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An Escherichia coli (E. coli) O2:K1 bacterial ghost was produced by controlled expression of bacteriophage PhiX 174 lysis gene E. Temperature controlled expression of this gene caused tunnels and holes in the cell wall of E. coli O2:K1 bacterium, leading to loss of cytoplasmic contents. Formation of E. coli O2:K1 ghost was confirmed by scanning electron microscopy and determination of colony forming units. To evaluate the efficiency of this bacterial ghost vaccine to elicit cellular and humoral immune responses, 85 one day old chickens from Ross 308 breed were divided into the following 5 groups; group 1 (non-immunized control), group 2 (vaccine administered by injection of E. coli O2:K1 killed vaccine), group 3 (vaccine administered by injection of E. coli O2:K1 ghost), group 4 (vaccine administered by inhalation of E. coli O2:K1 ghost), and group 5 (neither immunized, nor challenged as negative control). The groups of 2, 3, and 4 were received vaccines at days 7, 14, and 22. Groups 1 to 4 were challenged with the wild type at day 33. Evaluation of post-mortem lesions and immune responses in all groups showed that chicken injected with the killed vaccine and the bacterial ghost had the best protection. These findings suggest that this bacterial ghost has the potential to be used as a poultry colibacillosis vaccine.

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.

Urinary tract infections (UTIs) are recognized as one of the most common infectious diseases in the world that can be divided to different types. Uropathogenic Escherichia coli (UPEC) strains are the most prevalent causative agent of UTIs that applied different virulence factors such as fimbriae, capsule, iron scavenger receptors, flagella, toxins, and lipopolysaccharide for their pathogenicity in the urinary tract. Despite the high pathogenicity of UPEC strains, host utilizes different immune systems such as innate and adaptive immunity for eradication of them from the urinary tract. The routine therapy of UTIs is based on the use of antibiotics such as β-lactams, trimethoprim, nitrofurantoin and quinolones in many countries. Unfortunately, the widespread and misuse of these antibiotics resulted in the increasing rate of resistance to them in the societies. Increasing antibiotic resistance and their side effects on human body show the need to develop alternative strategies such as vaccine against UTIs. Developing a vaccine against UTI pathogens will have an important role in reduction the mortality rate as well as reducing economic costs. Different vaccines based on the whole cells (killed or live-attenuated vaccines) and antigens (subunits, toxins and conjugatedvaccines) have been evaluated against UTIs pathogens. Furthermore, other therapeutic strategies such as the use of probiotics and antimicrobial peptides are considered against UTIs. Despite the extensive efforts, limited success has been achieved and more studies are needed to reach an alternative of antibiotics for treatment of UTIs.

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.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common bacterial causes of mortalities in developing countries due to diarrhea. Since mucosal immune responses to CFs can prevent the disease, a chimeric protein containing ETEC's CFA/I (CfaE) tip subunits and CS2 (CotD) sub-structural units is developed to produce effective vaccine. Using bioinformatics tools, the chimeric construct was analyzed and then the optimized gene was synthesized and expressed in E. coli. The recombinant protein was expressed and purified by the Ni-NTA chromatography column and confirmed by anti-his tag antibody by western blotting. Mice were immunized with recombinant protein, and the IgG and IgA antibodies' titrations of the sera were analyzed by ELISA. In addition, the immunogenicity and protective efficacy against the live ETEC bacteria in the challenge test were determined. Western blot analysis verified the chimeric protein expression of CotD-CfaE. The outcome of ELISA was a substantial improvement in the IgG antibody titer in immunized mice. In a live ETEC challenge, the survival percentage of 30% was shown for immunized mice. The developed recombinant chimeric protein could be suggested as an effective component in producing an efficient vaccine against Enterotoxigenic E. coli with other crucial subunits, different immunization route, and other factors.

