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To assess the pattern of multiple human papillomavirus infection to predict the type replacement postvaccination. A total of 7372 women aged 18-45y from a phase III trial of an Escherichia coli -produced HPV-16/18 vaccine were analyzed at enrollment visit before vaccination. Hierarchical multilevel logistic regression was used to evaluate HPV vaccine type and nonvaccine-type interactions with age as a covariate. Binary logistic regression was construed to compare multiple infections with single infections to explore the impact of multiple-type infections on the risk of cervical disease. Multiple HPV infections were observed in 25.2% of HPV-positive women and multiple infections were higher than expected by chance. Statistically significant negative associations were observed between HPV16 and 52, HPV18 and HPV51/52/58, HPV31 and HPV39/51/52/53/54/58, HPV33 and HPV52/58, HPV58 and HPV52, HPV6 and HPV 39/51/52/53/54/56/58. Multiple HPV infections increased the risk of CIN2+ and HSIL+, with the ORs of 2.27(95%CI: 1.41, 3.64) and 2.26 (95%CI: 1.29, 3.95) for multiple oncogenic HPV infection separately. However, no significant evidence for the type-type interactions on risk of CIN2+ or HSIL+. There is possibility of type replacement between several pairs of vaccine and nonvaccine HPV type. Multiple HPV infection increased the risk of cervical disease, but coinfection HPV types seem to follow independent disease processes. Continued post-vaccination surveillance for HPV 51/52/58 types and HPV 39/51 types separately was essential after the first and second generation of HPV vaccination implementation in China.

Avian pathogenic Escherichia coli (APEC) is primarily responsible for causing septicemia, pneumonitis, peritonitis, swollen head syndrome, and salpingitis in poultry, leading to significant losses in the poultry sector, particularly within the broiler industry. The removal of the lpxL and lpxM genes led to an eightfold decrease in the endotoxin levels of wild APEC strains. In this study, mutant strains of lpxL/lpxM and their O1, O2, and O78 wild-type strains were developed for an inactivated vaccine (referred to as the mutant vaccine and the wild-type vaccine, respectively), and the safety and effectiveness of these two prototype vaccines were assessed in white Leghorn chickens. Findings indicated that chickens immunized with the mutant vaccine showed a return of appetite sooner post-immunization and experienced earlier disappearance of nodules at the injection site compared to those immunized with the wild-type vaccine. Pathological examinations revealed that lesions were still present in the liver, lung, and injection site in chickens vaccinated with the wild-type vaccine 14 days post-vaccination (dpv), whereas no lesions were found in chickens vaccinated with the mutant vaccine at 14 dpv. There were no significant differences in antibody levels on the challenge day or in mortality or lesion scores between challenged birds immunized with either the mutant vaccine or the wild-type vaccine at the same dose. In this study, the safety of a single dose or overdose of the mutant vaccine and its efficacy at one dose were evaluated in broilers, and the results showed that the mutant vaccine had no adverse effects on or protected vaccinated broilers from challenge with the APEC O1, O2, or O78 strains. These results demonstrated that the mutant polyvalent inactivated vaccine is a competitive candidate against APEC O1, O2, and O78 infection compared to the wild-type vaccine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Enterotoxigenic Escherichia coli (ETEC) is an important type of pathogenic bacteria that causes diarrhea in pigs. The objective of this study was to prepare a novel tetravalent vaccine to effectively prevent piglet diarrhea caused by E. coli. In order to realize the production of K88ac-K99-ST 1 -LT B tetravalent inactivated vaccine, the biological characteristics, stability, preservation conditions, and safety of the recombinant strain BL21(DE3) (pXKKSL4) were studied, and the vaccine efficacy and minimum immune dose were measured. The results indicated that the biological characteristics, target protein expression, and immunogenicity of the 1st to 10th generations of the strain were stable. Therefore, the basic seed generation was preliminarily set as the 1st to 10th generations. The results of the efficacy tests showed that the immune protection rate could reach 90% with 1 minimum lethal dose (MLD) virulent strain attack in mice. The immunogenicity was stable, and the minimum immune dose was 0.1 mL per mouse. Our research showed that the genetically engineered vaccine developed in this way could prevent piglet diarrhea caused by enterotoxigenic E. coli through adhesin and enterotoxin. In order to realize industrial production of the vaccine as soon as possible, we conducted immunological tests and production process research on the constructed K88ac-K99-ST 1 -LT B tetravalent inactivated vaccine. The results of this study provide scientific experimental data for the commercial production of vaccines and lay a solid foundation for their industrial production., Competing Interests: Competing interests: The authors declare that there are no conflicts of interest associated with the publication of this article., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

