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The mortality and morbidity burden estimation of diarrheal diseases (DD), and Shigella and Enterotoxigenic E. Coli (ETEC) varies among different studies and by the models used for producing these estimates. Understanding the real burden of these important pathogens will guide public health and policy makers to prioritize resources for accelerating interventions against these enteric infections. In addition, long term effects, in the form of growth faltering, cognitive impairment and decreased school performance are important aspects of burden that has not been well captured. Efforts to incorporate these effects and refine their estimation, in the form of Disability Adjusted Life years (DALYs) are very important to inform the burden of diarrheal diseases and Shigella and ETEC specifically. The Institute for Health Metrics and Evaluation (IHME) at the University of Washington conducted a workshop at the VASE 2018 meeting to discuss IHME Global Burden of Diseases (GBD) modelling methods for diarrheal diseases, with a focus on ETEC and Shigella estimates in relation to other pathogens, including limitations, areas of improvements, and IHME plans for future GBD iterations.

Diarrhea remains one of the top five causes of disease and death among young children in developing nations. Fortunately, scientists are making progress developing vaccines against enterotoxigenic E. coli (ETEC) and Shigella, two of the leading diarrhea pathogens. As vaccine developers start to consider field efficacy trials of these vaccines, they should be aware of the importance of evaluating not only vaccine direct effects on the immunized, but also the herd effects that vaccination can afford to the unimmunized in a community. In a workshop held at the conference titled "Vaccines against Shigella and ETEC (VASE)", we described to participants what herd effects are and we presented on methods used in cholera and rotavirus studies that could be useful for future ETEC and Shigella vaccine trials conducted in low and middle-income nations. We also presented evidence on the effects of vaccine herd effects for estimates of vaccine cost-effectiveness.

Enterotoxigenic Escherichia coli (ETEC) is the most frequent bacterial cause of diarrhea particularly reported in children of developing countries and also travelers. Enterotoxins and colonization factor antigens (CFAs) are two major virulence factors in ETEC pathogenesis. Colonization factor antigen I (CFA/I) includes major pilin subunit CfaB, and a minor adhesive subunit (CfaE), and enterotoxins consisting of heat-labile toxin subunit B (LTB) and heat-stable toxin (ST). Chimeric proteins (CCL) carrying epitopes and adjuvant sequences increase the possibility of eliciting a broad cellular or effective immune response. In the present study, a chimeric candidate vaccine containing CfaB*ST, CfaE, and LTB (CCL) was designed via in silico techniques. This chimeric gene was synthesized by using codon usage of E. coli for increasing the expression of the recombinant protein. After designing the chimeric construct, it showed a high antigenicity index estimated by the vaxiJen server. Linear and conformational B-cell epitopes were identified and indicated suitable immunogenicity of this multimeric recombinant protein. Thermodynamic analyses for mRNA structures revealed the appropriate folding of the RNA representative good stability of this molecule. In silico scanning was done to predict the 3D structure of the protein, and modeling was validated using the Ramachandran plot analysis. The chimeric protein (rCCL) was expressed in a prokaryotic expression system (E. coli), purified, and analyzed for their immunogenic properties. It was revealed that the production of a high titer of antibody produced in immunized mice could neutralize the ETEC using the rabbit ileal loop tests. The results indicated that the protein inferred from the recombinant protein (rCCL) construct could act as a proper vaccine candidate against three critical causative agents of diarrheal bacteria at the same time.

Diarrhoea infection is a major global health public problem and is caused by many organisms including diarrheagenic Escherichia coli pathotypes. The common problem with diarrhoea is the drug resistance of pathogenic bacteria, the most promising alternative means of preventing drug resistance is vaccination. However, there has not been any significant success in the prevention of diarrhoea caused by E. coli through vaccination. Epitope-based vaccine is gaining more attention due to its safety and specificity. Sequence variation of protective antigens of the pathogen has posed a new challenge in the development of epitope-based vaccines against the infection, leading to the necessity of multiepitope based design. In this study, immunoinformatics tools were used to design multiepitope vaccine candidates from plasmid genome sequences of multiple pathotypes of E. coli species involved in diarrhoea infections. The ability of the identified epitopes to be used as a cross-protect multiepitope vaccine was achieved by identifying conserved, immunogenic and antigenic peptides that can elicit CD4+ T-cell, CD8+ T-cell and B-cell and bind to MHC I and II HLA alleles. The molecular docking results of T-cell epitopes showed their well binding affinity to receptive protein and with a wider population coverage. The different multiepitope-based vaccines (MEVCs) candidates were constructed and based on the types of epitope linker they contained. The MEVCs exhibited very good binding interactions with the human immune receptor. Among multiepitope vaccines constructed, MEVC6, MEVCA and MEVCB are more promising as potential vaccine candidates for cross-protection against gastrointestinal infections according to the computational study. It is also hoped that after validation and testing, the predicted multiepitope-based vaccine candidates will probably resolve the challenge of immunological heterogeneity facing enteric vaccine development.

Robust and well-established immunological assays and firm immune correlates of protection that can predict disease outcome and/or vaccine efficacy are essential to adequately assess human immune responses to infection and vaccination. The availability of reagents and calibrated controls is also critically important to standardize assays and generate comparable results among different laboratories. The workshop "Human Immune Responses against Shigella and ETEC: Current Advances and the Path Forward" held during the VASE meeting provided an opportunity to disseminate and discuss recent advances in the field of Shigella and ETEC immunology, identify research needs, and propose collaborative activities to advance the field. Four presentations featured current knowledge on humoral and cellular immune responses to Shigella and ETEC during infection and vaccination. A discussion followed on immunological methods relevant for clinical studies, immune parameters associated with protection, harmonization of assays among laboratories, and availability of reagents and standards. Specific recommendations proposed to facilitate "the path forward" included supporting communication among scientists, harmonization of assays and sharing of protocols, the creation of a repository of reagents and calibrated controls and distribution of such material to the research community, and expansion of exploratory studies to better understand the interactions between these pathogens and the human immune system and the ensuing responses.