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

BACKGROUND Enterotoxigenic Escherichia coli (ETEC) infection is a primary cause of livestock diarrhea. Therefore, effective vaccines are needed to reduce the incidence of ETEC infection. OBJECTIVES Our study aimed to develop a multivalent ETEC vaccine targeting major virulence factors of ETEC, including enterotoxins and fimbriae. METHODS SLS (STa-LTB-STb) recombinant enterotoxin and fimbriae proteins (F4, F5, F6, F18, and F41) were prepared to develop a multivalent vaccine. A total of 65 mice were immunized subcutaneously by vaccines and phosphate-buffered saline (PBS). The levels of specific immunoglobulin G (IgG) and pro-inflammatory cytokines were determined at 0, 7, 14 and 21 days post-vaccination (dpv). A challenge test with a lethal dose of ETEC was performed, and the survival rate of the mice in each group was recorded. Feces and intestine washes were collected to measure the concentrations of secretory immunoglobulin A (sIgA). RESULTS Anti-SLS and anti-fimbriae-specific IgG in serums of antigen-vaccinated mice were significantly higher than those of the control group. Immunization with the SLS enterotoxin and multivalent vaccine increased interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) concentrations. Compared to diarrheal symptoms and 100% death of mice in the control group, mice inoculated with the multivalent vaccine showed an 80% survival rate without any symptom of diarrhea, while SLS and fimbriae vaccinated groups showed 60 and 70% survival rates, respectively. CONCLUSIONS Both SLS and fimbriae proteins can serve as vaccine antigens, and the combination of these two antigens can elicit stronger immune responses. The results suggest that the multivalent vaccine can be successfully used for preventing ETEC in important livestock.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain is known as one of the major human foodborne pathogens. Lack of effective clinical treatment for human diarrheal diseases confirms the need for vaccine production against enteric bacteria such as E.coli O157:H7. Shiga-like toxin (Stx), EscC, and Intimin are the main important virulent factors of this enteric pathogen. In the present study, a comparative Omics analysis was conducted to identify most invasion EHEC antigenic factors as a potential immunogen. SEI (Stx-EscC-Intimin) trivalent chimeric protein was designed from the exposed and epitope rich part of these virulence factors. Sequence optimization, physicochemical properties, mRNA folding, three-dimensional structure and immunoinformatics data were investigated. The chimeric gene was synthesized with codon bias of E. coli. Recombinant protein was expressed and confirmed by western blot analysis. To evaluate the immunogenicity of the designed protein, the protein was administered to BALB/c mice and the serum IgG was determined by ELISA. Based on the Ramachandran plot, the validation data showed that 90.1 % of residues lie in the favored region. The high antigenicity of the multimeric protein was predicted by the immunoinformatic analysis. Epitope prediction had shown the proper distribution of linear and conformational B-cell epitopes and the competition of T-cell epitopes to bind MHC molecules too. Recombinant ESI Protein with 74.5 kDa was expressed in E. coli. Western blot analysis by anti-Stx antibody, confirmed a single band of chimeric protein. Consequently, the chimeric gene was designed and constructed after assessments. From in silico approach, the protein deduced from this cassette can be an immunogen candidate, and act against toxicity and adherence of EHEC.

One of the major bacterial infectious diseases in the poultry industry is avian pathogenic Escherichia coli (APEC), which causes colibacillosis in chickens. To develop a novel nucleic acid-free bacterial ghost (BG) vaccine against the O78:K80 serotype of APEC, in this study we constructed a plasmid that harbored E-lysis and S nuclease (SNUC). Following the expression, the O78:K80 bacteria lost all of their cytoplasmic content and nucleic acids by enzymatic digestion. The functionality of these two proteins in the production procedure of bacterial ghosts was confirmed by monitoring the number of colonies, scanning electron microscopy imaging, gel electrophoresis of genomic DNA, and qPCR on the plasmid content of bacterial ghosts. The protective efficacy of the ghost vaccine generated from O78:K80 serotype of APEC was tested in chickens by injection and inhalation routes and compared with that in chickens that received the injection of a killed vaccine. The O78:K80 BG vaccine candidate, used as injection and inhalation, in comparison with the killed vaccine, triggered higher proinflammatory cytokine expression including IL-6, IL-1β, and TNFSF15; a higher level of antibody-dependent humoral (IgY and IgA) and cellular immune responses (IFNγ and lymphocyte proliferation); and lower lesion scores. According to the results of this study, we suggest that the bacterial ghost technology has the potential to be applied for the development of novel vaccines against avian colibacillosis. This technology provides an effective and reliable approach to make multivalent vaccines for more prevalent APEC strains involved in the establishment of this infectious disease in the poultry industry.

