted by Gram-negative bacteria, and have gained much attention for developing effective vaccine candidates. Recent report has demonstrated that OMVs of APEC O78 can induce protective immunity in chickens. Here, a novel multi-serogroup OMVs (MOMVs) vaccine was developed to achieve cross-protection against APEC infection in broiler chickens. Results In this study, OMVs produced by three APEC strains were isolated, purified and prepared into MOMVs by mixing these three OMVs. By using SDS-PAGE and LC–MS/MS, 159 proteins were identified in MOMVs and the subcellular location and biological functions of 20 most abundant proteins were analyzed. The immunogenicity of MOMVs was evaluated, and the results showed that MOMVs could elicit innate immune responses, including internalization by chicken macrophage and production of immunomodulatory cytokines. Vaccination with MOMVs induced specific broad-spectrum antibodies as well as Th1 and Th17 immune responses. The animal experiment has confirmed that immunization with an appropriate dose of MOMVs could not cause any adverse effect and was able to reduce bacteria loads and pro-inflammatory cytokines production, thus providing effective cross-protection against lethal infections induced by multi-serogroup APEC strains in chickens. Further experiments indicated that, although vesicular proteins were able to induce stronger protective efficiency than lipopolysaccharide, both vesicular proteins and lipopolysaccharide are crucial in MOMVs-mediated protection. Conclusions The multi-serogroup nanovesicles produced by APEC strains will open up a new way for the development of next generation vaccines with low toxicity and broad protection in the treatment and control of APEC infection.

Summary Background 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. Methods 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 . Findings 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. Interpretation 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. Funding PATH (Bill & Melinda Gates Foundation and the UK's Department for International Development), the Swedish Research Council, and The Swedish Foundation for Strategic Research.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease among children in developing countries, and there are no licensed vaccines to protect against ETEC. Passive immunization by oral delivery of ETEC-specific secretory IgAs (sIgAs) could potentially provide an alternative approach for protection in targeted populations. In this study, a series of physiochemical techniques and an in vitro gastric digestion model were used to characterize and compare key structural attributes and stability profiles of 3 anti–heat-labile enterotoxin mAbs (sIgA1, sIgA2, and IgG1 produced in CHO cells). The mAbs were evaluated in terms of primary structure, N-linked glycan profiles, size and aggregate content, relative apparent solubility, conformational stability, and in vitro antigen binding. Compared to IgG1 mAb, sIgA1 and sIgA2 mAbs showed increased sample heterogeneity, especially in terms of N-glycan composition and the presence of higher molecular weight species. The sIgA mAbs showed overall better physical stability and were more resistant to loss of antigen binding activity during incubation at low pH, 37°C with pepsin. These results are discussed in terms of future challenges to design stable, low-cost formulations of sIgA mAbs as an oral supplement for passive immunization to protect against enteric diseases in the developing world.

There are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading bacterial cause of children’s diarrhea and travelers’ diarrhea. MecVax, a multivalent E. coli vaccine candidate composed of two epitope- and structure-based polyvalent proteins (toxoid fusion 3xSTa(N12S)-mnLT(R192G/L211A) and colonization factor antigen [CFA]/I/II/IV multiepitope fusion antigen [MEFA]), is designed to induce broad antiadhesin and antitoxin antibodies against heterogeneous ETEC pathovars. When administered intraperitoneally or intramuscularly, MecVax was shown to induce antibodies against seven ETEC adhesins (CFA/I and CS1 to CS6) produced by ETEC pathovars that cause over 60% of ETEC-associated diarrheal cases and moderate-to-severe cases and both toxins (heat-labile toxin [LT] and heat-stable toxin [STa]) expressed by all ETEC strains. To further characterize the immunogenicity of this protein-based injectable subunit vaccine candidate and to explore other parenteral administration routes for the product, in this study we immunized mice intradermally (i.d.) with MecVax and measured antigen-specific antibody responses and further antibody functional activities against the adhesins and toxins targeted by the vaccine. Data showed that mice immunized i.d. with MecVax developed robust anti-CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, LT and anti-STa IgG responses. Furthermore, antibodies derived from MecVax administered via the i.d. route inhibited the adherence of ETEC or E. coli strains expressing any of the seven target adhesins (CFA/I and CS1 to CS6) and neutralized the enterotoxicity of LT and STa. These results confirmed broad immunogenicity of MecVax and suggested that this multivalent ETEC subunit vaccine candidate can be effectively delivered via the i.d. route. IMPORTANCE ETEC is a leading bacterial cause of diarrhea in children living in developing countries and international travelers. Developing an effective vaccine for ETEC diarrhea has been hampered because of the challenges of virulence heterogeneity and the difficulties of inducing neutralizing antibodies against the key toxin STa. MecVax, a subunit vaccine candidate carrying two polyvalent protein antigens, for the first time induces functional antibodies against the most important ETEC adhesins, which are associated with a majority of diarrheal cases and moderate-to-severe cases, and also against the enterotoxicity of LT and more importantly STa, which plays a key role in children’s diarrhea and travelers’ diarrhea, potentially leading to the development of a truly effective ETEC vaccine. Data from this study may also indicate that this ETEC subunit vaccine can be administered effectively via the i.d. route, expanding clinical administration options for this vaccine product.