The global public health nonprofit organization PATH hosted the third Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference in Washington, DC, from November 29 to December 1, 2022. This international gathering focused on cutting-edge research related to the development of vaccines against neglected diarrheal pathogens including Shigella, enterotoxigenic Escherichia coli (ETEC), Campylobacter, and non-typhoidal Salmonella. In addition to the conference's plenary content, the agenda featured ten breakout workshops on topics of importance to the enteric vaccine field. This unique aspect of VASE Conferences allows focused groups of attendees to engage in in-depth discussions on subjects of interest to the enteric vaccine development community. In 2022, the workshops covered a range of topics. Two focused on the public health value of enteric vaccines, with one examining how to translate evidence into policy and the other on the value proposition of potential combination vaccines against bacterial enteric pathogens. Two more workshops explored new tools for the development and evaluation of vaccines, with the first on integrating antigen/antibody technologies for mucosal vaccine and immunoprophylactic development, and the second on adjuvants specifically for Shigella vaccines for children in low- and middle-income countries. Another pair of workshops covered the status of vaccines against two emerging enteric pathogens, Campylobacter and invasive non-typhoidal Salmonella. The remaining four workshops examined the assessment of vaccine impact on acute and long-term morbidity. These included discussions on the nature and severity of intestinal inflammation; cellular immunity and immunological memory in ETEC and Shigella infections; clinical and microbiologic endpoints for Shigella vaccine efficacy studies in children; and intricacies of protective immunity to enteric pathogens. This article provides a brief summary of the presentations and discussions at each workshop in order to share these sessions with the broader enteric vaccine field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The global nonprofit organization PATH hosted the third Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference in Washington, DC, on November 29 to December 1, 2022. With a combination of plenary sessions and posters, keynote presentations, and breakout workshops, the 2022 VASE Conference featured key updates on research related to the development of vaccines against neglected diarrheal pathogens including Shigella, enterotoxigenic Escherichia coli (ETEC), Campylobacter, and Salmonella. The presentations and discussions highlighted the significant impact of these diarrheal pathogens, particularly on the health of infants and young children in low- and middle-income countries, reflecting the urgent need for the development and licensure of new enteric vaccines. Oral and poster presentations at the VASE Conference explored a range of topics, including: the global burden and clinical presentation of disease, epidemiology, and the impact of interventions; the assessment of the value of vaccines against enteric pathogens; preclinical evaluations of vaccine candidates and models of enteric diseases; vaccine candidates in clinical trials and human challenge models; host parameters and genomics that predict responses to infection and disease; the application of new omics technologies for characterization of emerging pathogens and host responses; novel adjuvants, vaccine delivery platforms, and immunization strategies; and strategies for combination/co-administered vaccines. The conference agenda also featured ten breakout workshop sessions on topics of importance to the enteric vaccine field, which are summarized separately. This article reviews key points and highlighted research presented in each of the plenary conference sessions and poster presentations at the 2022 VASE Conference., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

There are no vaccines licensed against enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea and the most common cause of travelers' diarrhea. Multivalent vaccine candidate MecVax unprecedentedly targets two ETEC enterotoxins (heat-stable toxin, STa; heat-labile toxin, LT) and the seven most prevalent ETEC adhesins (colonization factor antigen, CFA/I, coli surface antigens, CS1-CS6) and has been demonstrated preclinically to protect against STa- and LT-mediated ETEC clinical diarrhea and prevent intestinal colonization from ETEC strain H10407 (CFA/I, STa, LT). However, it is unattested whether MecVax broadly protects against intestinal colonization from ETEC strains producing the other six adhesins (CS1-CS6) also targeted by this product. In this study, we immunized rabbits with MecVax and challenged them with heterogeneous ETEC strains that express CS1-CS6 adhesins to evaluate MecVax's efficacy against bacterial intestinal colonization, thus providing broad vaccine protection against ETEC infection. Data revealed that rabbits intramuscularly immunized with MecVax developed robust responses to both ETEC enterotoxins (STa, LT) and seven adhesins (CFA/I, CS1-CS6), and when challenged with ETEC isolates expressing CS1/CS3, CS2/CS3, CS4/CS6, CS5/CS6, or CS6 adhesin, the immunized rabbits prevented over two logs (>99%) of bacteria from colonization in small intestines. Additionally, compared to a CFA-toxoid fusion protein, which is another potential ETEC vaccine antigen to target two ETEC enterotoxins and the seven adhesins, MecVax exhibited better protection against ETEC intestinal colonization. These results, in conjunction with the protection data from early studies, evidenced that MecVax is broadly protective, validating MecVax's candidacy as an effective vaccine against ETEC-associated diarrhea and accelerating ETEC vaccine development., Competing Interests: The senior author has the patent for the development of a protein-based vaccine for ETEC.