PATH hosted the inaugural Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference in Washington, DC in June 2016, bringing together experts from around the world for a highly collaborative forum to discuss progress in the development of new enteric vaccines. Diarrheal disease and long-term sequelae caused by infections with the bacterial pathogens Shigella and enterotoxigenic E. coli (ETEC) pose a significant public health burden in low-income communities. There are currently no licensed vaccines against these pathogens, and the global health community has recently prioritized their development. The 2016 VASE Conference aimed to accelerate communication and progress among those working in the enteric vaccine field to make Shigella and ETEC vaccines a reality as quickly as possible. Research presented in oral and poster presentations at the VASE Conference covered a range of topics, including: the global burden of disease and public health case for Shigella and ETEC vaccines; current vaccine candidates in development; immunology and host responses to the pathogens; and the rationale for and status of combined Shigella-ETEC vaccine candidates. This article reviews key points and highlighted research presented in each of the plenary conference sessions and poster presentations at the 2016 conference. Planning for the 2018 VASE Conference is underway and will likely provide an important platform for sharing the latest updates on Shigella and ETEC vaccine research efforts and maintaining the momentum for accelerating this work. It is also expected that the VASE Conference will continue to provide a unique opportunity for those in the enteric vaccine field to share ideas, make connections, and create workable plans to make Shigella and ETEC vaccines a reality. (Updates available at: www.vaseconference.org.).

We previously developed attenuated rabbit enteropathogenic E. coli (rEPEC) strains which are effective oral vaccines against their parent pathogens by deleting le r, a global regulator of virulence genes. To use these strains as orally administered vectors to deliver other antigens we incorporated the B subunit of shiga-like toxin 1(Stx1) into the passenger domain of the autotransporter EspP expressed on a plasmid. Native EspP enters the periplasm where its passenger domain is exported to the bacterial surface through an outer membrane channel formed by its translocator domain, then cleaved and secreted. Since antigen localization may determine immunogenicity, we engineered derivatives of EspP expressing Stx1B- passenger domain fusions: 1. in cytoplasm 2. in periplasm, 3. surface-attached or 4. secreted. To determine which construct was most immunogenic, rabbits were immunized with attenuated O103 E. coli strain (E22 Δler) alone or expressing Stx1B in each of the above four cellular locations. IgG responses to Stx1B, and toxin-neutralizing antibodies were measured. Animals were challenged with a virulent rabbit Enterohemorrhagic E. coli (EHEC) strain of a different serogroup (O15) than the vaccine strain expressing Stx1 (RDEC-H19) and their clinical course observed. IgG responses to Stx1B subunit were induced in all animals vaccinated with the strain secreting Stx1B, in some vaccinated with surface-expressed Stx1B, but in not animals immunized with periplasmic or cytoplasmic Stx1B. Robust protection was observed only in the group immunized with the vaccine secreting Stx1B. Taken together, our data suggest that secretion of Stx1B, or other antigens, via an autotransporter, may maximize the protective response to live attenuated oral vaccine strains.

Uropathogenic Escherichia coli (UPEC) are among the most prevalent agents of urinary tract infections (UTIs). Antibiotic resistance reaches the need for alternative treatment approaches such as vaccination against UTIs. There is no ideal vaccine against UTIs, thus there is a need to evaluate different targets of uropathogens against UTIs. Ferric scavenger receptor FyuA in UPEC has the properties of an ideal vaccine candidate against UTIs. In the present study, the prevalence of FyuA among UPEC isolates, its immunogenicity with and without alum adjuvant, and its efficacy against experimental UTI were assessed. Totally, fyuA gene was present in 77% of the UPEC isolates tested. Alignments of FyuA exhibited a high degree of conservation among different submitted UPEC isolates in GenBank. The bioinformatics studies showed the high confidence value and stability of the FyuA structure. SDS-PAGE and Western blot confirmed the purification of FyuA with high yield by nickel resins. Mice vaccinated subcutaneously with the FyuA induced a significantly higher humoral response (total IgG, IgG1 and IgG2a) than control mice that alum enhanced these responses. The FuyA alone showed the ability to reduce the colonization of UPEC in bladder and kidney of mice as compared to the control group. But the addition of alum to FyuA increased the protection level against UPEC in these organs. Since, FyuA induced significant IgG1 (Th2) and IgG2a (Th1) responses and protected the mice against experimental UTI, it could be a promising target against UPEC infections.

Autogenous Escherichia coli vaccines to prevent the E. coli peritonitis syndrome (EPS) in laying hens are often used in the field, although their effectiveness has not been demonstrated yet. Therefore, in this study, which consisted of two experiments, their efficacy was assessed. In the first experiment, the EPS-inducing ability of three E. coli isolates originating from bone marrow of hens that died due to EPS and with different Pulsed-Field Gel Electrophoresis patterns, was examined by intravenous inoculation of the isolates in 17-week-old brown layers. Based on the results one isolate was chosen for the preparation of the vaccines and for homologous challenge and another one for heterologous challenge performed in the second experiment. In the named experiment, groups of laying hens which had been vaccinated intramuscularly at 14 and 18 weeks of age with inactivated vaccine either formulated as aqueous suspension or as water-in-oil emulsion were homologously or heterologously challenged per aerosol at 30 weeks of age. The vaccines contained ≥10