EnterotoxigenicEscherichia coli(ETEC) strains are a leading cause of children’s and travelers’ diarrhea. Developing effective vaccines against this heterologous group has proven difficult due to the varied nature of toxins and adhesins that determine their pathology. A multivalent candidate vaccine was developed using a multi-epitope fusion antigen (MEFA) vaccinology platform and shown to effectively elicit broad protective antibody responses in mice and pigs. However, direct protection against ETEC colonization of the small intestine was not measured in these systems. Colonization of ETEC strains is known to be a determining factor in disease outcomes and is adhesin-dependent. In this study, we developed a non-surgical rabbit colonization model to study immune protection against ETEC colonization in rabbits. We tested the ability for the MEFA-based vaccine adhesin antigen, in combination with dmLT adjuvant, to induce broad immune responses and to protect from ETEC colonization of the rabbit small intestine. Our results indicate that the candidate vaccine MEFA antigen elicits antibodies in rabbits that react to seven adhesins included in its construction and protects against colonization of a challenge strain that consistently colonized naïve rabbits.

RESEARCH HIGHLIGHTSPan-RV analysis was used for the first time in the discovery of APEC-protective proteins.A total of 53 potential protective proteins were screened out.Four proteins were verified as potential vaccine candidates using western blotting.

Avian pathogenic Escherichia coli (APEC) can cause colibacillosis in poultry, characterised by localised or systemic infections. Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carcass condemnations. A live attenuated Escherichia coli O78 aroA gene mutant is widely used to prevent disease. However, no effective strategies to differentiate the vaccine strain from field strains are available, hampering follow-up of vaccination campaigns. In the current study, we report a PCR-based method to simultaneously detect the vaccine strain by targeting the vaccine-specific mutation in the aroA gene, as well as the wild type E. coli strains by targeting the xanQ gene. The specificity of this PCR was evaluated using 123 E. coli isolates, form which 5 WT aroA auxotrophic strains (WT strains with a natural aroA deficiency), as well as 7 non-Escherichia isolates. The PCR showed 100% sensitivity of the xanQ primers for E. coli detection and 100% sensitivity of the ΔaroA primers for the vaccine strain. In order to allow quantification of the vaccine strain in complex samples containing many different E. coli strains and other related organisms, such as chicken faeces, a probe-based duplex qPCR was developed. The limit of detection (LOD) of this duplex qPCR method was 8.4*103 copies/g faeces. The specificity of the duplex qPCR was confirmed by determining both the vaccine strain levels, and the total E. coli load in intestinal digesta from both vaccinated and non-vaccinated birds. E. coli could be detected in both vaccinated and non-vaccinated birds. The duplex qPCR was specific for the vaccine strain as this strain was detected in all vaccinated birds, whereas no signal was detected in non-vaccinated birds. The duplex qPCR is helpful in monitoring colonization and shedding of the vaccine strain.

Vaccination against coliform mastitis has become part of mastitis control programs in the past 3 decades, as a means of reducing the severity of clinical mastitis. Our study objective was to evaluate the effect of 2 commercially available vaccines on clinical, behavioral, and antibody response following Escherichia coli intramammary challenge in cows near peak lactation. Cows (n = 12 per group) were vaccinated with vaccine 1 (V1) or vaccine 2 (V2) at dry-off, 21 d pre-calving, and 14 d post-calving. Twelve cows served as unvaccinated controls (CTL). Cows were challenged with E. coli in a rear quarter at approximately 100 d in milk. Milk samples were collected pre- and post-challenge to enumerate E. coli and determine somatic cell count. Serum was collected before each vaccination and at d 0, 1, 2, 3, 6, 30, and 60 relative to challenge, to study antibody response. Milk IgA and tumor necrosis factor-α concentrations were determined in whey. Vaginal temperature, cow activity, and milk yield and components were monitored post-challenge. Bacterial count, somatic cell score, milk yield and component decline, vaginal temperature, activity measures, and antibody and cytokine response were analyzed for treatment differences. The effects of parity, breed, and a repeated measure of time were also tested. Seven cows had to be removed from the study post-challenge for antibiotic treatment (CTL and V1, n = 3 each; V2, n = 1), 2 of which were euthanized (both CTL). Vaccinated cows exhibited fever (vaginal temperature ≥39.4°C) 3 h earlier than CTL cows, but we found no differences between treatments for bacterial count, somatic cell score, or milk yield reduction. Vaccinated cows spent more time lying per rest bout 2 d post-challenge, but total daily lying time was not different from CTL cows during the 7 d post-challenge. The vaccines differed in antibody response: V1 cows had greater serum IgG1 and IgG2 post-challenge. A parity effect was also evident: primiparous cows had lower bacterial counts, somatic cell score and a smaller milk yield decline than multiparous cows, but also had lower antibody production. Immunization with either J5 bacterin did not reduce clinical signs of mastitis in cows challenged at 100 d in milk, demonstrating that the effects of J5 vaccination had diminished at peak lactation.