Enterotoxigenic Escherichia coli (ETEC) is one of the leading bacterial causes of diarrhoea, especially among children in low-resource settings, and travellers and military personnel from high-income countries. WHO's primary strategic goal for ETEC vaccine development is to develop a safe, effective, and affordable ETEC vaccine that reduces mortality and morbidity due to moderate-to-severe diarrhoeal disease in infants and children under 5 years of age in LMICs, as well as the long-term negative health impact on infant physical and cognitive development resulting from infection with this enteric pathogen. An effective ETEC vaccine will also likely reduce the need for antibiotic treatment and help limit the further emergence of antimicrobial resistance bacterial pathogens. The lead ETEC vaccine candidate, ETVAX, has shown field efficacy in travellers and has moved into field efficacy testing in LMIC infants and children. A Phase 3 efficacy study in LMIC infants is projected to start in 2024 and plans for a Phase 3 trial in travellers are under discussion with the U.S. FDA. Licensing for both travel and LMIC indications is projected to be feasible in the next 5-8 years. Given increasing recognition of its negative impact on child health and development in LMICs and predominance as the leading etiology of travellers' diarrhoea (TD), a standalone vaccine for ETEC is more cost-effective than vaccines targeting other TD pathogens, and a viable commercial market also exists. In contrast, combination of an ETEC vaccine with other vaccines for childhood pathogens in LMICs would maximize protection in a more cost-effective manner than a series of stand-alone vaccines. This 'Vaccine Value Profile' (VVP) for ETEC is intended to provide a high-level, holistic assessment of available data to inform the potential public health, economic and societal value of pipeline vaccines and vaccine-like products. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public private partnerships, and multi-lateral organizations. All contributors have extensive expertise on various elements of the ETEC VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in children in developing countries and in travelers. WHO has affirmed ETEC as a priority vaccine target, but there is no licensed ETEC vaccine available yet. We here describe recent, promising developments of different live, inactivated, and subunit ETEC candidate vaccines expressing or containing nontoxic enterotoxin and/or colonization factor antigens with a focus on oral vaccines. Many of the ETEC candidate vaccines have been tested in clinical trials for safety and immunogenicity and some of them also for protective efficacy in field trials or in challenge studies., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships that may be considered as potential competing interests: A.M. S. is a shareholder of the biotech company Gotovax AB that may receive a small royalty on sales of ETVAX if it becomes a commercial product., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Enterotoxigenic Escherichia coli (ETEC) is an important cause of children's and travelers' diarrhea, with no licensed vaccine. This study aimed to explore the role of cellular immunity in protection against human ETEC infection. Nine volunteers were experimentally infected with ETEC, of which six developed diarrhea. Lymphocytes were collected from peripheral blood buffy coats, before and 3, 5, 6, 7, 10, and 28 days after dose ingestion, and 34 phenotypic and functional markers were examined by mass cytometry. Thirty-three cell populations, derived by manually merging 139 cell clusters from the X-shift unsupervised clustering algorithm, were analyzed. Initially, the diarrhea group responded with increased CD56dim CD16 + natural killer cells, dendritic cells tended to rise, and mucosal-associated invariant T cells decreased. On day 5-7, an increase in plasmablasts was paralleled by a consistent rise in CD4 + Th17-like effector memory and regulatory cell subsets. CD4 + Th17-like central memory cells peaked on day 10. All Th17-like cell populations showed increased expression of activation, gut-homing, and proliferation markers. Interestingly, in the nondiarrhea group, these same CD4 + Th17-like cell populations expanded earlier, normalizing around day 7. Earlier development of these CD4 + Th17-like cell populations in the nondiarrhea group may suggest a recall response and a potential role in controlling ETEC infections., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

There are no licensed vaccines for enterotoxigenic Escherichia coli (ETEC), a common cause of children's diarrhea and travelers' diarrhea. ETEC strains producing enterotoxins (heat-labile toxin, LT; heat-stable toxin, STa) and adhesins CFA/I, CFA/II (CS1-CS3) or CFA/IV (CS4-CS6) attributed to a majority of ETEC-associated diarrheal cases, thus the two toxins (STa, LT) and the seven adhesins (CFA/I, CS1 to CS6) are historically the primary targets in ETEC vaccine development. Recent studies, however, revealed that ETEC strains with adhesins CS14, CS21, CS7, CS17, and CS12 are also prevalent and cause moderate-to-severe diarrhea; these adhesins are now considered antigen targets as well for ETEC vaccines. In this study, we applied the epitope- and structure-based multiepitope-fusion-antigen (MEFA) vaccinology platform and constructed a polyvalent protein to present immuno-dominant continuous