F4- and F18-positive enterotoxigenic E. coli strains (F4-ETEC and F18-ETEC) are important causes of post-weaning diarrhea (PWD) in pigs. F4 (antigenic variant ac) and F18 (ab and ac) fimbriae are major antigens that play an important role in the early stages of infection. Herein, the efficacy of a live oral vaccine consisting of two non-pathogenic E. coli strains, one F4ac- and one F18ac-positive, was evaluated using F4ac-ETEC and F18ab-ETEC challenge models. A randomized, masked, placebo-controlled, block design, parallel-group confirmatory study with two different vaccination-challenge intervals (7 and 21 days) was conducted for each challenge model. The vaccine was administered in one dose, to ≥18-day-old piglets via drinking water. Efficacy was assessed by evaluating diarrhea, clinical observations, weight gain and fecal shedding of F4-ETEC or F18-ETEC. Anti-F4 and anti-F18 immunoglobulins in blood were measured. The vaccination resulted in significant reductions in clinical PWD and fecal shedding of F4-ETEC and F18-ETEC after the 7- and 21-day-post-vaccination heterologous challenges, except for after the 21-day-post-vaccination F4-ETEC challenge, when the clinical PWD was too mild to demonstrate efficacy. A significant reduction of mortality and weight loss by vaccination were observed following the F18-ETEC challenge. The 7-day protection was associated with induction of anti-F4 and anti-F18 IgM, whereas the 21-day protection was mainly associated with anti-F4 and anti-F18 IgA. The 7-day onset and 21-day duration of protection induced by this vaccine administered once in drinking water to pigs of at least 18days of age were confirmed by protection against F4-ETEC and F18-ETEC, and induction of F4 and F18-specific immunity. Cross protection of the vaccine against F18ab-E. coli was demonstrated for both the 7- and 21-day F18-ETEC challenges.

BACKGROUND Escherichia coli infections are increasing worldwide in community and hospital settings. The E coli O-antigen is a promising vaccine target. We aimed to assess the safety and immunogenicity of a bioconjugate vaccine containing the O-antigens of four E coli serotypes (ExPEC4V). METHODS In this multicentre phase 1b, first-in-human, single-blind, placebo-controlled trial, we randomly assigned (1:1) healthy adult women with a history of recurrent urinary tract infection (UTI) to receive a single injection of either intramuscular ExPEC4V or placebo. The primary outcome was the incidence of adverse events among vaccine and placebo recipients throughout the study. Secondary outcomes included immunogenicity and antibody functionality, and the incidence of UTIs caused by E coli vaccine serotypes in each group. This study is registered with ClinicalTrials.gov, number NCT02289794. FINDINGS Between Jan 20, 2014, and Aug 27, 2014, 93 women received target-dose ExPEC4V and 95 received placebo. The vaccine was well tolerated: no vaccine-related serious adverse events occurred. Overall, 56 (60%) target-dose vaccines and 47 (49%) placebo recipients experienced at least one adverse event that was possibly, probably, or certainly related to injection. Vaccination induced significant IgG responses for all serotypes: at day 30 compared with baseline, O1A titres were 4·6 times higher, O2 titres were 9·4 times higher, O6A titres were 4·9 times higher, and O25B titres were 5·9 times higher (overall p

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic uremic syndrome in humans. Due to the risks associated with antibiotic treatment against EHEC O157:H7 infection, vaccines represent a promising method for prevention of EHEC O157:H7 infection. Therefore, we constructed the novel bivalent antigen EspA-Tir-M as a candidate EHEC O157:H7 subunit vaccine. We then evaluated the immunogenicity of this novel EHEC O157:H7 subunit vaccine. Immune responses to the fusion protein administered by intranasal and subcutaneous routes were compared in mice. Results showed higher levels of specific mucosal and systemic antibody responses induced by intranasal as compared to subcutaneous immunization. Intranasal immunization enhanced the concentration of interleukin-4, interleukin-10, and interferon-γ, while subcutaneous immunization enhanced only the latter two. In addition, intranasal immunization protected against EHEC O157:H7 colonization and infection in mice at a rate of 90%.Histopathological analysis revealed that vaccination reduced colon damage, especially when administered intranasally. In contrast, subcutaneous immunization elicited a weak immune response and exhibited a low protection rate. These findings demonstrate that intranasal immunization with the fusion protein induces both humoral and cellular immune (Th1/Th2) responses in mice. The novel EspA-Tir-M novel fusion protein therefore represents a promising subunit vaccine against EHEC O157:H7 infection.

CS21 pili of enterotoxigenic Escherichia coli (ETEC) is one of the most prevalent ETEC colonization factors. CS21 major subunit, LngA, mediates ETEC adherence to intestinal cells, and contributes to ETEC pathogenesis in a neonatal mouse infection model. The objectives of this work were to evaluate LngA major subunit purified protein and CS21 purified pili on immunogenicity and protection against ETEC colonization of mice intestine. Recombinant LngA purified protein or purified CS21 pili from E9034A ETEC strain were evaluated for immunogenicity after immunization of C57BL/6 mice. Specific anti-LngA antibodies were detected from mice serum, feces, and intestine fluid samples by ELISA assays. Protection against gut colonization was evaluated on immunized mice orally challenged with wild type E9034A ETEC strain and by subsequent quantification of bacterial colony forming units (CFU) recovered from feces. Recombinant LngA protein and CS21 pili induced specific humoral and mucosal anti-LngA antibodies in the mouse model. CS21 combined with CT delivered intranasally as well as LngA combined with incomplete Freund adjuvant delivered intraperitoneally inhibited ETEC gut colonization in a mouse model. In conclusion, both LngA purified protein and CS21 pili from ETEC are highly immunogenic and may inhibit ETEC intestinal shedding. Our data on immunogenicity and immunoprotection indicates that CS21 is a suitable vaccine candidate for a future multivalent vaccine against ETEC diarrhea.