Multiple diarrheagenic enteric bacterial infections cause global morbidity and mortality. A combination vaccine is needed to combat different diarrhea-causing organisms. In our present work, we formulated a combination of antigens from three different diarrheagenic Escherichia coli strains and three different Vibrio cholerae strains. We demonstrated that our newly formulated combination immunogen was able to raise species-specific immunogenicity. This formulation also gave protection against different diarrheagenic E. coli strains in the removable intestinal tie-adult rabbit diarrhea model. However, protective efficacy was not found against the V. cholerae El Tor Ogawa Haitian variant, but challenged with V. cholerae El Tor Inaba or O139 showed protection in rabbits. This is the first report of a single formulated nonliving heat-killed combination immunogen from different diarrheagenic E. coli and V. cholerae that could bestow protection against different bacteria in an animal model.

Background: Caused by bacterial, viral, and parasitic pathogens, diarrhea is the second leading cause of death among children under five. Two strains of E. coli, namely Enterotoxigenic, ETEC and Enterohemorrhagic EHEC are the most important causes of this disease in developing countries. EHEC is a major causative agent of bloody diarrhea and hemorrhagic uremic syndrome, while ETEC is the most important cause of diarrhea in neonates and travelers. Objective: To evaluate the immunologic properties of a subunit vaccine candidate comprising the main immunogenic epitopes from these two bacterial strains. Methods: The construct comprised of LTB and CfaB antigens from ETEC, and Intimin and Stx2B antigens from EHEC, was designed, analyzed and synthesized using bioinformatics methods. The chimeric gene was sub-cloned in the expression vector and expressed in E. coli host. The purified chimera protein was injected subcutaneously into the experimental animals. The production of specific antibodies was confirmed by immunological methods, and the protection capacity was evaluated by the challenge of immunized mice with the pathogenic bacteria. Results: Chimeric recombinant protein was able to increase IgG titer. Neutralization assay indicated that the antibodies generated against LtB moiety were able to neutralize ETEC toxin. In animal challenge study, all non-immune mice died within 3 days after the injection of toxin, but all immunized mice survived from Stx toxin. Conclusion: The immunity to both ETEC and EHEC bacteria is significant, and this structure can be considered as a candidate for vaccine production against these bacterial strains.

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.

Highlights • The killed oral ETEC vaccine ETVAX ± dmLT adjuvant was safe in Bangladeshi adults. • All vaccinees responded to all 5 primary vaccine antigens in ALS specimens. • A majority of vaccinees responded to ≥4 antigens in plasma specimens. • A sensitive electrochemiluminescence assay was established for small sample volumes. • ALS responses measured by electrochemiluminescence and ELISA assays correlated well., 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

Well-established, validated and clinically meaningful primary and secondary endpoints are critical in advancing vaccines through proof of principal studies, licensure and pre-qualification. To that end, the field of vaccine development for Shigella, enterotoxigenic Escherichia coli (ETEC) as well as other enteric pathogens would benefit greatly from a focused review of clinical endpoints and the use of common endpoints across the field to enable study-to-study comparisons as well as comparative assessments between vaccine candidates. A workshop was conducted to review clinical endpoints from controlled human challenge studies, field studies in naive adult travelers and pediatric studies in low-middle income countries and to develop a consensus on clinical endpoints for future vaccine trials. Following sequential presentations on different study designs (CHIM, travelers’ efficacy and pediatric efficacy), workshop participants broke into three simultaneous workgroups focused on those study designs to discuss a number of topics key to clinical endpoints specific to each study design. Previously utilized endpoints were reviewed with an eye towards potentially novel endpoints for future studies and consideration of the disease parameters and spectrum of disease targeted for prevention. The strength of support among workshop participants for the use of various endpoints is summarized as are recommendations for additional endpoints to be considered in future studies. It is anticipated that this report will facilitate endpoint determination in future efficacy trials of vaccine candidates.

In the absence of good animal models, Controlled Human Infection Models (CHIMs) are useful to assess efficacy of new vaccine candidates against Enterotoxic Escherichia coli (ETEC), as well as other preventive or therapeutic interventions. At the 2018 Vaccines Against Shigella and ETEC (VASE) conference, a workshop was held to further review and discuss new challenge model developments and key issues related to further model standardization. During the workshop, invited speakers briefly summarized for attendees recent developments and main agenda issues before workshop participants were divided into four groups for more focused discussions. The main issues discussed were: (1) whether there is a need for more ETEC strains to test a diversity of vaccine candidates, and if so, what criteria/qualities are desirable in strain selection; (2) how ETEC CHIMs could be more standardized to better support ETEC vaccine development; (3) how volunteer selection criteria and screening should be performed, and; (4) how an expanded sample collection schema and collaborative analysis plan may facilitate a more in-depth assessment of the role of antigen-specific humoral and cellular immune responses in ETEC infection, and provide better insights into ETEC pathogenesis and correlates of protection. The workshop concluded that additional challenge strains may need to be developed to better support new vaccines and therapeutics that are advancing in the development pipeline. In this regard, the need for a well characterized ST-only expressing ETEC strain was highlighted as a priority given that promising new heat stable toxoid based vaccine candidates are on the horizon. In addition, further standardization of the ETEC CHIMs was strongly encouraged, noting that it may not be realistic to standardize across all strains. Also, intensified volunteer screening may result in higher attack rates, although more stringent eligibility criteria may contribute to a more limited application of the model and diminish its representativeness. Finally, a sampling schedule and priority list for minimum set of samples was also proposed. Future workshops could be held to further refine standards for ETEC CHIMS and to facilitate more collaborative work on stored sample sets from previous and future ETEC CHIMs to maximize the contribution of these trials to our understanding of ETEC pathogenesis and our development of better prevention and control measures for this important pathogen. publishedVersion