Avian pathogenic Escherichia coli (APEC) O

Recent efforts to develop an enterotoxigenic Escherichia coli (ETEC) vaccine have focused on the antigenically conserved tip adhesins of colonization factors. We showed previously that intranasal immunization with dsc(19)CfaE, a soluble variant of the in cis donor strand-complemented tip adhesin of a colonization factor of the class 5 family (CFA/I) fimbria, is highly immunogenic and protects against oral challenge with CFA/I-positive (CFA/I(+)) ETEC strain H10407 in the Aotus nancymaae nonhuman primate. We also reported a cholera toxin (CT)-like chimera (called dsc(19)CfaE-CTA2/CTB) in which the CTA1 domain of CT was replaced by dsc(19)CfaE that was strongly immunogenic when administered intranasally or orogastrically in mice. Here, we evaluate the immunogenicity and protective efficacy (PE) of a refined and more stable chimera comprised of a pentameric B subunit of ETEC heat-labile toxin (LTB) in lieu of the CTB pentamer and a donor strand truncation (dsc(14)) of CfaE. The refined chimera, dsc(14)CfaE-sCTA2/LTB, was highly immunogenic in mice when administered intranasally or intradermally, eliciting serum and fecal antibody responses against CfaE and LTB, as well as strong hemagglutination inhibition titers, a surrogate for neutralization of intestinal adhesion mediated by CfaE. Moreover, the chimera was safe and highly immunogenic when administered intradermally to guinea pigs. In A. nancymaae, intradermal (i.d.) immunization with chimera plus single-mutant heat-labile toxin [LT(R192G)] elicited strong serum anti-CfaE and anti-LTB antibody responses and conferred significant reduction of diarrhea compared to phosphate-buffered saline (PBS) controls (PE = 84.1%; P

A series of studies was undertaken in specific-pathogen-free white leghorn chickens for the development of a chicken model of avian pathogenic Escherichia coli (APEC) peritonitis. Once established, this model was then used to measure the effectiveness of a siderophore receptor and porin proteins (SRP®) APEC vaccine. Initially, five pilot studies were performed to compare the E. coli serotype, challenge route, and dose of inoculum that resulted in pathologies characteristic of the peritonitis observed in commercial layer facilities, such as widespread organ infection, atrophy, discoloration, corrugation of yolk sacs, and the presence of caseous exudate. Isolates of serotypes O1, O2, and O78 were tested by intravenous, intravaginal, intratracheal, and intraperitoneal routes and were compared at various levels of challenge inoculum. Daily observations of mortality and morbidity were made, and at necropsy, gross lesion scores were collected and bacterial colonization of internal organs determined. Outcomes varied from a complete lack of mortality or detectable pathology and low, or no, organ colonization in the case of intravaginal and intratracheal routes with each E. coli serotype to moderate to high levels of mortality, pathology, and colonization after challenge via the intravenous and intraperitoneal routes with O2 and O78 serotypes, respectively. The O78 serotype was found to result in pathologies consistent with field observations of peritonitis, and therefore, subsequent studies were performed only with O78. In addition to the relative failure with both the intratracheal and intravaginal routes of challenge, the intravenous route was found to be inconsistent and often resulted in lameness not observed with the intraperitoneal route. A final pilot study confirmed that the dose (∼ 8 log 10 CFU) administered by the intraperitoneal route replicated peritonitis, and therefore, all vaccination/challenge studies were conducted in this manner. Five vaccination/challenge studies are reported here in which variables of chicken age, vaccination interval, and vaccination to challenge interval were examined. In all studies, vaccine effectiveness was dramatic and was shown to completely protect against mortality and substantially against tissue colonization and pathology typical of APEC infections. The vaccine elicited a rapid onset of immunity with both narrow and broad vaccination intervals and in both young and mature chickens. Additionally, the vaccine was demonstrated to sustain robust effectiveness against mortality over 3 months. The SRP APEC vaccine should provide effective protection of young and mature chickens from E. coli under broadly flexible conditions of use in commercial operations.