B-cell epitopes of these five adhesins (also an STa toxoid); we then characterized this protein antigen's (termed as adhesin MEFA-II) broad immunogenicity and evaluated antibody functions against each targeted adhesin and STa toxin. Data showed that mice intramuscularly immunized with adhesin MEFA-II protein developed robust IgG to the targeted adhesins and toxin STa. Importantly, the antigen-derived antibodies significantly inhibited adherence of ETEC bacteria expressing adhesin CS7, CS12, CS14, CS17, or CS21 and reduced STa enterotoxicity. These results indicated that adhesin MEFA-II protein is broadly immunogenic and induces cross-functional antibodies, suggesting adhesin MEFA-II can be an effective ETEC vaccine antigen; if included in an ETEC vaccine candidate, adhesin MEFA-II can expand vaccine coverage and increase efficacy against ETEC-associated children's diarrhea and travelers' diarrhea. IMPORTANCE An effective vaccine is lacking against ETEC, a primary cause of children's diarrhea and traveler's diarrhea and a threat to global health. The key challenge in ETEC vaccine development is that ETEC bacteria express heterogeneous virulence determinants (>25 adhesins and two toxins). While the current strategy to target the seven most prevalent ETEC adhesins (CFA/I, CS1 to CS6) potentially lead to a vaccine against many clinical cases, the prevalence of ETEC strains shifts chronically and geographically, and ETEC expressing other adhesins, mainly CS7, CS12, CS14, CS17, and CS21, also cause moderate-to-severe diarrhea. However, it is impossible to develop an ETEC vaccine to target as many as 12 adhesins under conventional approaches. This study used a unique vaccinology platform to create a polyvalent antigen and demonstrated the antigen's broad immunogenicity and functions against the targeted ETEC adhesins, enabling the development of a broadly protective vaccine essentially against all of the important ETEC strains., Competing Interests: The authors declare no conflict of interest.

Background: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea through two enterotoxins, a heat-labile toxin and a heat-stable toxin. These toxins alter the cellular signaling pathways, ultimately triggering an increase in chloride secretion and watery diarrhea., Objective: For the development of an ETEC vaccine, we attempted to construct a peptide-specific monoclonal antibody library against heat-labile enterotoxin A subunit (LT-A) by epitope mapping using synthetic peptides., Methods: Sera produced by five mice immunized with recombinant LT-A protein were examined for specific recognition with synthetic 15-mer and 34-mer peptides of LT-A proteins using enzyme-linked immunosorbent assay. The analysis revealed that the synthetic peptides number 8, 16, 24, 33, 36, 38, and 39 reacted with an anti-LT-A polyclonal antibody. For the possible prediction of LT-A epitopes, each full-length protein sequence was subjected to BCPreds analysis and three-dimensional protein structure analysis. The data showed that three peptides (synthetic peptide numbers: 33, 36, and 38-39) have identical antigenic specificities with LT-A protein, suggesting the usefulness of these linear peptide epitopes., Results: Based on these peptides, we produced monoclonal antibodies to improve the specificity of LT-A detection. Monoclonal antibodies produced from two peptides (numbers 33 and 36) showed affinity for an LT-A recombinant antigen. Moreover, peptide epitope prediction analysis showed that the sites of the three peptides were identical to those exhibiting actual antigenicity. Also, it was confirmed that the amino acid sequence that actually showed antigenicity was included in the peptide predicted only by ETEC-LT-A-33. Also, the specificity of the antibody for ETEC-LT-A-33 was validated using bacterial cells, and the neutralizing effect of the antibody was determined by assessing cytokine release in infected HCT-8 cells., Conclusion: The monoclonal antibodies produced in this study are useful toolsfor vaccine production against ETEC and can be used to identify peptide antigencandidates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Park and Cho.)

Oral immunization is an effective strategy for inducing protective immunity against mucosal enteric pathogens. Although live-attenuated as well as subunit approaches have been explored for vaccination against enteric pathogens, inactivated whole bacterial cells may also be effective in introducing protective immunity. Successfully accomplishing this goal with inactivated whole bacterial cells will require that a complex antigenic repertoire be presented in controlled immunogenic amounts, in a safe and relatively simple and self-contained delivery format. The benefit from immunization with whole cell vaccines can be further enhanced through genetic engineering to over-express selected antigens and also by the use of mucosal adjuvants to direct a more robust immunologic response. These steps are being taken for the development of ETVAX, the most clinically advanced vaccine candidate against the major enteric pathogen, enterotoxigenic Escherichia coli (ETEC) with significant positive impact., Competing Interests: RW and AB are employees of PATH which has used various grants to fund portions of the development of ETVAX as part of PATH’s role in promoting the development of vaccines against enteric diseases., (Copyright © 2023 Walker and Bourgeois.)