A new HPV-16/18 bivalent vaccine expressed by the Escherichia coli has been proven to be efficacious in adult women. A randomized, immunogenicity noninferiority study of this candidate vaccine was conducted in December 2015 in China. Girls aged 9–14 years were randomized to receive 2 doses at months 0 and 6 (n=301) or 3 doses at months 0, 1 and 6 (n=304). Girls aged 15–17 years (n=149) and women aged 18–26 years (n=225) received 3 doses. The objectives included noninferiority analysis of the IgG geometric mean concentration (GMC) ratio (95% CI, lower bound>0.5) to HPV-16 and HPV-18 at month 7 in girls compared with women. In the per-protocol set, the GMC ratio of IgG was noninferior for girls aged 9–17 years receiving 3 doses compared with women (1.76 (95% CI, 1.56, 1.99) for HPV-16 and 1.93 (95% CI, 1.69, 2.21) for HPV-18) and noninferior for girls aged 9–14 years receiving 2 doses compared with women (1.45 (95% CI, 1.25, 1.62) for HPV-16 and 1.17 (95% CI, 1.02, 1.33) for HPV-18). Noninferiority was also demonstrated for neutralizing antibodies. The immunogenicity of the HPV vaccine in girls receiving 3 or 2 doses was noninferior compared with that in young adult women. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s11427-019-9547-7 and is accessible for authorized users.

Summary Previous epidemiological studies have shown that enterotoxins from enterotoxigenic Escherichia coli (ETEC) appear to be the most important causes of neonatal piglet and porcine post‐weaning diarrhoea (PWD). Thus, it is necessary to develop an effective vaccine against ETEC infection. In the present study, the Kil cassette was inserted into the pseudogene yaiT by homologous recombination to create an attenuated E. coli double selection platform O142(yaiT‐Kil). After that, PRPL‐Kil was replaced with a fusion gene (LTA1‐STa13‐STb‐LTA2‐LTB‐STa13‐STb) to establish oral vaccines O142(yaiT::LTA1‐STa13‐STb‐LTA2‐LTB‐STa13‐STb) (ER‐T). Subsequently, BALB/c mice were orally immunized with ER‐T. Results showed that serum IgG and faecal sIgA responded against all ETEC enterotoxins and induced F41 antibody in BALB/c mice by orogastrically inoculation with recombinant E. coli ER‐T. Moreover, the determination of cellular immune response demonstrated that the stimulation index (SI) was significantly higher in immunized mice than in control mice, and a clear trend in the helper T‐cell (Th) response was Th2‐cell (IL‐4) exceed Th1‐cell (IFN‐γ).Our results indicated that recombinant E. coli ER‐T provides effective protection against ETEC infection.

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

Enterotoxigenic Escherichia coli (ETEC) is a major cause of travelers’ diarrhea and of diarrhea among young children in developing countries. Experimental challenge studies in adult volunteers have played a pivotal role in establishing ETEC as an enteric pathogen, elucidating its pathogenesis by identifying specific virulence attributes, characterizing the human immune response to clinical and sub-clinical ETEC infection and assessing preliminarily the clinical acceptability, immunogenicity and efficacy of prototype ETEC vaccines. This review provides a historical perspective of experimental challenge studies with ETEC. It summarizes pioneering early studies carried out by investigators at the University of Maryland School of Medicine to show how those studies provided key information that influenced the directions taken by many research groups to develop vaccines to prevent ETEC. In addition, key experimental challenge studies undertaken at other institutions will also be cited.

