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14 pages, 1599 KiB

Open AccessArticle

Validation of a Human Challenge Model Using an LT-Expressing Enterotoxigenic E. coli Strain (LSN03-016011) and Characterization of Potential Amelioration of Disease by an Investigational Oral Vaccine Candidate (VLA1701)

by Kawsar R. Talaat, Chad K. Porter, Subhra Chakraborty, Brittany L. Feijoo, Jessica Brubaker, Brittany M. Adjoodani, Barbara DeNearing, Michael G. Prouty, Steven T. Poole, A. Louis Bourgeois, Madison Billingsley, David A. Sack, Susanne Eder-Lingelbach and Christian Taucher

Microorganisms 2024, 12(4), 727; https://doi.org/10.3390/microorganisms12040727 - 03 Apr 2024

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Abstract

Controlled human infection models are important tools for the evaluation of vaccines against diseases where an appropriate correlate of protection has not been identified. Enterotoxigenic Escherichia coli (ETEC) strain LSN03-016011/A (LSN03) is an LT enterotoxin and CS17-expressing ETEC strain useful for evaluating vaccine [...] Read more.

Controlled human infection models are important tools for the evaluation of vaccines against diseases where an appropriate correlate of protection has not been identified. Enterotoxigenic Escherichia coli (ETEC) strain LSN03-016011/A (LSN03) is an LT enterotoxin and CS17-expressing ETEC strain useful for evaluating vaccine candidates targeting LT-expressing strains. We sought to confirm the ability of the LSN03 strain to induce moderate-to-severe diarrhea in a healthy American adult population, as well as the impact of immunization with an investigational cholera/ETEC vaccine (VLA-1701) on disease outcomes. A randomized, double-blinded pilot study was conducted in which participants received two doses of VLA1701 or placebo orally, one week apart; eight days after the second vaccination, 30 participants (15 vaccinees and 15 placebo recipients) were challenged with approximately 5 × 10⁹ colony-forming units of LSN03. The vaccine was well tolerated, with no significant adverse events. The vaccine also induced serum IgA and IgG responses to LT. After challenge, 11 of the placebo recipients (73.3%; 95%CI: 48.0–89.1) and 7 of the VLA1701 recipients (46.7%; 95%CI: 24.8–68.8) had moderate-to-severe diarrhea (p = 0.26), while 14 placebo recipients (93%) and 8 vaccine recipients (53.3%) experienced diarrhea of any severity, resulting in a protective efficacy of 42.9% (p = 0.035). In addition, the vaccine also appeared to provide protection against more severe diarrhea (p = 0.054). Vaccinees also tended to shed lower levels of the LSN03 challenge strain compared to placebo recipients (p = 0.056). In addition, the disease severity score was lower for the vaccinees than for the placebo recipients (p = 0.046). In summary, the LSN03 ETEC challenge strain induced moderate-to-severe diarrhea in 73.3% of placebo recipients. VLA1701 vaccination ameliorated disease severity, as observed by several parameters, including the percentage of participants experiencing diarrhea, as well as stool frequency and ETEC severity scores. These data highlight the potential value of LSN03 as a suitable ETEC challenge strain to evaluate LT-based vaccine targets (NCT03576183). Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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14 pages, 3892 KiB

Open AccessArticle

Application of a Novel Proteomic Microarray Reveals High Exposure to Diarrhoeagenic Escherichia coli among Children in Zambia Participating in a Phase I Clinical Trial

by Kapambwe Mwape, Cynthia Mubanga, Obvious Nchimunya Chilyabanyama, Kennedy Chibesa, Caroline Cleopatra Chisenga, Suwilanji Silwamba, Arlo Randall, Xiaowu Liang, Tobias George Barnard, Michelo Simuyandi and Roma Chilengi

Microorganisms 2024, 12(3), 420; https://doi.org/10.3390/microorganisms12030420 - 20 Feb 2024

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Abstract

Diarrhoeagenic E. coli (DEC) significantly contributes to the burden of diarrhoea among children. Currently, there is no approved vaccine against DEC, but several vaccines against the enterotoxigenic E. coli (ETEC) pathotype are in advanced clinical trial stages, including the ETVAX^® vaccine, undergoing [...] Read more.