A key aspect to vaccine efficacy is formulation stability. Biochemical evaluations provide information on optimal compositions or thermal stability but are routinely validated by ex vivo analysis and not efficacy in animal models. Here we assessed formulations identified to improve or reduce stability of the mucosal adjuvant dmLT being investigated in polio and enterotoxigenic E. coli (ETEC) clinical vaccines. We observed biochemical changes to dmLT protein with formulation or thermal stress, including aggregation or subunit dissociation or alternatively resistance against these changes with specific buffer compositions. However, upon injection or mucosal vaccination with ETEC fimbriae adhesin proteins or inactivated polio virus, experimental findings indicated immunization route and co-administered antigen impacted vaccine immunogenicity more so than dmLT formulation stability (or instability). These results indicate the importance of both biochemical and vaccine-derived immunity assessment in formulation optimization. In addition, these studies have implications for use of dmLT in clinical settings and for delivery in resource poor settings., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Aim: Production of IgY antibodies against CfaB-EtpA-LTB (CEL) chimeric protein and evaluation of its protective effects against enterotoxigenic Escherichia coli (ETEC) by in vivo and in vitro investigation., Methods and Results: Indirect ELISA and immunoblotting methods were applied to assess the immunogenicity and specificity of IgYs and also to evaluate the efficacy of IgYs in binding prevention and neutralizing the heat-labile (LT) toxin of ETEC bacteria. The results indicated that the anti-CEL IgY at a concentration of 2 mg ml-1 could decrease the bacterial adhesion to HT-29 cells by 74% compared to the control group.At a concentration of 750 μg ml-1, the IgY antibody managed to neutralize the disruptive LT toxin effect on the Y1 cell line. At a concentration of 2 mg ml-1, 81% reduction was observed in the fluid accumulation in the ileal loop assay., Conclusion: According to our findings, passive immunotherapy with anti-CEL IgY can prevent bacterial colonization and toxicity, thus facilitating in controlling the enteric diseases caused by ETEC infection., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Avian pathogenic Escherichia coli, a causative agent of avian colibacillosis, has been causing serious economic losses in the poultry industry. The increase in multidrug-resistant isolates and the complexity of the serotypes of this pathogen, especially the recently reported emergence of a newly predominant serogroup of O145, make the control of this disease difficult. To address this challenge, a high-throughput screening approach, called Pan-RV (Reverse vaccinology based on pangenome analysis), is proposed to search for universal protective antigens against the three traditional serogroups and the newly emerged O145. Using this approach, a total of 61 proteins regarded as probable antigens against the four important serogroups were screened from the core genome of 127 Avian pathogenic Escherichia coli (APEC) genomes, and six were verified by Western blots using antisera. Overall, our research will provide a foundation for the development of an APEC subunit vaccine against avian colibacillosis. Given the exponential growth of whole-genome sequencing (WGS) data, our Pan-RV pipeline will make screening of bacterial vaccine candidates inexpensive, rapid, and efficient. IMPORTANCE With the emergence of drug resistance and the newly predominant serogroup O145, the control of Avian pathogenic Escherichia coli is facing a serious challenge; an efficient immunological method is urgently needed. Here, for the first time, we propose a high-throughput screening approach to search for universal protective antigens against the three traditional serogroups and the newly emerged O145. Importantly, using this approach, a total of 61 proteins regarded as probable antigens against the four important serogroups were screened, and three were shown to be immunoreactive with all antisera (covering the four serogroups), thereby providing a foundation for the development of APEC subunit vaccines against avian colibacillosis. Further, our Pan-RV pipeline will provide immunological control strategies for pathogens with complex and variable genetic backgrounds such as Escherichia coli and will make screening of bacterial vaccine candidates more inexpensive, rapid, and efficient.