E. coli infections in avian species have become an economic threat to the poultry industry worldwide. The objective of this study is to determine whether the immunization using commercially available living Escherichia coli vaccines as Nisseiken Avian Colibacillosis Vaccine (Nisseiken Co., Ltd., Ome, Tokyo, Japan) and O78 aroA deleted vaccine (Poulvac ® E.coli, Zoetis) are protective against APEC challenges or not. Ninety eight chicks (Arbor, Acres) of both sexes were divided into seven groups (14 birds/each); two groups were vaccinated at day 1 of age by spray route using Nisseiken Avian colibacillosis Vaccine, then one of them challenged intratracheally with homologous E.coli O78 and the other with heterologus O1 at day 14, the other two groups were vaccinated at day 1 of age by eye drop route using Poulvac ® E.coli, Zoetis vaccine then one of them challenged intratracheally with homologus E.coli O78 and the other with heterologus O1 at day 14. The other two groups were positive control (challenged, unvaccinated); one challenged with O78 and the other with O1 at day 14 using intratracheal route. The last group served as environmental control (non vaccinated, non challenged). At day 28, birds were necropised and examined to evaluate the efficacy of both of the two different vaccines. The best obtained results were recorded to the vaccinated challenged groups with the homologous and heterologous strains and vaccinated by spraying and eye drop methods which showed a decrease in organ lesion scores in comparison to the other groups (non-vaccinated, challenged broilers). These results suggest that the two different vaccines used in our study are efficient in reducing lesion scores against homologous and heterologous challenge using spray and eye drop methods that could lead to minimizing the time for treatment and cases of condemnation in processing plants.

Background There is no licensed vaccine against enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhea-associated morbidity and mortality among infants and children in low-income countries and travelers. The results of this vaccination/challenge study demonstrate strong protection by an attenuated ETEC vaccine candidate, ACE527, when co-administered with a mucosal adjuvant, the double-mutant heat-labile toxin (dmLT) of ETEC. Methods Sixty healthy adults participated in a randomized, placebo-controlled, double-blind study with three doses of lyophilized ACE527 (∼3 × 109 of each strain per dose) administered orally with or without dmLT adjuvant (25 µg/dose). Six months later, 36 of these volunteers and a control group of 21 unvaccinated volunteers were challenged with virulent ETEC strain H10407. The primary outcome was severe diarrhea, defined as passing >800 g of unformed stools during the inpatient period following challenge. Findings The vaccine was well tolerated and induced robust immune responses to key antigens. The protective efficacy (PE) against the primary outcome of severe diarrhea was 65.9% (95% confidence interval [CI] 5.4–87.7, p = 0.003). Among subjects receiving the adjuvanted vaccine, the attack rate of severe diarrhea was 23.1, while in unimmunized controls it was 67.7%. The PE against diarrhea of any severity was 58.5% (95% CI 3.8– 82.1, p = 0.016). There was a strong inverse correlation between shedding of the vaccine strain after either of the first two doses and absence of severe diarrhea upon challenge (RR = 0.29, 95% CI 0.08–1.05, p = 0.041). Challenge strain shedding was 10-fold lower in those receiving the adjuvant than in those receiving vaccine alone. The unadjuvanted vaccine was not protective (PE = 23.1%). Interpretation The results of this study support further development of ACE527 + dmLT as a vaccine for children in endemic countries and travelers. This is the first clinical demonstration that dmLT can contribute significantly to vaccine efficacy and may warrant testing with other oral vaccines. ( ClinicalTrials.gov registration: NCT01739231).

International audience; Emergence and dissemination of multidrug resistance among pathogenic Escherichia coli have posed a serious threat to public health across developing and developed countries. In combination with a flexible repertoire of virulence mechanisms, E. coli can cause a vast range of intestinal (InPEC) and extraintestinal (ExPEC) diseases but only a very limited number of antibiotics still remains effective against this pathogen. Hence, a broad spectrum E. coli vaccine could be a promising alternative to prevent the burden of such diseases, while offering the potential for covering against several InPEC and ExPEC at once. SslE, the Secreted and Surface-associated Lipoprotein of E. coli, is a widely distributed protein among InPEC and ExPEC. SslE functions ex vivo as a mucinase capable of degrading mucins and reaching the surface of mucus-producing epithelial cells. SslE was identified by reverse vaccinology as a protective vaccine candidate against an ExPEC murine model of sepsis, and further shown to be cross-effective against other ExPEC and InPEC models of infection. In this study, we aimed to gain insight into the immune response to antigen SslE and identify an immunization strategy suited to generate robust mucosal and systemic immune responses. We showed, by analyzing T cell and antibody responses, that mice immunized with SslE via an intranasal prime followed by two intramuscular boosts developed an enhanced overall immune response compared to either intranasal-only or intramuscular-only protocols. Importantly, we also report that this regimen of immunization did not impact the richness of the murine gut microbiota, and mice had a comparable cecal microbial composition, whether immunized with SslE or PBS. Collectively, our findings further support the use of SslE in future vaccination strategies to effectively target both InPEC and ExPEC while not perturbing the resident gut microbiota.