In poultry production Escherichia coli autogenous vaccines are often used. However, the efficacy of autogenous E. coli vaccinations has not been evaluated experimentally in chickens after start of lay. The aim of the present study was to evaluate the protective effect of an autogenous E. coli vaccine in broiler breeders. Three groups of 28 weeks old broiler breeders (unvaccinated, vaccinated once and twice, respectively) were challenged with a homologous E. coli strain (same strain as included in the vaccine) or a heterologous challenge strain in an experimental ascending model. The clinical outcome was most pronounced in the unvaccinated group; however, the vast majority of chickens in the vaccinated groups had severe pathological manifestations similar to findings in the unvaccinated group after challenge with a homologous as well as a heterologous E. coli strain. Although significant titer rises in IgY antibodies were observed in the twice vaccinated group, antibodies did not confer significant protection in terms of pathological impact. Neither could transfer of maternal derived antibodies to offspring be demonstrated. In conclusion, with the use of the present model for ascending infection, significant protection of an autogenous E. coli vaccine against neither a homologous nor a heterologous E. coli challenge could not be documented.

Small ruminants have been implicated in outbreaks of Escherichia coli O157:H7 at livestock exhibitions throughout the United States. Additionally, goat meat or milk may serve as a reservoir for foodborne transmission of the organism. These associations highlight the public health importance of an effective strategy to reduce E. coli O157:H7 shedding in goats. We examined the efficacy of the SRP® vaccine in goats orally challenged with E. coli O157:H7. Mixed-breed goats (n = 14) were randomly allocated into vaccinated and unvaccinated treatments (n = 7 per treatment). Goats were housed with a vaccinated and unvaccinated animal in each pen. Feces were collected for 3 weeks, then at necropsy, gastrointestinal contents were collected to determine the concentration of E. coli O157:H7. Three isolates per positive sample were saved and evaluated by pulsed-field gel electrophoresis (PFGE) to assess strain persistence over time. The mean concentration of E. coli O157:H7 in the feces of goats was numerical...

Oral administration of vaccines is simpler and more acceptable than injection via needle and syringe, particularly for infants (Fig. 1) This route is promising for new vaccines in development against enterotoxigenic Escherichia coli (ETEC) and Shigella that cause childhood diarrhea with devastating consequences in low-resource countries. However, vaccine antigens and adjuvants given orally need buffering against the degradative effects of low stomach pH, and the type and volume of antacid buffer require special attention for infants. In addition, container/closure systems must be compatible with vaccine formulations, protect against water and gas transfer, and have minimal impact on the cold chain. Health care workers in demanding low-resource settings need an administration device that is easy to use, yet will accurately measure and safely deliver the correct vaccine dose. Developers must consider manufacturing capabilities, and immunization program managers want affordable vaccines. As new combination enteric vaccine candidates advance into clinical evaluation, features of the final vaccine presentation—liquid or dry format, diluent, buffer, primary and secondary packaging, and administration device—should be taken into account early in product development to achieve the greatest possible impact for the vaccine.

Vaccination-induced Escherichia coli O157:H7-specific immune responses have been shown to reduce E. coli O157:H7 shedding in cattle. Although E. coli O157:H7 colonization is correlated with perturbations in intestinal microbial diversity, it is not yet known whether vaccination against E. coli O157:H7 could cause shifts in bovine intestinal microbiota. To understand the impact of E. coli O157:H7 vaccination and colonization on intestinal microbial diversity, cattle were vaccinated with two doses of different E. coli O157:H7 vaccine formulations. Six weeks post-vaccination, the two vaccinated groups (Vx-Ch) and one non-vaccinated group (NonVx-Ch) were orally challenged with E. coli O157:H7. Another group was neither vaccinated nor challenged (NonVx-NonCh). Fecal microbiota analysis over a 30-day period indicated a significant (FDR corrected, p0.05) association of bacterial community structure with vaccination until E. coli O157:H7 challenge. Shannon diversity index and species richness were significantly lower in vaccinated compared to non-vaccinated groups after E. coli O157:H7 challenge (p0.05). The Firmicutes:Bacteroidetes ratio (p0.05) was not associated with vaccination but the relative abundance of Proteobacteria was significantly lower (p0.05) in vaccinated calves after E. coli O157:H7 challenge. Similarly, Vx-Ch calves had higher relative abundance of Paeniclostridium spp. and Christenellaceae R7 group while Campylobacter spp., and Sutterella spp. were more abundant in NonVx-Ch group post-E. coli O157:H7 challenge. Only Vx-Ch calves had significantly higher (p0.001) E. coli O157:H7-specific serum IgG but no detectable E. coli O157:H7-specific IgA. However, E. coli O157:H7-specific IL-10-producing T cells were detected in vaccinated animals prior to challenge, but IFN-γ-producing T cells were not detected. Neither E. coli O157:H7-specific IgG nor IgA were detected in blood or feces, respectively, of NonVx-Ch and NonVx-NonCh groups prior to or post vaccinations. Both Vx-Ch and NonVx-Ch animals shed detectable levels of challenge strain during the course of the study. Despite the lack of protection with the vaccine formulations there were detectable shifts in the microbiota of vaccinated animals before and after challenge with E. coli O157:H7.

Enterotoxigenic Escherichia coli (ETEC) is the most common agent of diarrhea morbidity in developing countries. ETEC adheres to host intestinal epithelial cells via various colonization factors. The CooD and CotD proteins play a significant role in bacteria binding to the intestinal epithelial cells as adhesin tip subunits of CS1 and CS2 pili. The purpose here was to design a new construction containing cooD and cotD genes and use several types of bioinformatics software to predict the structural and immunological properties of the designed antigen. The fusion gene was synthesized with codon bias of E. coli in order to increase the expression level of the protein. The amino acid sequences, protein structure, and immunogenicity properties of potential antigens were analyzed in silico. The chimeric protein was expressed in E. coliBL21 (DE3). The antigenicity of the recombinant proteins was verified by Western blotting and ELISA. In order to assess the induced immunity, the immunized mice were challenged with wild-type ETEC by an intraperitoneal route. Immunological analyses showed the production of a high titer of IgG serum with no sign of serum-mucosal IgA antibody response. The result of the challenge assay showed that 30% of immunized mice survived. The results of this study showed that CooD-CotD recombinant protein can stimulate immunity against ETEC. The designed chimera could be a prototype for the subunit vaccine, which is worthy of further consideration.