Diarrhoeagenic E. coli (DEC) significantly contributes to the burden of diarrhoea among children. Currently, there is no approved vaccine against DEC, but several vaccines against the enterotoxigenic E. coli (ETEC) pathotype are in advanced clinical trial stages, including the ETVAX^® vaccine, undergoing evaluation in Zambia. This study reports on the reactivity of antibodies from ETVAX^® vaccine and placebo recipients in a phase I clinical trial to proteins derived from (DEC) other than ETEC. Plasma samples collected at two time points (prior to any vaccination and post-third dose vaccination) from 16 vaccinated and 4 placebo participants in a phase 1 clinical trial examining the safety, tolerability, and immunogenicity of ETVAX^® with dmLT adjuvant were evaluated for IgG response to E. coli antigens other than ETEC using the Pan-DEC protein microarray. This was the first field application of the novel pan-DEC array as a new tool in assessing the antigenic breadth of antibody responses induced by the ETVAX vaccine, as well as to assess early life exposure to DEC pathotypes and other bacterial enteric pathogens. We observed that plasma obtained from ETVAX^® and placebo recipients had high antibody reactivity to Ipa, SseC and EspB proteins. These findings suggest that there is high exposure early in life to DEC pathogens, like EPEC, EHEC, EAEC and EIEC in addition to ETEC, in the Zambian population. These immunological observations are consistent with the results of recent epidemiological studies assessing the etiology of diarrheal disease among infants and young children in Zambia. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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15 pages, 3159 KiB

Open AccessArticle

Epidemiology of Enterotoxigenic Escherichia coli among Children and Adults Seeking Care at Hospitals in Two Geographically Distinct Rural Areas in Bangladesh

by Subhra Chakraborty, Fatema-Tuz Johura, Marzia Sultana, Xueyan Zhang, Abdus Sadique, Christine M. George, Shirajum Monira, David A. Sack, Richard Bradley Sack and Munirul Alam

Microorganisms 2024, 12(2), 359; https://doi.org/10.3390/microorganisms12020359 - 09 Feb 2024

Cited by 1 | Viewed by 714

Abstract

Enterotoxigenic Escherichia coli (ETEC) infections undeniably continue to have substantial morbidity and mortality in younger children; however, limited data are available on the disease burden of older children and adults and on ETEC epidemiology by geographical location at the subnational level. Facility-based surveillance [...] Read more.

Enterotoxigenic Escherichia coli (ETEC) infections undeniably continue to have substantial morbidity and mortality in younger children; however, limited data are available on the disease burden of older children and adults and on ETEC epidemiology by geographical location at the subnational level. Facility-based surveillance over the years was established to identify patients with ETEC diarrhea in two geographically distinct areas in rural Bangladesh, Chhatak in the north and Mathbaria in the southern coastal area. ETEC was highly prevalent in both areas, while the proportions, toxin types and colonization factors varied by location, season and age groups. Children < 5 years old and adults between 20 and 60 years old were at the highest risk of ETEC diarrhea which required urgent care. This study underscores the importance of capturing subnational and seasonal variations in ETEC epidemiology. ETEC vaccine developers and public health stakeholders may need to target adults between 20 and 60 years of age in addition to young children as new vaccines currently under development become licensed and introduction begins. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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15 pages, 2548 KiB

Open AccessArticle

The Antimicrobial Resistance of Enterotoxigenic Escherichia coli from Diarrheal Patients and the Environment in Two Geographically Distinct Rural Areas in Bangladesh over the Years

by Fatema-Tuz Johura, Marzia Sultana, Abdus Sadique, Shirajum Monira, David A. Sack, Richard Bradley Sack, Munirul Alam and Subhra Chakraborty

Microorganisms 2024, 12(2), 301; https://doi.org/10.3390/microorganisms12020301 - 31 Jan 2024

Cited by 1 | Viewed by 830

Abstract

Antimicrobial resistance (AMR) is an unprecedented global health challenge, involving the transfer of bacteria and genes between humans and the environment. We simultaneously and longitudinally determined the AMR of enterotoxigenic Escherichia coli (ETEC) strains isolated from diarrheal patients and an aquatic environment over [...] Read more.