We tested an oral enterotoxigenic Escherichia coli (ETEC) vaccine, ETVAX, consisting of inactivated E. coli overexpressing the most prevalent ETEC colonization factors (CFs) and a toxoid (LCTBA), in Bangladeshi children for capacity to induce mucosal and plasma immune responses against O78 lipopolysaccharide (LPS) expressed on the vaccine strains. The vaccine was given ± double-mutant heat-labile toxin (dmLT) adjuvant. We evaluated the impact of dmLT on anti-O78 LPS immune responses and whether such responses can predict responses against the CFs as a marker for vaccine "take". Two fractionated doses of ETVAX ± different amounts of dmLT were administered biweekly to groups of children 24-59 (n = 125), 12-23 (n = 97) and 6-11 (n = 158) months of age. Immune responses were evaluated in antibody in lymphocyte supernatants (ALS), fecal extracts and plasma. ALS IgA responses against O78 LPS were induced in 44-49% of the children aged 12-59 months. The magnitudes of the ALS responses were significantly higher in children receiving a half-dose (5 × 1010 bacteria) of ETVAX ± dmLT than in placebo recipients.

Introduction Enterotoxigenic Escherichia coli (ETEC) is a common cause of infectious diarrhoea and a leading cause of morbidity and mortality in children living in resource-limited settings. It is also the leading cause of travellers’ diarrhoea among civilian and military travellers. Its dual importance in global public health and travel medicine highlights the need for an effective vaccine. ETEC express colonization factors (CFs) that mediate adherence to the small intestine. An epidemiologically prevalent CF is coli surface antigen 6 (CS6). We assessed the safety and immunogenicity of a CS6-targeted candidate vaccine, CssBA, co-administered intramuscularly with the double-mutant heat-labile enterotoxin, dmLT [LT(R192G/L211A)]. Methods This was an open-label trial. Fifty subjects received three intramuscular injections (Days 1, 22 and 43) of CssBA alone (5 µg), dmLT alone (0.1 µg) or CssBA (5, 15, 45 µg) + dmLT (0.1 and 0.5 µg). Subjects were actively monitored for adverse events for 28 days following the third vaccination. Antibody responses (IgG and IgA) were characterized in the serum and from lymphocyte supernatants (ALS) to CS6 and the native ETEC heat labile enterotoxin, LT. Results Across all dose cohorts, the vaccine was safe and well-tolerated with no vaccine-related severe or serious adverse events. Among vaccine-related adverse events, a majority (98%) were mild with 79% being short-lived vaccine site reactions. Robust antibody responses were induced in a dose-dependent manner with a clear dmLT adjuvant effect. Response rates in subjects receiving 45 µg CssBA and 0.5 µg dmLT ranged from 50 to 100% across assays. Conclusion This is the first study to demonstrate the safety and immunogenicity of CssBA and/or dmLT administered intramuscularly. Co-administration of the two components induced robust immune responses to CS6 and LT, paving the way for future studies to evaluate the efficacy of this vaccine target and development of a multivalent, subunit ETEC vaccine.

There are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading cause of diarrhea for children in developing countries and international travelers. Virulence heterogeneity among strains and difficulties identifying safe antigens for protective antibodies against STa, a potent but poorly immunogenic heat-stable toxin which plays a key role in ETEC diarrhea, are challenges in ETEC vaccine development. To overcome these challenges, we applied a toxoid fusion strategy and a novel epitope- and structure-based multiepitope fusion antigen (MEFA) vaccinology platform to construct two chimeric multivalent proteins, toxoid fusion 3xSTa(N12S)-mnLT(R192G/L211A) and adhesin CFA/I/II/IV MEFA, and demonstrated that the proteins induced protective antibodies against STa and heat-labile toxin (LT) produced by all ETEC strains or the seven most important ETEC adhesins (CFA/I and CS1 to CS6) expressed by the ETEC strains causing 60 to 70% of diarrheal cases and moderate to severe cases. Combining two proteins, we prepared a protein-based multivalent ETEC vaccine, MecVax. MecVax was broadly immunogenic; mice and pigs intramuscularly immunized with MecVax developed no apparent adverse effects but had robust antibody responses to the target toxins and adhesins. Importantly, MecVax-induced antibodies were broadly protective, demonstrated by significant adherence inhibition against E. coli bacteria producing any of the seven adhesins and neutralization of STa and cholera toxin (CT) enterotoxicity. Moreover, MecVax protected against watery diarrhea and provided over 70% and 90% protection against any diarrhea from an STa-positive or an LT-positive ETEC strain in a pig challenge model. These results indicated that MecVax induces broadly protective antibodies and prevents diarrhea preclinically, signifying that MecVax is potentially an effective injectable vaccine for ETEC. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) bacteria are a top cause of children’s diarrhea and travelers’ diarrhea and are responsible for over 220 million diarrheal cases and more than 100,000 deaths annually. A safe and effective ETEC vaccine can significantly improve public health, particularly in developing countries. Data from this preclinical study showed that MecVax induces broadly protective antiadhesin and antitoxin antibodies, becoming the first ETEC vaccine candidate to induce protective antibodies inhibiting adherence of the seven most important ETEC adhesins and neutralizing the enterotoxicity of not only LT but also STa toxin. More importantly, MecVax is shown to protect against clinical diarrhea from STa-positive or LT-positive ETEC infection in a pig challenge model, recording protection from antibodies induced by the protein-based, injectable, subunit vaccine MecVax against ETEC diarrhea and perhaps the possibility of intramuscularly administered protein vaccines for protection against intestinal mucosal infection.