Colibacillosis is one of the most common and economically devastating infectious diseases in poultry production worldwide. Innovative universal vaccines are urgently needed to protect chickens from the infections caused by genetically diverse avian pathogenic Escherichia coli (APEC). Enterobactin (Ent) is a highly conserved siderophore required for E. coli iron acquisition and pathogenesis. The Ent-specific antibodies induced by a novel Ent conjugate vaccine significantly inhibited the in vitro growth of diverse APEC strains. In this study, White Leghorn chickens were immunized with the Ent conjugate vaccine using a crossed design with two variables, vaccination (with or without) and APEC challenge (O1, O78, or PBS control), resulting in six study groups (9 to 10 birds/group). The chickens were subcutaneously injected with the vaccine (100 μg per bird) at 7 days of age, followed by booster immunization at 21 days of age. The chickens were intratracheally challenged with an APEC strain (10 8 CFU/bird) or PBS at 28 days of age. At 5 days post infection, all chickens were euthanized to examine lesions and APEC colonization of the major organs. Immunization of chickens with the Ent vaccine elicited a strong immune response with a 64-fold increase in the level of Ent-specific IgY in serum. The hypervirulent strain O78 caused extensive lesions in lung, air sac, heart, liver, and spleen with significantly reduced lesion scores observed in the vaccinated chickens. Interestingly, the vaccination did not significantly reduce APEC levels in the examined organs. The APEC O1 with low virulence only caused sporadic lesions in the organs in both vaccination and control groups. The Ent conjugate vaccine altered the bacterial community of the ileum and cecum. Taken together, the findings from this study showed the Ent conjugate vaccine could trigger a strong specific immune response and was promising to confer protection against APEC infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In silico techniques are highly suited for both the discovery of new and development of available vaccines. Escherichia coli O157: H7, a main cause of food poisoning can infect humans through the consumption of contaminated water or food. Vaccination is a choice strategy to combat the bacterium. In the present study, we designed, expressed and purified a chimeric protein comprising two antigens of Escherichia coli O157: H7, including intimin and flagellin proteins, as a vaccine candidate and evaluated its immunization ability in mice. Thein silicoresults showed that the proposed antigen has a high antigenicity and conformation to be used as a potent vaccine candidate. The protein was successfully expressed in E. coli expression system with a proper level of expression (0/8g/L). Immunization evaluation showed that the protein is able to evoke the mice's humoral immunity and can confer a protective immunity against E. coli O157:H7, so that 80 % of the immunized animals were survived following the intraperitoneal injection of 100 LD 50 of the live bacteria. Shedding analysis also showed the protectivity power of the protein. Bacterial excretion in control animals remained stable at about 10 8 CFU after 15 days, while the excreted bacteria in the feces of immunized mice's decreased to about 10 2 after the same time. According to the results, the proposed protein is able to stimulate the immune responses of mice and protect them against E. coli O157:H7., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors thank Imam Hossein University for its support and research facilities., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Avian pathogenic Escherichia coli (APEC) can cause colibacillosis in poultry, characterised by localised or systemic infections. Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carcass condemnations. A live attenuated Escherichia coli O78 aroA gene mutant is widely used to prevent disease. However, no effective strategies to differentiate the vaccine strain from field strains are available, hampering follow-up of vaccination campaigns. In the current study, we report a PCR-based method to simultaneously detect the vaccine strain by targeting the vaccine-specific mutation in the aroA gene, as well as the wild type E. coli strains by targeting the xanQ gene. The specificity of this PCR was evaluated using 123 E. coli isolates, form which 5 WT aroA auxotrophic strains (WT strains with a natural aroA deficiency), as well as 7 non-Escherichia isolates. The PCR showed 100% sensitivity of the xanQ primers for E. coli detection and 100% sensitivity of the ΔaroA primers for the vaccine strain. In order to allow quantification of the vaccine strain in complex samples containing many different E. coli strains and other related organisms, such as chicken faeces, a probe-based duplex qPCR was developed. The limit of detection (LOD) of this duplex qPCR method was 8.4*103 copies/g faeces. The specificity of the duplex qPCR was confirmed by determining both the vaccine strain levels, and the total E. coli load in intestinal digesta from both vaccinated and non-vaccinated birds. E. coli could be detected in both vaccinated and non-vaccinated birds. The duplex qPCR was specific for the vaccine strain as this strain was detected in all vaccinated birds, whereas no signal was detected in non-vaccinated birds. The duplex qPCR is helpful in monitoring colonization and shedding of the vaccine strain., Competing Interests: This work was supported by a research grant from Zoetis. This funder provided support in the form of salaries for authors K.L. and D.V. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2022 Leurs et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The study assessed long-term immunopersistence and safety of the Escherichia coli ( E. coli )-produced HPV-16/18 bivalent vaccine. In total, 979 participants in the initial immunogenicity noninferiority study, including girls aged 9-14 years who were randomized in a 1:1 ratio 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) who received 3 doses of the vaccine, were invited to participate in follow-up to 30 months post vaccination (NCT03206255). Serum samples were collected at months 18 and 30, and anti-HPV-16/18 IgG antibodies were measured by enzyme-linked immunosorbent assay. Serious adverse events (SAEs) occurred from month 7 through month 30 were recorded. At month 30, in the per-protocol set, all participants remained seropositive, except for one girl in the 9-14 years (2 doses) group who seroconverted to negative for HPV-18. HPV-16 and HPV-18 antibody levels were higher in girls aged 9-17 years who received 3 doses (125.3 and 60.2 IU/ml) than in women aged 18-26 years who received 3 doses (72.6 and 28.3 IU/ml), and those in girls aged 9-14 years who received 2 doses (73.2 and 24.9 IU/ml) were comparable to those in women aged 18-26 years who received 3 doses. No SAEs were reported to be causally related to vaccination. The E. coli -produced bivalent HPV-16/18 vaccine is safe and induces persistent protective antibodies for up to 30 months after vaccination in girls aged 9-17 years receiving 2 or 3 doses.