The present study was performed on 180-day-old commercial Cobb chicks to assess the effects of the probiotic candidate Enterococcus faecalis-1, the Poulvac Escherichia coli vaccine, and their combination on growth parameters, intestinal microbial composition, immune response, and protection against challenge with the avian pathogen E. coli O78. The experimental groups were as follows: G1, basal diet; G2, basal diet and challenge with O78 at 28 days of growth; G3, basal diet, vaccination with Poulvac (1 and 15 days), and challenge with O78 at 28 days of growth; G4, basal diet, E. faecalis-1 supplementation for the first 3 days of growth, and challenge with O78 at 28 days of growth; G5, basal diet, E. faecalis-1 supplementation for the first 3 days of growth, vaccination with Poulvac (1 and 15 days), and challenge with O78 at 28 days of growth; G6, basal diet and E. faecalis-1 supplementation for the first 3 days of growth. The results showed that E. faecalis-1 in drinking water significantly improved the growth performance and immune response, increased the total Enterococcus counts, reduced the mortality, and decreased the visceral invasion by O78 in challenged broilers. While the effect of the Poulvac vaccine alone or with E. faecalis-1 was not significant compared with that of the E. faecalis-1 supplement, the vaccine improved the growth rate and decreased the mortality and visceral invasion by APEC O78 in challenged broilers. These results showed that E. faecalis-1 supplementation and routine vaccination with the Poulvac vaccine could improve the growth performance and immune response of broiler chickens and protect against challenge with APEC O78.

The objective of the present study was to evaluate the efficacy of a J-5 Escherichia coli vaccine in a mild to moderate inflammatory challenge model using primiparous dairy cows for inoculation only. We hypothesized a clinical difference between placebo and J-5 E. coli vaccinated animals with the mild to moderate inflammatory challenge model. In case the null hypothesis could not be confirmed, the alternate hypothesis was no clinical difference between both treatment groups. Therefore, 23 primiparous cows in mo 7 of pregnancy were randomly assigned to 1 of 2 treatment groups: J-5 E. coli vaccine (n = 12) or placebo (n = 11). Animals were vaccinated 3 times at 56 (±7) and 28 (±7) d before expected calving date and within 14 d postcalving (DIM 5 ± 3). All cows were challenged by infusion with E. coli P4:O32 into 2 left mammary quarters between 14 and 28 d postparturition, at least 10 d after the 3rd vaccination, immediately after the morning milking. Clinical observations and blood and milk samples were collected at several time points from 7 d pre-challenge until 13 d post-challenge. Primiparous cows responded mild to moderately to intramammary E. coli challenge with little clinical difference between treatment groups. Rectal temperature increased earlier in the vaccinated animals, which also eliminated bacteria faster during the early hours after intramammary E. coli challenge. At post-infusion hour 9, the bacterial population was significantly lower in the infected quarters of the vaccinated animals. Blood leukocyte number was only numerically higher in the vaccinated animals, in combination with a numerically higher percentage of late immature polymorphonuclear leukocytes (band cells) in circulation. Even in the nonvaccinated animals, the E. coli challenge in the primiparous cows elicited only a mild to moderate response. The absence of a pronounced clinical difference between vaccinated and nonvaccinated animals was therefore not surprising.

Enterotoxigenic Escherichia coli (ETEC) causes diarrhoea by secreting enterotoxins into the small intestine. Human ETEC strains may secrete any combination of three enterotoxins: the heat-labile toxin (LT) and the heat-stable toxins (ST), of which there are two variants, called human ST (STh) and porcine ST (STp). Strains expressing STh, either alone or in combination with LT and/or STp, are among the four most important diarrhoea-causing pathogens affecting children in low- and middle-income countries. ST is therefore an attractive target for ETEC vaccine development. To produce a safe ST-based vaccine, several challenges must be solved. ST must be rendered immunogenic and non-toxic, and antibodies elicited by an ST vaccine should neutralize ST but not cross-react with the endogenous ligands uroguanylin and guanylin. Virus-like particles (VLPs) tend to be highly immunogenic and are increasingly being used as carriers for presenting heterologous antigens in new vaccines. In this study, we have coupled native STh and the STh-A14T toxoid to the coat protein of Acinetobacter phage AP205 by using the SpyCatcher system and immunized mice with these VLPs without the use of adjuvants. We found that both STs were efficiently coupled to the VLP, that both the STh and STh-A14T VLPs were immunogenic in mice, and that the resulting serum antibodies could completely neutralize the toxic activities of native STh. The serum antibodies showed a high degree of immunological cross-reaction to STp, while there was little or no unwanted cross-reaction to uroguanylin and guanylin. Moreover, compared to native STh, the STh-A14T mutation did not seem to negatively impact the immunogenicity of the construct or the neutralizing ability of the resulting sera. Taken together, these findings demonstrate that VLPs are suitable carriers for making STs immunogenic, and that the STh-A14T-coupled AP205 VLP represents a promising ETEC vaccine candidate. publishedVersion

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.

The generation of robust systemic and mucosal antibody and cell-mediated immune (CMI) responses that are protective, long-lasting, and can quickly be recalled upon subsequent re-exposure to the cognate antigen is the key to the development of effective vaccine candidates. These responses, whether they represent mechanistic or non-mechanistic immunological correlates of protection, usually entail the activation of T cell memory and effector subsets (T-CMI) and induction of long-lasting memory B cells. However, for ETEC and Shigella, the precise role of these key immune cells in primary and secondary (anamnestic) immune responses remains ill-defined. A workshop to address immune correlates for ETEC and Shigella, in general, and to elucidate the mechanistic role of T-cell subsets and B-cells, both systemically and in the mucosal microenvironment, in the development of durable protective immunity against ETEC and Shigella was held at the recent 2nd Vaccines against Shigella and ETEC (VASE) conference in June 2018. This report is a summary of the presentations and the discussion that ensued at the workshop. (C) 2019 The Authors. Published by Elsevier Ltd.