Summary Environmental enteric dysfunction (EED) is a gastrointestinal inflammatory disease caused by malnutrition and chronic infection. EED is associated with stunting in children and reduced efficacy of oral vaccines. To study the mechanisms of oral vaccine failure during EED, we developed a microbiota- and diet-dependent mouse EED model. Analysis of E. coli-labile toxin vaccine-specific CD4+ T cells in these mice revealed impaired CD4+ T cell responses in the small intestine and but not the lymph nodes. EED mice exhibited increased frequencies of small intestine-resident RORγT+FOXP3+ regulatory T (Treg) cells. Targeted deletion of RORγT from Treg cells restored small intestinal vaccine-specific CD4 T cell responses and vaccine-mediated protection upon challenge. However, ablation of RORγT+FOXP3+ Treg cells made mice more susceptible to EED-induced stunting. Our findings provide insight into the poor efficacy of oral vaccines in EED and highlight how RORγT+FOXP3+ Treg cells can regulate intestinal immunity while leaving systemic responses intact.

With advancements in sequencing technologies, vast amount of experimental data has accumulated. Due to rapid progress in the development of bioinformatics tools and the accumulation of data, immunoinformatics or computational immunology emerged as a special branch of bioinformatics which utilizes bioinformatics approaches for understanding and interpreting immunological data. One extensively studied aspect of applied immunology involves using available databases and tools for prediction of B- and T-cell epitopes. B and T cells comprise two arms of adaptive immunity.This chapter first reviews the methodology we used for computational identification of B- and T-cell epitopes against enterotoxigenic Escherichia coli (ETEC). Then we discuss other databases of epitopes and analysis tools for T-cell and B-cell epitope prediction and vaccine design. The predicted peptides were analyzed for conservation and population coverage. HLA distribution analysis for predicted epitopes identified efficient MHC binders. Epitopes were further tested using computational docking studies to bind in MHC-I molecule cleft. The predicted epitopes were conserved and covered more than 80% of the world population.

Earlier studies have demonstrated the use of inactivated recombinant E. coli (bacterins), to protect against Clostridium spp. in vaccinated animals. These bacterins have a simpler, safer, and faster production process. However, these bacterins carry expression plasmids, containing antibiotic resistance gene, which could be assimilate accidentally by environmental microorganisms. Considering this, we aimed to impair this plasmids using formaldehyde at different concentrations. This compound inactivated the highest density of cells in 24 h. KanR cassette amplification was found to be impaired with 0.8% for 24 h or 0.4% for 72 h. Upon electroporation, E. coli DH5α ultracompetent cells were unable to acquire the plasmids extracted from the bacterins after inactivation procedure. Formaldehyde-treated bacterins were incubated with other viable strains of E. coli, leading to no detectable gene transfer. We found that this compound is effective as an inactivation agent. Here we demonstrate the biosafety involving antibiotic resistance gene of recombinant E. coli vaccines allowing to industrial production and animal application.

Objectives: Lectin-like adhesins of enteric bacterial pathogens such as Escherichia coli are an attractive target for vaccine or drug development. Here, we have developed e-Membranome as a database of genome-wide putative adhesins in Escherichia coli (E. coli). Methods: The outer membrane adhesins were predicted from the annotated genes of Escherichia coli strains using the PSORTb program. Further analysis was performed using Interproscan and the String database. The candidate proteins can be investigated for homology modeling of the Three-Dimensional (3D) structure (I-TASSER version 5.1), epitope region (ABCpred), and the glycan array. Results: e-Membranome is implemented using the Django (version 2.2.5) framework. The Web Application Server Apache Tomcat 6.0 is integrated into the platform on Ubuntu Linux (version 16.04). MySQL database (version 5.7) is used as a database engine. The information on homology model of the 3D structure, epitope region, and affinity information from the glycan array will be stored in the e- Membranome database. As a case study, we performed a genome-wide screening of outer membraneembedded proteins from the annotated genes of E. coli using the e-Membranome pipeline. Conclusion: This platform is expected to be a valuable resource for advancing research of outer membrane proteins for the construction of lectin-glycan interaction network of E. coli. In addition, the e- Membranome pipeline can be extended to other similar biological systems that need to address hostpathogen interactions.