Escherichia coli is one of the most common bacterial pathogens in cattle. Prophylactic vaccines are considered promising strategies with the potential to reduce the incidence of colibacillosis. Some studies suggested that bacterial ghosts may serve as a novel approach for preventing bacterial infections. However, the roles of administration route on vaccine immunogenicity and efficacy have not been investigated. In this study, the efficacy of vaccination via different immune routes in generating humoral and cellular immune response was compared through subcutaneous (SC), intramuscular (IM), and oral (O) administration in female BALB/c mice with bacterial ghosts prepared using wild type Escherichia coli isolates CE9, while phosphate buffer saline (PBS) and inactivated vaccines containing aluminum adjuvants (Killed) were used as control. Our results showed that the plasmid pBV220- E- aa- SNA containing E. coli was efficiently cleaved at 42°C with 94.8% positive ratio as assessed by colony counts. Transmission electron microscopy (TEM) confirmed bacteria retained intact surface structure while devoid of cytoplasmic component. We found that total IgG titers in killed, IM and SC groups showed significant increase on 7, 14, 21 and 28 days post-immunization. The IgA level of the IM group was higher than that of all other groups on the 28th day. Meanwhile, four experimental groups showed a significant difference in IgA levels compared with PBS control. In the IM group, an increase in the relative percentages of CD3+CD4+ T cells was accompanied by an increase in the relative percentages of splenic CD3+CD8+ T cells. In comparison with the inactivated vaccine, intramuscular CE9 ghosts immunization elicited higher levels of IL-1β, IL-2, IL-6 and IL-12. Subcutaneous and intramuscular immunizations were significantly associated with improved survival in comparison with oral route, traditional vaccine and the control. Pathologic assessment revealed that less severe tissue damage and inflammation were found in lung, kidney, and intestine of IM group compared with other groups. The results above demonstrate that immunization of Escherichia coli CE9 ghosts via intramuscular injection elicits a more robust antigen-specific immune response in mice to prevent the Escherichia coli infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mu, Lei, Zheng, Li, Li, Fu, Wang and Liu.)

Avian pathogenic Escherichia coli (APEC) cause extra-intestinal infections called colibacillosis, which is the dominant bacterial disease in broilers. To date, given the diversity of APEC strains and the need for an acceptable level of protection in day-old chicks, no satisfactory commercial vaccine is available. As part of a French nationwide project, we selected three representative strains among several hundred APEC that cause colibacillosis disease. We first performed experiments to develop colibacillosis in vivo models, using an inoculum of 3 × 10 7 CFU of each E. coli strain per chick. Two APEC strains (19-381 and 19-383-M1) were found to be highly virulent for day-old chicks, whereas the third strain (19-385-M1) induced no mortality nor morbidity.We then produced an autogenous vaccine using the (Llyod, 1982; MaCQueen, 1967) 19-381 and 19-383-M1 APEC strains and a passive immunization trial was undertaken. Specific-pathogen-free Leghorn hens were vaccinated twice 2 weeks apart, the control group receiving a saline solution. The vaccinated and control hens exhibited no clinical signs, and egg production and fertility of both groups were similar. Fertile eggs were collected for 2 weeks after the second vaccination and chicks were obtained. After challenge with each APEC (19-381 and 19-383-M1), chicks appeared to be partially protected from infection with the 19-383-M1 strain, with 40% mortality compared with 80% for the non-vaccinated chicks. No protection was found when the chicks were challenged with the 19-381 strain. Now, further work is needed to consider some aspects: severity of the pathogen challenge model, persistence of the protection, number of APEC strains in the autogenous vaccine, choice of adjuvants, and heterologous protection by the vaccine made from strain 19-383-M1. RESEARCH HIGHLIGHTS Three APEC strains were characterized and selected to develop in vivo models of colibacillosis.A bivalent autogenous vaccine was produced and a passive immunization trial was carried out.Protection of chicks was demonstrated when challenged with the 19-383-M1 APEC strain (homologous challenge).Further work is needed in particular to evaluate the protection against heterologous challenge.

