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Abstract Background No licensed human vaccines are available against enterotoxigenic Escherichia coli (ETEC), a major diarrhoeal pathogen affecting children in low- and middle-income countries and foreign travellers alike. ETVAX®, a multivalent oral whole-cell vaccine containing four inactivated ETEC strains and the heat-labile enterotoxin B subunit (LTB), has proved promising in Phase 1 and Phase 1/ 2 studies. Methods We conducted a Phase 2b double-blinded, randomized, placebo-controlled trial amongst Finnish travellers to Benin, West Africa. This report presents study design and safety and immunogenicity data. Volunteers aged 18–65 years were randomized 1:1 to receive ETVAX® or placebo. They visited Benin for 12 days, provided stool and blood samples and completed adverse event (AE) forms. IgA and IgG antibodies to LTB and O78 lipopolysaccharide (LPS) were measured by electrochemiluminescence. Results The AEs did not differ significantly between vaccine (n = 374) and placebo (n = 375) recipients. Of the solicited AEs, loose stools/diarrhoea (26.7/25.9%) and stomach ache (23.0/20.0%) were reported most commonly. Of all possibly/probably vaccine-related AEs, the most frequent were gastrointestinal symptoms (54.0/48.8%) and nervous system disorders (20.3/25.1%). Serious AEs were recorded for 4.3/5.6%, all unlikely to be vaccine related. Amongst the ETVAX® recipients, LTB-specific IgA antibodies increased 22-fold. For the 370/372 vaccine/placebo recipients, the frequency of ≥2-fold increases against LTB was 81/2.4%, and against O78 LPS 69/2.7%. The majority of ETVAX® recipients (93%) responded to either LTB or O78. Conclusions This Phase 2b trial is the largest on ETVAX® undertaken amongst travellers to date. ETVAX® showed an excellent safety profile and proved strongly immunogenic, which encourages the further development of this vaccine.

Diarrheagenic E. coli pathotypes are major foodborne pathogens causing gastrointestinal tract infections leading to hemolytic uremic syndrome and hemorrhagic colitis. Consumption of raw vegetables is encouraged due to its nutrient content and antioxidant properties, although their ingestion is linked to a series of foodborne disease outbreaks. The aim of this study was to evaluate the prevalence of Enterotoxigenic, Enteropathogenic, and Shiga toxin-producing E. coli pathotypes in raw vegetables and ready-to-eat salad for the development of better risk management. A total of 260 vegetable and salad mix (cucumber, lettuce, spinach, and carrot) samples were collected from commercial food markets in Southern districts of Khyber Pakhtunkhawa, Pakistan. About 34 % vegetable samples were contaminated with E. coli strains. 32.4 % E. coli strains from vegetable sample were identified as diarrheagenic E. coli pathotypes. Similarly, 26.7 % of salad samples were contaminated with E. coli pathotypes. 15 % [two Enterotoxigenic E. coli and one Enteropathogenic E. coli] were isolated from spinach salad samples. 25 % [three Enterotoxigenic E. coli, one Enteropathogenic E. coli and one Shiga toxin-producing E. coli] were isolated from mixed salad type A. 40 % [four Enterotoxigenic E. coli, two Enteropathogenic E. coli and two Shiga toxin-producing E. coli] were isolated from mixed salad type B. 92 % diarrheagenic E. coli pathotypes showed resistance against Tetracycline and 87 % to Ampicillin. This study showed that fresh vegetable and their products were contaminated with multidrug-resistant E. coli pathotypes.