Double-mutant heat-labile toxin (dmLT, LTR192G/L211A) of enterotoxigenic Escherichia coli (ETEC) is an effective mucosal adjuvant. Recent studies have shown that dmLT also exhibits adjuvanticity for antigens administered parenterally. In this study, we subcutaneously (SC) immunized mice with the ETEC adhesin-based vaccine, CFA/I/II/IV MEFA (multiepitope fusion antigen), adjuvanted with dmLT and examined the impact of dmLT on antibody responses specific to the seven adhesins in the vaccine construction [CFA/I, CFA/II (CS1, CS2, CS3) and CFA/IV (CS4, CS5, CS6)]. Mice were immunized with a fixed dose of CFA/I/II/IV MEFA and ascending doses of dmLT adjuvant (0, 0.05, 0.1, 0.5 or 1.0 µg) to assess the potential dmLT dose response relationship. Data showed that dmLT enhanced systemic antibody responses to all seven antigens (CFA/I, CS1-CS6) targeted by MEFA in a dose-dependent way. The adjuvant effect of dmLT on the MEFA construct plateaued at a dose of 0.1 µg. Results also indicated that dmLT is an effective parenteral adjuvant when given by the SC route with the ETEC adhesin MEFA vaccine and that antibody enhancement was achieved with relatively low doses. These observations suggest the potential usefulness of dmLT for parenteral ETEC vaccine candidates and also perhaps for vaccines against other pathogens.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of infectious diarrhea in children, travelers, and deployed military personnel. As such, development of a vaccine would be advantageous for public health. One strategy is to use subunits of colonization factors combined with antigen/adjuvant toxoids as an ETEC vaccine. Here, we investigated the intradermal (i.d.) or sublingual (s.l.) delivery of CFA/I fimbrial antigens, including CfaEB and a CfaE-heat-labile toxin B subunit (LTB) chimera admixed with double mutant heat-labile toxin (LT) LT-R192G/L211A (dmLT). In addition, we compared dmLT with other LT proteins to better understand the generation of adjuvanted fimbrial and toxoid immunity as well as the influence on any local skin reactogenicity. We demonstrate that immunization with dmLT admixed with CfaEB induces robust serum and fecal antibody responses to CFA/I fimbriae and LT but that i.d. formulations are not optimal for s.l. delivery. Improved s.l. vaccination outcomes were observed when higher doses of dmLT (1 to 5 μg) were admixed with CfaEB or, even better, when a CfaE-LTB chimera antigen was used instead. Serum anti-CFA/I total antibodies, detected by enzyme-linked immunosorbent assay, were the best predictor of functional antibodies, based on the inhibition of red blood cell agglutination by ETEC. Immunization with other LT proteins or formulations with altered B-subunit binding during i.d. immunization (e.g., by addition of 5% lactose, LTA1, or LT-G33D) minimally altered the development of antibody responses and cytokine recall responses but reduced skin reactogenicity at the injection site. These results reveal how formulations and delivery parameters shape the adaptive immune responses to a toxoid and fimbria-derived subunit vaccine against ETEC.

Enterotoxigenic Escherichia coli (ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against various ETEC virulence determinants has proven challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we identified B-cell immunodominant epitopes in silico from the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell line IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While conventional vaccinology approaches encounter difficulties at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from various heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide.

Shiga toxin-producing Escherichia coli O157:H7 (O157) can cause mild to severe gastrointestinal disease in humans. Cattle are the primary reservoir for O157, which colonizes the intestinal tract without inducing any overt clinical symptoms. Parenteral vaccination can reduce O157 shedding in cattle after challenge and limit zoonotic transmission to humans, although the impact of vaccination and vaccine formulation on cellular and mucosal immune responses are undetermined. To better characterize the cattle immune response to O157 vaccination, cattle were vaccinated with either water-in-oil-adjuvanted, formalin-inactivated hha deletion mutant of Shiga toxin 2 negative (stx2−) O157 (Adj-Vac); non-adjuvanted (NoAdj-Vac); or non-vaccinated (NoAdj-NoVac) and peripheral T cell and mucosal antibody responses assessed. Cattle in Adj-Vac group had a higher percentage of O157-specific IFNγ producing CD4+ and γδ+ T cells in recall assays compared to the NoAdj-Vac group. Furthermore, O157-specific IgA levels detected in feces of the Adj-Vac group were significantly lower in NoAdj-Vac group. Extracts prepared only from Adj-Vac group feces blocked O157 adherence to epithelial cells. Taken together, these data suggest parenteral administration of adjuvanted, inactivated whole-cell vaccines for O157 can induce O157-specific cellular and mucosal immune responses that may be an important consideration for a successful vaccination scheme.

The safety of the live Escherichia coli vaccine Poulvac® E. coli was tested with a flock (10,000) of layer parents aged 30 weeks. Three and 7 days after vaccination, 60 whole unbroken eggs, the egg white and yolk of 60 eggs and 60 cloacal swabs were enriched in MacConkey broth. At both sampling times, 6 out of 60 whole eggs were found positive for coliform bacteria. None of the enriched samples of yolk + egg white were positive for coliform bacteria. Three and seven days after vaccination 44 and 37, respectively out of 60 swabs were positive for coliform bacteria in MacConkey broth. All coliform isolates collected from whole eggs and cloacal swabs were tested in parallel for growth on minimal agar and blood agar to identify the vaccine strain. Some isolates showed reduced growth on minimal agar compared to blood agar and they were tested further with a PCR for the aroA gene mutation and all were found with the wild type version of the gene. Only two isolates did not grow on minimal agar but grew on blood agar and they were tested both with PCR and PFGE. They also showed the wild type version of the aroA gene and their PFGE profile was different from the vaccine strain of Poulvac® E. coli. In conclusion, the Poulvac® E. coli vaccine strain of E. coli was not identified at the detection limit of one CFU on one egg or in the content of one egg or from a cloacal swab of one hen with at least 95 % probability on flock level. The use of the vaccine is safe for hens in lay with lack of survival of the vaccine strain and lack of negative effects on the hens including egg production.