Vaccines against enteric diseases could improve global health. Despite this, only a few oral vaccines are currently available for human use. One way to facilitate such vaccine development could be to identify a practical and relatively low cost biomarker assay to assess oral vaccine induced primary and memory IgA immune responses in humans. Such an IgA biomarker assay could complement antigen-specific immune response measurements, enabling more oral vaccine candidates to be tested, whilst also reducing the work and costs associated with early oral vaccine development. With this in mind, we take a holistic systems biology approach to compare the transcriptional signatures of peripheral blood mononuclear cells isolated from volunteers, who following two oral priming doses with the oral cholera vaccine Dukoral®, had either strong or no vaccine specific IgA responses. Using this bioinformatical method, we identify TNFRSF17, a gene encoding the B cell maturation antigen (BCMA), as a candidate biomarker of oral vaccine induced IgA immune responses. We then assess the ability of BCMA to reflect oral vaccine induced primary and memory IgA responses using an ELISA BCMA assay on a larger number of samples collected in clinical trials with Dukoral® and the oral enterotoxigenic Escherichia coli vaccine candidate ETVAX. We find significant correlations between levels of BCMA and vaccine antigen-specific IgA in antibodies in lymphocyte secretion (ALS) specimens, as well as with proportions of circulating plasmablasts detected by flow cytometry. Importantly, our results suggest that levels of BCMA detected early after primary mucosal vaccination may be a biomarker for induction of long-lived vaccine specific memory B cell responses, which are otherwise difficult to measure in clinical vaccine trials. In addition, we find that ALS-BCMA responses in individuals vaccinated with ETVAX plus the adjuvant double mutant heat-labile toxin (dmLT) are significantly higher than in subjects given ETVAX only. We therefore propose that as ALS-BCMA responses may reflect the total vaccine induced IgA responses to oral vaccination, this BCMA ELISA assay could also be used to estimate the total adjuvant effect on vaccine induced-antibody responses, independently of antigen specificity, further supporting the usefulness of the assay.

Despite intensive research, mastitis remains an important disease in dairy cattle with a significant impact on animal welfare, use of antibiotics, and, in the end, the economy of dairy farms. Although vaccines available so far have shown limited efficacy against coliform mastitis, vaccination is considered one of the measures that could limit the consequences of mastitis., Escherichia coli is the leading cause of severe mastitis in dairy farms. As E. coli mastitis is refractory to the hygienic control measures adapted to contagious mastitis, efficient vaccines are in demand. Existing mastitis vaccines, based on the use of killed rough E. coli J5 as the antigen, aim at inducing phagocytosis by neutrophils. We assessed the binding of J5-induced antibodies to isogenic rough and smooth strains along with a panel of mastitis-associated E. coli. Analysis by enzyme-linked immunosorbent assay revealed that antibodies to OmpA or killed J5 bind readily to rough E. coli but poorly to smooth strains. Flow cytometry analysis indicated that immunization with J5 induced antibodies that cross-reacted with rough E. coli strains but with only a small subpopulation of smooth strains. We identified type 1 fimbriae as the target of most antibodies cross-reacting with the smooth strains. These results suggest that the O-polysaccharide of lipopolysaccharide shields the outer membrane antigens and that only fiber antigens protruding at the bacterial surface can elicit antibodies reacting with mastitis-associated E. coli. We evaluated J5-induced antibodies in an opsonophagocytic killing assay with bovine neutrophils. J5 immune serum was not more efficient than preimmune serum, showing that immunization did not improve on the already high efficiency of naturally acquired antibodies to E. coli. In conclusion, it is unlikely that the efficiency of J5 vaccines is related to the induction of opsonic antibodies. Consequently, other research directions, such as cell-mediated immunity, should be explored to improve E. coli mastitis vaccines. IMPORTANCE Despite intensive research, mastitis remains an important disease in dairy cattle with a significant impact on animal welfare, use of antibiotics, and, in the end, the economy of dairy farms. Although vaccines available so far have shown limited efficacy against coliform mastitis, vaccination is considered one of the measures that could limit the consequences of mastitis. One reason for the lack of efficiency of current vaccines likely stems from the current evaluation of vaccines that relies mostly on measuring antibody production against vaccine antigens. This report clearly shows that vaccine-induced antibodies fail to bind to most mastitis-associated E. coli strains because of the presence of an O-antigen and, thus, do not allow for improved phagocytosis of pathogens. As a consequence, this report calls for revised criteria for the evaluation of vaccines and suggests that cell-mediated immunity should be targeted by new vaccinal strategies. More generally, these results could be extended to other vaccine development strategies targeting coliform bacteria.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrheal illness in the military, travelers, and children living in low- to middle-income countries. Increased antibiotic resistance, the absence of a licensed vaccine, and the lack of broadly practical therapeutics perpetuate the significant health and financial burden resulting from ETEC infection. A critical step in the evaluation of vaccines and therapeutics is preclinical screening in a relevant animal disease model that closely replicates human disease. We previously developed a diarrheal model of class 5a colonization factor (CF) CFA/I-expressing ETEC in the New World owl monkey species Aotus nancymaae using ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407. In order to broaden the use of the model, we report here on the development of A. nancymaae models of ETEC expressing the class 5b CFs CS17 and CS19 with strains LSN03-016011/A and WS0115A, respectively. For both models, we observed diarrheal attack rates of ≥80% after oral inoculation with 5 × 1011 CFU of bacteria. These models will aid in assessing the efficacy of future ETEC vaccine candidates and therapeutics.