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

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

•ETEC is a top cause of diarrhea in children and travelers to low-resource countries.•Models suggest ETEC vaccines would be cost-effective and have a beneficial impact.•ETVAX is the most advanced ETEC candidate; a subunit approach is also in clinical development.•Ongoing research is evaluating the impact of adjuvants and new “omics” technologies.•Limited financing for ETEC vaccine may be enhanced by combined vaccine formulations. Enterotoxigenic Escherichia coli (ETEC) is one of the most common bacterial causes of diarrhea-associated morbidity and mortality, particularly among infants and young children in developing countries. Still, the true impact on child and traveler health is likely underestimated. There are currently no licensed vaccines for ETEC, but studies indicate high public health impact, cost-effectiveness, and feasibility of immune protection through vaccination. ETEC vaccine development remains a World Health Organization priority. Traditionally, ETEC vaccine development efforts have focused on inducing antitoxin and anticolonization antigen immunity, as studies indicate that antibodies against both antigen types can contribute to protection and thus have potential for vaccines. Leading cellular vaccine candidates are ETVAX (a mixture of four inactivated strains) and ACE527 (a mixture of three live attenuated strains), both of which have been found to be safe and immunogenic in Phase 1/2 trials. ETVAX is the furthest along in development with descending-age studies already underway in Bangladesh. Other ETEC vaccine candidates based on protein subunits, toxoids (both LT and ST), or novel, more broadly conserved ETEC antigens are also under development. Of these, a protein adhesin-based subunit approach is the most advanced. Impact and economic models suggest favorable vaccine cost-effectiveness, which may help expand market interest in ETEC vaccines. Combination vaccine formulations may help improve the economic case for development and use, and better point-of-care diagnostics will help to raise awareness of the true health burden of ETEC and highlight the potential public health benefit of ETEC vaccine introduction. Better diagnostics and vaccine demand forecasting will also improve vaccine development financing and support accelerated uptake once a licensed vaccine becomes available.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease worldwide, particularly in children in low- and middle-income countries. Its ability to rapidly colonize the intestinal tract through diverse colonization factors and toxins underpins its significant public health impact. Despite extensive research and several vaccine candidates reaching clinical trials, no licensed vaccine exists for ETEC. This review explores the temporal and spatial coordination of ETEC virulence factors, focusing on the interplay between adherence mechanisms and toxin production as critical targets for therapeutic intervention. Advancements in molecular biology and host–pathogen interaction studies have uncovered species-specific variations and cross-reactivity between human and animal strains. In particular, the heat-labile (LT) and heat-stable (ST) toxins have provided crucial insights into molecular mechanisms and intestinal disruption. Additional exotoxins, such as EAST-1 and hemolysins, further highlight the multifactorial nature of ETEC pathogenicity. Innovative vaccine strategies, including multiepitope fusion antigens (MEFAs), mRNA-based approaches, and glycoconjugates, aim to enhance broad-spectrum immunity. Novel delivery methods, like intradermal immunization, show promise in eliciting robust immune responses. Successful vaccination against ETEC will offer an effective and affordable solution with the potential to greatly reduce mortality and prevent stunting, representing a highly impactful and cost-efficient solution to a critical global health challenge.

Purpose of ReviewReview recent developments pertaining to the epidemiology, molecular pathogenesis, and sequelae of enterotoxigenic Escherichia coli (ETEC) infections in addition to discussion of challenges for vaccinology.Recent FindingsETEC are a major cause of diarrheal illness in resource poor areas of the world where they contribute to unacceptable morbidity and continued mortality particularly among young children; yet, precise epidemiologic estimates of their contribution to death and chronic disease have been difficult to obtain. Although most pathogenesis studies, and consequently vaccine development have focused intensively on canonical antigens, more recently identified molecules unique to the ETEC pathovar may inform our understanding of ETEC virulence, and the approach to broadly protective vaccines.SummaryETEC undeniably continue to have a substantial impact on global health; however, further studies are needed to clarify the true impact of these infections, particularly in regions where access to care may be limited. Likewise, our present understanding of the relationship of ETEC infection to non-diarrheal sequelae is presently limited, and additional effort will be required to achieve a mechanistic understanding of these diseases and to fulfill Koch’s postulates on a molecular level. Precise elucidation of the role played by novel virulence factors, the global burden of acute illness, and the contribution of these pathogens and/or their toxins to non-diarrheal morbidity remain important imperatives.