Surface-expressed colonization factors and their subunits are promising candidates for inclusion into a multivalent vaccine targeting enterotoxigenic Escherichia coli (ETEC), a leading cause of acute bacterial diarrhea in developing regions. However, soluble antigens are often poorly immunogenic in the absence of an adjuvant. We show here that the serum immune response to CfaE, the adhesin of the ETEC colonization factor CFA/I, can be enhanced in BALB/c mice by immunization with a chimeric antigen containing CfaE and pentameric cholera toxin B subunit (CTB) of cholera toxin from Vibrio cholerae. We constructed this antigen by replacing the coding sequence for the A1 domain of the cholera toxin A subunit (CTA) with the sequence of donor strand complemented CfaE (dscCfaE) within the cholera toxin operon, resulting in a dscCfaE-CTA2 fusion. After expression, via non-covalent interactions between CTA2 and CTB, the fusion and CTB polypeptides assemble into a complex containing a single dscCfaE-CTA2 protein bound to pentameric CTB (dscCfaE-CTA2/CTB). This holotoxin-like chimera retained the GM1 ganglioside binding activity of CTB, as well as the ability of CfaE to mediate the agglutination of bovine red blood cells when adsorbed to polystyrene beads. When administered intranasally to mice, the presence of CTB in the chimera significantly increased the serum immune response to CfaE compared to dscCfaE alone, stimulating a response similar to that obtained with a matched admixture of dscCfaE and CTB. However, by the orogastric route, immunization with the chimera elicited a superior functional immune response compared to an equivalent admixture of dscCfaE and CTB, supporting further investigation of the chimera as an ETEC vaccine candidate.

Bacterial pathogen infections are fast-growing public health threats and worldwide problems. Glycoconjugate vaccines are among the most effective means in combating such infections. Recent advances in bacterial protein glycan coupling technology (PGCT) have revolutionized the production of glycoconjugate vaccines and drawn enormous attention from both researchers and pharmaceutical companies. Cloning of bacterial surface polysaccharide gene cluster is a prerequisite for the application of PGCT. In this study, we applied the RecET direct cloning strategy for rapid and efficient cloning of O-antigen polysaccharide gene clusters from Escherichia coli serotypes O25b, O26, and O55 in a high-fidelity manner. Then, these gene clusters were applied in PGCT to produce corresponding glycoconjugates. Subsequent immunological studies verified the abilities of glycoconjugate vaccine candidates O25-maltose-binding protein (MBP), O26-MBP, and O55-MBP to generate serotype-specific antibodies and confer protection against E. coli infections. The combination of RecET direct cloning and PGCT makes the rapid production of glycoconjugate vaccines against fast-expanding bacterial pathogens possible.

One of the strategies used to improve the immunogenicity of purified protein antigens has relied on their association with synthetic nanocarriers, which, in general, have functioned as simple antigen containers. Here, we present a more advanced strategy based on the design of an antigen nanocarrier at the molecular level. The nanocarrier is composed of a vitamin E oily core, surrounded by two layers: a first layer of chitosan and a second of dextran sulphate. The selected antigen, IutA protein from Escherichia coli, was harboured between the two polymeric layers. The final bilayer nanocapsules had a nanometric size (≈ 200 nm), a negative zeta potential (< -40 mV) and a good antigen association efficiency (≈ 70%). The bilayer architecture led to an improvement on the formulation stability and the controlled release of the associated antigen. Remarkably, after being administered to mice, bilayer nanocapsules elicited higher IgG levels than those obtained with antigen precipitated with Alum. Moreover, freeze-dried nanocapsules were stable at room temperature for, at least, 3 months. These promising data, in addition to their contribution to the development of an uropathogenic E. coli vaccine, has allowed us to validate these novel bilayer nanocapsules as adequate platforms for the delivery of protein antigens.