Currently, there are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea in developing countries and the most common cause of travelers' diarrhea. A vaccine preventing ETEC bacteria from colonization at small intestines and neutralizing enterotoxin toxicity is expected to be effective against ETEC diarrhea. Protein-based multivalent vaccine candidate MecVax was demonstrated recently to induce antibodies neutralizing heat-labile toxin (LT) and heat-stable toxin (STa) enterotoxicity and inhibiting adherence of seven ETEC adhesins (CFA/I, CS1 to CS6) but also to protect against ETEC toxin-mediated clinical diarrhea in a pig challenge model. To further evaluate MecVax preclinical efficacy against ETEC colonization at small intestines, in this study, we intramuscularly immunized adult rabbits with MecVax, challenged rabbits with ETEC strain H10407 (CFA/I, LT, STa), and examined prevention of bacteria intestinal colonization. Data showed that rabbits immunized with MecVax developed antibodies to both ETEC toxins (LT, STa) and seven adhesins (CFA/I, CS1 to CS6) and had over 99.9% reduction of H10407 intestinal colonization, indicating that the broadly immunogenic ETEC vaccine candidate MecVax is protective against ETEC H10407 intestinal colonization. This study also confirmed that parenteral administration of a protein-based vaccine can prevent bacteria intestinal colonization. Protection against ETEC intestinal colonization demonstrated by this rabbit study, in conjugation with protection against ETEC enterotoxin-mediated clinical diarrhea from a previous pig challenge study, suggested that MecVax can potentially be an effective ETEC vaccine and a combined pig and rabbit challenge model can evaluate ETEC vaccine preclinical efficacy. IMPORTANCE An effective ETEC vaccine would prevent hundreds of millions of diarrhea clinical cases and save nearly 100,000 lives annually. MecVax, a protein-based injectable multivalent ETEC vaccine candidate, has been shown for the first time to induce functional antibodies against both ETEC enterotoxins (STa, LT) produced by all ETEC strains and seven ETEC adhesins (CFA/I, CS1 to CS6) expressed by ETEC strains causing a majority of ETEC diarrhea clinical cases and the moderate-to-severe cases. Moreover, MecVax was demonstrated to protect against ETEC STa or LT toxin-mediated diarrhea in a pig model. If it also protects against ETEC intestinal colonization, MecVax can be validated as an effective ETEC vaccine candidate. This adult rabbit colonization model study showed that intramuscular administration of MecVax effectively prevented intestinal colonization by H10407, perhaps the most virulent ETEC strain, affirming MecVax vaccine candidacy and accelerating vaccine development against ETEC children's diarrhea and travelers' diarrhea.

Enterotoxigenic Escherichia coli is the most important bacterial agent causing traveller's diarrhea in developing countries. Enterotoxins (LT & ST) and colonization factors (CFs) are two important factors in the ETEC pathogenesis. In the present study, a recombinant four-part fusion protein containing CFAB*ST, CFAE, and LTB (CCL) was expressed in hairy roots of Nicotiana tabacum. The synthetic gene sequence and gene order were designed based on bioinformatics analysis that predicted the best arrangement for antigenicity and stimulation of the immune response. Codon usage was optimized for expression in tobacco plant, under the control of promoter CaMV35S in plasmid pBI121. CCL was efficiently expressed in tobacco hairy roots to yield 1.11% soluble protein as determined by quantitative ELISA and Western blot. In this study, mice were immunized with purified CCL via the oral and subcutaneous route. Humoral immunity especially mucosal immunity with antigen specific IgG and IgA detected in serum and feces. The ability of CCL to elicit neutralizing antibodies was evaluated in the rabbit ileal loop model, using anti-CCL antibodies derived from immunized mice, and co-incubated with ETEC strains. A decrease in fluid accumulation in the intestinal lumen of rabbit ileal loops challenged with ETEC LT and ST positive strains, correlated with the presence of anti-CCL antibodies capable of toxin neutralization. The ability of these antibodies to neutralize toxin confirmed the recognition of epitopes, either linear or conformational, displayed by the recombinant chimeric protein expressed in transgenic tobacco hairy roots. Transgenic plants containing multivalent immunogenic vaccine candidates have the potential to be used for immunization with protection against gastrointestinal pathogens like ETEC., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Abstract: Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in developing countries, and perennially the most common cause of traveller''s diarrhea. ETEC constitute a diverse pathotype that elaborate heat-labile and/or heat-stable enterotoxins. Recent molecular pathogenesis studies reveal sophisticated pathogen–host interactions that might be exploited in efforts to prevent these important infections. While vaccine development for these important pathogens remains a formidable challenge, extensive efforts that attempt to exploit new genomic and proteomic technology platforms in discovery of novel targets are presently ongoing. [Copyright &y& Elsevier]

Abstract: Enterotoxigenic Escherichia coli (ETEC) remain a leading cause diarrheal illness, prompting a search for vaccine targets that led to the recent discovery of EtpA, a secreted adhesin of ETEC that acts by bridging flagella and host cells. In a murine model, immunization with recombinant EtpA glycoprotein inhibited colonization by two EtpA-producing human ETEC strains, H10407 and E24377A. In addition, vaccination with recombinant flagellin (serotype H11) generated antibodies that specifically recognized the tips of flagella from E24377A expressing a heterologous flagellar serotype (H28) and afforded significant protection against colonization. EtpA and/or flagellin could be valuable subunit antigens in the formulation of a broadly protective ETEC vaccine. [Copyright &y& Elsevier]