Subcutaneous vaccination of cattle for enterohemorrhagic Escherichia coli O157:H7 reduces the magnitude and duration of fecal shedding, but the often-required, repeated cattle restraint can increase costs, deterring adoption by producers. In contrast, live oral vaccines may be repeatedly administered in feed, without animal restraint. We investigated whether oral immunization with live stx-negative LEE(+) E. coli O157:H7 reduced rectoanal junction (RAJ) colonization by wild-type (WT) E. coli O157:H7 strains after challenge. Two groups of cattle were orally dosed twice weekly for 6 weeks with 3 × 10(9) CFU of a pool of three stx-negative LEE(+) E. coli O157:H7 strains (vaccine group) or three stx-negative LEE(−) non-O157:H7 E. coli strains (control group). Three weeks following the final oral dose, animals in both groups were orally challenged with a cocktail of four stx(+) LEE(+) E. coli O157:H7 WT strains. Subsequently, WT strains at the RAJ were enumerated weekly for 4 weeks. Serum antibodies against type III secretion protein (TTSP), the translocated intimin receptor (Tir), and EspA were determined by enzyme-linked immunosorbent assay (ELISA) at day 0 (preimmunization), day 61 (postimmunization, prechallenge), and day 89 (postchallenge). Vaccine group cattle had lower numbers of WT strains at the RAJ than control group cattle on postchallenge days 3 and 7 (P ≤ 0.05). Also, vaccine group cattle shed WT strains for a shorter duration than control group cattle. All cattle seroconverted to TTSP, Tir, and EspA, either following immunization (vaccine group) or following challenge (control group). Increased antibody titers against Tir and TTSP postimmunization were associated with decreased numbers of WT E. coli O157:H7 organisms at the RAJ. IMPORTANCE The bacterium E. coli O157:H7 causes foodborne disease in humans that can lead to bloody diarrhea, kidney failure, vascular damage, and death. Healthy cattle are the main source of this human pathogen. Reducing E. coli O157:H7 in cattle will reduce human disease. Using a randomized comparison, a bovine vaccine to reduce carriage of the human pathogen was tested. A detoxified E. coli O157:H7 strain, missing genes that cause disease, was fed to cattle as an oral vaccine to reduce carriage of pathogenic E. coli O157:H7. After vaccination, the cattle were challenged with disease-causing E. coli O157:H7. The vaccinated cattle had decreased E. coli O157:H7 during the first 7 days postchallenge and shed the bacteria for a shorter duration than the nonvaccinated control cattle. The results support optimization of the approach to cattle vaccination that would reduce human disease.

Background Increasing rates of antimicrobial resistance among uropathogens led, among other efforts, to the application of subtractive reverse vaccinology for the identification of antigens present in extraintestinal pathogenic E. coli (ExPEC) strains but absent or variable in non-pathogenic strains, in a quest for a broadly protective Escherichia coli vaccine. The protein coded by locus c5321 from CFT073 E. coli was identified as one of nine potential vaccine candidates against ExPEC and was able to confer protection with an efficacy of 33% in a mouse model of sepsis. c5321 (known also as EsiB) lacks functional annotation and structurally belongs to the Sel1-like repeat (SLR) family. Herein, as part of the general characterization of this potential antigen, we have focused on its structural properties. Results We report the 1.74 Å-resolution crystal structure of c5321 from CFT073 E. coli determined by Se-Met SAD phasing. The structure is composed of 11 SLR units in a topological organisation that highly resembles that found in HcpC from Helicobacter pylori, with the main difference residing in how the super-helical fold is stabilised. The stabilising effect of disulfide bridges in HcpC is replaced in c5321 by a strengthening of the inter-repeat hydrophobic core. A metal-ion binding site, uncharacteristic of SLR proteins, is detected between SLR units 3 and 4 in the region of the inter-repeat hydrophobic core. Crystal contacts are observed between the C-terminal tail of one molecule and the C-terminal amphipathic groove of a neighbouring one, resembling interactions between ligand and proteins containing tetratricopeptide-like repeats. Conclusions The structure of antigen c5321 presents a mode of stabilization of the SLR fold different from that observed in close homologs of known structure. The location of the metal-ion binding site and the observed crystal contacts suggest a potential role in regulation of conformational flexibility and interaction with yet unidentified target proteins, respectively. These findings open new perspectives in both antigen design and for the identification of a functional role for this protective antigen.

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA., Glycoconjugate Journal, 38 (4), ISSN:0282-0080, ISSN:1573-4986