Infection with enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea-related illness and death among children under 5 years of age in low– and middle-income countries (LMIC). Recent studies have found that it is the ETEC subtypes that produce the heat-stable enterotoxin (ST), irrespective of whether they also secrete the heat-labile enterotoxin (LT), which contribute most importantly to the disease burden in children from LMIC. Therefore, adding an ST toxoid would importantly complement ongoing ETEC vaccine development efforts. The ST’s potent toxicity, its structural similarity to the endogenous peptides guanylin and uroguanylin, and its poor immunogenicity have all complicated the advancement of ST-based vaccine development. Recent remarkable progress, however, including the unprecedented screening for optimal ST mutants, mapping of cross-reacting ST epitopes and improved ST-carrier coupling strategies (bioconjugation and genetic fusion), enables the rational design of safe, immunogenic, and well-defined ST-based vaccine candidates. Development of an enterotoxigenic Escherichia coli vaccine based on the heat-stable toxin

Bacteremia and healthcare-associated infections (HAIs) continue to rise in industrialized countries, in line with population aging and increased healthcare needs. Escherichia coli and Staphylococcus aureus are the most common pathogens causing HAIs and bacteremia.This review discusses risk factors in the healthcare setting contributing to the rise in bacteremia and other invasive bacterial HAIs in older populations in general, and attributed to E. coli and S. aureus. Treatment of infections is becoming increasingly difficult due to emerging antimicrobial resistance. Prophylactic vaccines may be the solution to lowering the burden of serious infections in the elderly.E. coli and S. aureus vaccines routinely administered to older adults and to people undergoing prescheduled procedures with high infection risks could prevent a significant proportion of severe disease, and could potentially also limit the further emergence of antimicrobial resistance.

Strains of Shiga toxin-producing Escherichia coli (STEC) can cause the severe Hemolytic Uremic Syndrome (HUS). Shiga toxins are protein toxins that bind and kill microvascular cells, damaging vital organs. No specific therapeutics or vaccines have been licensed for use in humans yet. The most common route of infection is by consumption of dairy or farm products contaminated with STEC. Domestic cattle colonized by STEC strains represent the main reservoir, and thus a source of contamination. Outer Membrane Vesicles (OMV) obtained after detergent treatment of gram-negative bacteria have been used over the past decades for producing many licensed vaccines. These nanoparticles are not only multi-antigenic in nature but also potent immunopotentiators and immunomodulators. Formulations based on chemical-inactivated OMV (OMVi) obtained from a virulent STEC strain (O157:H7 serotype) were found to protect against pathogenicity in a murine model and to be immunogenic in calves. These initial studies suggest that STEC-derived OMV has a potential for the formulation of both human and veterinary vaccines.

This Phase 1, randomized, double-blind, placebo-controlled study was conducted to evaluate the safety, tolerability and immunogenicity of different doses of ExPEC4V conjugate vaccine (4-16µg Polysaccharide [PS]/serotype) in healthy Japanese participants, stratified into younger (≥20 to

Background Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in the developing world. Enterotoxigenic E coli vaccinology has been challenged by genetic diversity and heterogeneity of canonical antigens. Examination of the antigenic breadth of immune responses associated with protective immunity could afford new avenues for vaccine development. Methods Antibody lymphocyte supernatants (ALS) and sera from 20 naive human volunteers challenged with ETEC strain H10407 and from 10 volunteers rechallenged 4-6 weeks later with the same strain (9 of whom were completely protected on rechallenge) were tested against ETEC proteome microarrays containing 957 antigens. Results Enterotoxigenic E coli challenge stimulated robust serum and mucosal (ALS) responses to canonical vaccine antigens (CFA/I, and the B subunit of LT) as well as a small number of antigens not presently targeted in ETEC vaccines. These included pathovar-specific secreted proteins (EtpA, EatA) as well as highly conserved E coli antigens including YghJ, flagellin, and pertactin-like autotransporter proteins, all of which have previously afforded protection against ETEC infection in preclinical studies. Conclusions Taken together, studies reported here suggest that immune responses after ETEC infection involve traditional vaccine targets as well as a select number of more recently identified protein antigens that could offer additional avenues for vaccine development for these pathogens.