Shigella and enterotoxigenic Escherichia coli (ETEC) are among the top four enteric pathogens that cause diarrheal illness in young children in developing countries and are major etiologic agents of travellers' diarrhoea. A single vaccine that could target both of these pathogens would have significant public health impact. In this review, we highlight the many pivotal contributions of Phillippe Sansonetti to the identification of molecular mechanisms of pathogenesis of Shigella that paved the way for the development of rationally designed, novel vaccines candidates. The CVD developed a series of live attenuated Shigella vaccine strains based on the most prevalent serotypes associated with disease. Shigella vaccine strains were engineered to express critical ETEC antigens to form a broadly protective Shigella-ETEC multivalent vaccine.

Urinary tract infections (UTIs) are the most widespread and annoying infections affecting millions of people every year annually. The biggest problems of urinary diseases are recurrences, increasing resistance of uropathogens to commonly used antibiotics, as well as the high health care costs of afflicted persons. Uropathogenic Escherichia coli strains (UPECs) are the most dominant etiologic agents of community-acquired infections of this type. During UTI pathogenesis, UPECs utilize various virulence factors, especially mono- and polyadhesive appendages of the chaperone-usher secretion pathway (CUP) required for adhesion, invasion and biofilm formation. Commonly used antibiotics for UTI treatment are usually effective, but their long-term utility may affect gut microbiota of the treated individuals and cause selection of drug resistant uropathogenic variants. Due to increasing resistance of UPEC strains to antibiotics via the evolution of specific defense mechanisms, there is a need to develop alternative methods and therapeutic strategies to fight UTIs (vaccines, receptor analogues, pilicides and curlicides, bacterial interference or phagotherapy). Such therapeutic approaches usually target processes enabling uropathogens to survive within the urinary tract and cause recurrent infections.

CS6, a prevalent surface antigen expressed in nearly 20% of clinical enterotoxigenic Escherichia coli (ETEC) isolates, is comprised of two major subunit proteins, CssA and CssB. Using donor strand complementation, we constructed a panel of recombinant proteins of 1 to 3 subunits that contained combinations of CssA and/or CssB subunits and a donor strand, a C-terminal extension of 16 amino acids that was derived from the N terminus of either CssA or CssB. While the entire panel of recombinant proteins could be obtained as soluble, folded proteins, it was observed that the proteins possessing a heterologous donor strand, derived from the CS6 subunit different from the C-terminal subunit, had the highest degree of physical and thermal stability. Immunological characterization of the proteins, using a murine model, demonstrated that robust anti-CS6 immune responses were generated from fusions containing both CssA and CssB. Proteins containing only CssA were weakly immunogenic. Heterodimers, i.e., CssBA and CssAB, were sufficient to recapitulate the anti-CS6 immune response elicited by immunization with CS6, including the generation of functional neutralizing antibodies, as no further enhancement of the response was obtained with the addition of a third CS6 subunit. Our findings here demonstrate the feasibility of including a recombinant CS6 subunit protein in a subunit vaccine strategy against ETEC.

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of childhood diarrhea in low- and middle-income countries, as well as of diarrhea among travelers to these countries. In children, ETEC strains secreting the heat-stable toxin (ST) are the most pathogenic, and there are ongoing efforts to develop vaccines that target ST. One important challenge for ST vaccine development is to construct immunogens that do not elicit antibodies that cross-react with guanylin and uroguanylin, which are endogenous peptides involved in regulating the activity of the guanylate cyclase-C (GC-C) receptor. We immunized mice with both human ST (STh) and porcine ST (STp) chemically coupled to bovine serum albumin, and the resulting sera neutralized the toxic activities of both STh and STp. This suggests that a vaccine based on either ST variant can confer cross-protection. However, several anti-STh and anti-STp sera cross-reacted with the endogenous peptides, suggesting that the ST sequence must be altered to reduce the risk of unwanted cross-reactivity. Epitope mapping of four monoclonal anti-STh and six anti-STp antibodies, all of which neutralized both STh and STp, revealed that most epitopes appear to have at least one amino acid residue shared with guanylin or uroguanylin. Despite this, only one monoclonal antibody displayed demonstrable cross-reactivity to the endogenous peptides, suggesting that targeted mutations of a limited number of ST residues may be sufficient to obtain a safe ST-based vaccine.

The establishment of an animal model that closely approximates enterotoxigenic Escherichia coli (ETEC) disease in humans is critical for the development and evaluation of vaccines against this enteropathogen. Here, we evaluated the susceptibility of Aotus nancymaae, a New World monkey species, to ETEC infection. Animals were challenged orogastrically with 109 to 1011 CFU of the human pathogenic CFA/I+ ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 and examined for evidence of diarrhea and fecal shedding of bacteria. A clear dose-range effect was obtained, with diarrheal attack rates of 40% to 80%, validated in a follow-on study demonstrating an attack rate of 80% with 1011 CFU of {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 ETEC. To determine whether this model is an effective approach for assessing ETEC vaccine candidates, we used it to evaluate the ability of the donor strand-complemented CFA/I adhesin CfaE (dscCfaE) to protect against {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 challenge. In a series of experiments, animals were intranasally vaccinated with dscCfaE alone, dscCfaE with either cholera toxin B-subunit (CTB) or heat-labile toxin (LTB), or phosphate-buffered saline (PBS) alone and then challenged with 1011 CFU of {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407. Control animals vaccinated with PBS had attack rates of 70 to 90% on challenge. Vaccination with dscCfaE, or dscCfaE admixed with CTB or LTB, resulted in a reduction of attack rates, with vaccine efficacies of 66.7% (P = 0.02), 77.7% (P = 0.006), and 42.9% (P = 0.370) to 83.3% (P = 0.041), respectively. In conclusion, we have shown the {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 ETEC challenge of A. nancymaae to be an effective, reproducible model of ETEC disease, and importantly, we have demonstrated that in this model, vaccination with the prototype vaccine candidate dscCfaE is protective against CF-homologous disease.