•To provide a comprehensive protection, the SLS (STa–LTB–STb) antigen and two fimbriae proteins were mixed into the novel vaccine, which could produce multiple antibodies against both fimbriae and enterotoxins.•Fifteen healthy pregnant sows and their 179 healthy suckling piglets were used as a pig model for this study, and three compared vaccines were introduced.•We introduced a sensory evaluation method to score the degree of diarrhea, and plotted a spider chart to visualized it, which have not been used yet. As one of the most challenging problems in swine industry, piglet diarrhea has caused huge economic loss globally. Currently, vaccination is the most effective way of controlling enterotoxigenic Escherichia coli (ETEC) diarrhea. However, existing commercial vaccines could not provide broad protection against different types of ETEC. In this study, we mixed a enterotoxin fusion protein SLS (STa–LTB–STb) with the main fimbrial F4ac and F5 antigens as a novel multivalent vaccine candidate. Then an overall evaluation of this vaccine candidate against ETEC was carried out in a pig model. We found that the IgG titers in serum as well as colostrum in all the vaccinated sows were significantly higher than that in the control group (P < 0.05). By using a sensory evaluation method, we demonstrated that piglets in the vaccinated group exhibited significantly healthier status than the unimmunized group. Moreover, in response to F41 + ETEC challenge, none of the piglets with the vaccine candidate experienced diarrhea, whereas 30% of the piglets suffered without vaccination. In conclusion, these results showed that the candidate vaccine could elicit multiple high-titer antibodies against all the main virulence factors and provide a broad and effective protection against ETEC diarrhea.

Diarrheal diseases caused by Shigella and enterotoxigenic Escherichia coli (ETEC) are significant health burdens, especially in resource-limited regions with high child mortality. In response to the lack of licensed vaccines and rising antibiotic resistance for these pathogens, this study developed a recombinant Shigella flexneri strain with the novel incorporation of the eltb gene for the heat-labile enterotoxin B (LTB) subunit of ETEC directly into Shigella’s genome, enhancing stability and consistent production. This approach combines the immunogenic potential of LTB with the antigen delivery properties of S. flexneri outer membrane vesicles (OMVs), aiming to provide cross-protection against both bacterial pathogens in a stable, non-replicating vaccine platform. We confirmed successful expression through GM1-capture ELISA, achieving levels comparable to ETEC. Additionally, proteomic analysis verified that the isolated vesicles from the recombinant strains contain the LTB protein and the main outer membrane proteins and virulence factors from Shigella, including OmpA, OmpC, IcsA, SepA, and Ipa proteins, and increased expression of Slp and OmpX. Thus, our newly designed S. flexneri OMVs, engineered to carry ETEC’s LTB toxin, represent a promising strategy to be considered as a subunit vaccine candidate against S. flexneri and ETEC.

Post-weaning diarrhea due to enterotoxigenic Escherichia coli (ETEC) is a common disease of piglets and causes great economic loss for the swine industry. Over the past few decades, decreasing effectiveness of conventional antibiotics has caused serious problems because of the growing emergence of multidrug-resistant (MDR) pathogens. Various studies have indicated that antimicrobial peptides (AMPs) have potential to serve as an alternative to antibiotics owing to rapid killing action and highly selective toxicity. Our previous studies have shown that AMP GW-Q4 and its derivatives possess effective antibacterial activities against the Gram-negative bacteria. Hence, in the current study, we evaluated the antibacterial efficacy of GW-Q4 and its derivatives against MDR ETEC and their minimal inhibition concentration (MIC) values were determined to be around 2~32 μg/mL. Among them, AMP Q4-15a-1 with the second lowest MIC (4 μg/mL) and the highest minimal hemolysis concentration (MHC, 256 μg/mL), thus showing the greatest selectivity (MHC/MIC = 64) was selected for further investigations. Moreover, Q4-15a-1 showed dose-dependent bactericidal activity against MDR ETEC in time–kill curve assays. According to the cellular localization and membrane integrity analyses using confocal microscopy, Q4-15a-1 can rapidly interact with the bacterial surface, disrupt the membrane and enter cytosol in less than 30 min. Minimum biofilm eradication concentration (MBEC) of Q4-15a-1 is 4× MIC (16 μg/mL), indicating that Q4-15a-1 is effective against MDR ETEC biofilm. Besides, we established an MDR ETEC infection model with intestinal porcine epithelial cell-1 (IPEC-1). In this infection model, 32 μg/mL Q4-15a-1 can completely inhibit ETEC adhesion onto IPEC-1. Overall, these results suggested that Q4-15a-1 may be a promising antibacterial candidate for treatment of weaned piglets infected by MDR ETEC.