Objective Enterotoxigenic Escherichia Coli (ETEC) is the cause of diarrhea and even death in humans and offspring of animals. Outer membrane vesicles (OMVs) of the ETEC was prepared and its potential as a vaccine candidate against enteric colibacillosis in neonatal mice was evaluated. Dam mice intradermally injected with ETEC-derived OMVs and OMVs plus an active form of vitamin D3 (avD3). Mucosal and systemic immune responses in mice and passive immunity protection against ETEC lethality in their offspring was investigated. Results Immunization of adult mice via ETEC-derived OMV alone and in formulation with avD3 protect offspring from ETEC-induced lethality. Nevertheless, avD3 did not indicate a positive effect on mucosal and systemic immune responses. Only the combination of OMV plus avD3 elicited a significant (P

Innumerable Escherichia coli of animal origin are identified, which are of economic significance, likewise, cattle, sheep and goats are the carrier of enterohaemorrhagic E. coli, which are less pathogenic, and can spread to people by way of direct contact and through the contamination of foodstuff or portable drinking water, causing serious illness. The immunization of ruminants has been carried out for ages and is largely acknowledged as the most economical and maintainable process of monitoring E. coli infection in ruminants. Yet, only a limited number of E. coli vaccines are obtainable. Mucosal surfaces are the most important ingress for E. coli and thus mucosal immune responses function as the primary means of fortification. Largely contemporary vaccination processes are done by parenteral administration and merely limited number of E. coli vaccines are inoculated via mucosal itinerary, due to its decreased efficacy. Nevertheless, aiming at maximal mucosal partitions to stimulate defensive immunity at both mucosal compartments and systemic site epitomises a prodigious task. Enormous determinations are involved in order to improve on novel mucosal E. coli vaccines candidate by choosing apposite antigens with potent immunogenicity, manipulating novel mucosal itineraries of inoculation and choosing immune-inducing adjuvants. The target of E. coli mucosal vaccines is to stimulate a comprehensive, effective and defensive immunity by specifically counteracting the antibodies at mucosal linings and by the stimulation of cellular immunity. Furthermore, effective E. coli mucosal vaccine would make vaccination measures stress-free and appropriate for large number of inoculation. On account of contemporary advancement in proteomics, metagenomics, metabolomics and transcriptomics research, a comprehensive appraisal of the immeasurable genes and proteins that were divulged by a bacterium is now in easy reach. Moreover, there exist marvellous prospects in this bourgeoning technologies in comprehending the host bacteria affiliation. Accordingly, the flourishing knowledge could massively guarantee to the progression of immunogenic vaccines against E. coli infections in both humans and animals. This review highlight and expounds on the current prominence of mucosal and systemic immunogenic vaccines for the prevention of E. coli infections in ruminants.

Infectious diarrheal diseases are the second leading cause of death in children under 5 years, making vaccines against these diseases a high priority. It is known that certain vaccine adjuvants, chiefly bacterial ADP-ribosylating enterotoxins, can induce mucosal antibodies when delivered parenterally. Based on this, we reasoned vaccine-specific mucosal cellular immunity could be induced via parenteral immunization with these adjuvants. Here, we show that, in contrast to the Toll-like receptor-9 agonist CpG, intradermal immunization with non-toxic double-mutant heat-labile toxin (dmLT) from enterotoxigenic Escherichia coli drove endogenous, antigen-specific CD4+ T cells to expand and upregulate the gut-homing integrin α4β7. This was followed by T-cell migration into gut-draining lymph nodes and both small and large intestines. We also found that dmLT produces a balanced T-helper 1 and 17 (Th1 and Th17) response, whereas T cells from CpG immunized mice were predominantly Th1. Immunization with dmLT preferentially engaged CD103+ dendritic cells (DCs) compared with CpG, and mice deficient in CD103+ DCs were unable to fully license antigen-specific T-cell migration to the intestinal mucosae following parenteral immunization. This work has the potential to redirect the design of existing and next generation vaccines to elicit pathogen-specific immunity in the intestinal tract with non-mucosal immunization.