Enterohemorrhagic E. coli (EHEC) is a major cause of large outbreaks worldwide associated with hemorrhagic colitis and hemolytic uremic syndrome. While vaccine development is warranted, a licensed vaccine, specific for human use, against EHEC is not yet available. In this study, the reverse vaccinology approach combined with genomic, transcriptional and molecular epidemiology data was applied on the EHEC O157:H7 genome to select new potential vaccine candidates. Twenty-four potential protein antigens were identified and one of them (MC001) was successfully expressed onto Generalized Modules for Membrane Antigens (GMMA) delivery system. GMMA expressing this vaccine candidate was immunogenic, raising a specific antibody response. Immunization with the MC001 candidate was able to reduce the bacterial load of EHEC O157:H7 strain in feces, colon and caecum tissues after murine infection. MC001 is homologue to lipid A deacylase enzyme (LpxR), and to our knowledge, this is the first study describing it as a potential vaccine candidate. Gene distribution and sequence variability analysis showed that MC001 is present and conserved in EHEC and in enteropathogenic E. coli (EPEC) strains. Given the high genetic variability among and within E. coli pathotypes, the identification of such conserved antigen suggests that its inclusion in a vaccine might represent a solution against major intestinal pathogenic strains.

Thanks to the modern sequencing era, the extent to which infectious disease imposes selective pressures on the worldwide human population is being revealed. This is aiding our understanding of the underlying immunological and host mechanistic defenses against these pathogens, as well as potentially assisting in the development of vaccines and therapeutics to control them. As a consequence, the workshop "How genomics can be used to understand host susceptibility to enteric infection, aiding in the development of vaccines and immunotherapeutic interventions" at the VASE 2018 meeting, aimed to discuss how genomics and related tools could be used to assist Shigella and ETEC vaccine development. The workshop featured four short presentations which highlighted how genomic applications can be used to assist in the identification of genetic patterns related to the virulence of disease, or host genetic factors that could contribute to immunity or successful vaccine responses. Following the presentations, there was an open debate with workshop attendees to discuss the best ways to utilise such genomic studies, to improve or accelerate the process of both Shigella and ETEC vaccine development. The workshop concluded by making specific recommendations on how genomic research methods could be strengthened and harmonised within the ETEC and Shigella research communities.

E. coli infection of broilers can result in systemic diseases and productivity losses. Use of antimicrobials against this condition is common but other approaches, such as vaccination, are gaining ground. Anecdotal field reports indicate that intestinal health is improved unspecifically following E. coli live vaccination. We hypothesized that the intestine may be an important site for the functionality of the vaccine. Vaccine effects on the intestine were assessed. Spray vaccination induced marked alterations of the caecum microbiota of broilers within 3 days, and this effect gradually waned. However, T cell activation occurred in the spleen, but not in caecal tonsils, and anti-E. coli IgA was concentrated in the respiratory mucosae. Accordingly, IL-6 mRNA was produced in the lungs following immunization. Overall, these data are an initial indication that any vaccine-induced effects on the intestine are greatly associated with the microbiota. However, immunity conferred by vaccination is not primarily induced in gut-associated lymphoid tissues.

Extraintestinal pathogenic Escherichia coli (ExPEC) has presented a major clinical infection emerged in the past decades. O-polysaccharide (OPS)-based glycoconjugate vaccines produced using the bacterial glycosylation machinery can be utilized to confer protection against such infection. However, constructing a low-cost microbial cell factory for high-efficient production of OPS-based glycoconjugate vaccines remains challenging. Here, we engineered a glyco-optimized chassis strain by reprogramming metabolic network. The yield was enhanced to 38.6 mg L-1, the highest level reported so far. MS analysis showed that designed glycosylation sequon was modified by target polysaccharide with high glycosylation efficiency of 90.7 % and 76.7 % for CTB-O5 and CTB-O7, respectively. The glycoconjugate vaccines purified from this biosystem elicited a marked increase in protection against ExPEC infection in mouse model, compared to a non-optimized system. The glyco-optimized platform established here is broadly suitable for polysaccharide-based conjugate production against ExPEC and other surface-polysaccharide-producing pathogens.

Molecular breeding via DNA shuffling directs the evolution of vaccines with desired traits. In the present study, polyvalent OmpA vaccines were generated by DNA shuffling of five ompA genes from four species of bacteria Vibrio parahaemolyticus, V. alginolyticus, Edwardsiella tarda and Escherichia coli. First, a new hybrid OmpA was constructed using VA0764 primers and used for construction of a prokaryotic expressing library PompAs-FV containing 84 ompAs, which were validated by PCR and SDS/PAGE. Then, the 84 ompAs were used to construct a eukaryotic expressing library EompAs-FV for preparing DNA vaccines. Third, extracellular bacterium V. alginolyticus challenge post active immunization using these DNA vaccines was carried out to identify genes with high immunoprotection. Among the 84 ompAs, 17 showed higher or equal immune protection against infection caused by V. alginolyticus than control VA0764. Finally, immune protection against infection caused by intracellular bacterium Edwardsiella tarda was assessed further using the top seven out of the 17 ompAs. This led to identification of three efficient polyvalent vaccines against infections caused by the extracellular bacterium V. alginolyticus and intracellular bacterium E. tarda. In addition, we sequenced genes for understanding mechanisms of the polyvalent vaccines, but association of immune protection with mutation of gene and amino acids is not determined. These results indicate that DNA shuffling is an efficient way to develop polyvalent vaccines against microbial infections.