Antimicrobial resistance (AMR) is an unprecedented global health challenge, involving the transfer of bacteria and genes between humans and the environment. We simultaneously and longitudinally determined the AMR of enterotoxigenic Escherichia coli (ETEC) strains isolated from diarrheal patients and an aquatic environment over two years from two geographically distinct locations, Coastal Mathbaria and Northern Chhatak in Bangladesh. A total of 60% and 72% of ETEC strains from the patients in Mathbaria and Chhatak, respectively, were multi-drug resistant (MDR) with a high proportion of ETEC resistant to nalidixic acid (80.7%), macrolides (49.1–89.7%), ampicillin (57.9–69%), and trimethoprim/sulfamethoxazole (55.2%). From the surface water, 68.8% and 30% of ETEC were MDR in Mathbaria and Chhatak, respectively, with a high proportion of ETEC strains resistant to macrolides (87.5–100%), ampicillin (50–75%), ceftriaxone (62.5%), and nalidixic acid (40%). Notably, 80–100% of the ETEC strains were susceptible to tetracycline and quinolones (ciprofloxacin and norfloxacin), both in clinical and aquatic ETEC. The AMR varied by the ETEC toxin types. The patterns of excessive or limited consumption of drugs to treat diarrhea over time in Bangladesh were reflected in the ETEC AMR from the patients and the environment. The high prevalence of MDR-ETEC strains in humans and the environment is of concern, which calls for vaccines and other preventative measures against ETEC. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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18 pages, 2326 KiB

Open AccessArticle

Efficacy Evaluation of an Intradermally Delivered Enterotoxigenic Escherichia coli CF Antigen I Fimbrial Tip Adhesin Vaccine Coadministered with Heat-Labile Enterotoxin with LT(R192G) against Experimental Challenge with Enterotoxigenic E. coli H10407 in Healthy Adult Volunteers

by Ramiro L. Gutiérrez, Chad K. Porter, Clayton Harro, Kawsar Talaat, Mark S. Riddle, Barbara DeNearing, Jessica Brubaker, Milton Maciel, Jr., Renee M. Laird, Steven Poole, Subra Chakraborty, Nicole Maier, David A. Sack and Stephen J. Savarino

Microorganisms 2024, 12(2), 288; https://doi.org/10.3390/microorganisms12020288 - 29 Jan 2024

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Abstract

Background. Enterotoxigenic E. coli (ETEC) is a principal cause of diarrhea in travelers, deployed military personnel, and children living in low to middle-income countries. ETEC expresses a variety of virulence factors including colonization factors (CF) that facilitate adherence to the intestinal mucosa. We [...] Read more.

Background. Enterotoxigenic E. coli (ETEC) is a principal cause of diarrhea in travelers, deployed military personnel, and children living in low to middle-income countries. ETEC expresses a variety of virulence factors including colonization factors (CF) that facilitate adherence to the intestinal mucosa. We assessed the protective efficacy of a tip-localized subunit of CF antigen I (CFA/I), CfaE, delivered intradermally with the mutant E. coli heat-labile enterotoxin, LTR192G, in a controlled human infection model (CHIM). Methods. Three cohorts of healthy adult subjects were enrolled and given three doses of 25 μg CfaE + 100 ng LTR192G vaccine intradermally at 3-week intervals. Approximately 28 days after the last vaccination, vaccinated and unvaccinated subjects were admitted as inpatients and challenged with approximately 2 × 10⁷ cfu of CFA/I+ ETEC strain H10407 following an overnight fast. Subjects were assessed for moderate-to-severe diarrhea for 5 days post-challenge. Results. A total of 52 volunteers received all three vaccinations; 41 vaccinated and 43 unvaccinated subjects were challenged and assessed for moderate-to-severe diarrhea. Naïve attack rates varied from 45.5% to 64.7% across the cohorts yielding an overall efficacy estimate of 27.8% (95% confidence intervals: −7.5–51.6%). In addition to reducing moderate–severe diarrhea rates, the vaccine significantly reduced loose stool output and overall ETEC disease severity. Conclusions. This is the first study to demonstrate protection against ETEC challenge after intradermal vaccination with an ETEC adhesin. Further examination of the challenge methodology is necessary to address the variability in naïve attack rate observed among the three cohorts in the present study. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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12 pages, 818 KiB

Open AccessArticle

Contribution of the Rapid LAMP-Based Diagnostic Test (RLDT) to the Evaluation of Enterotoxigenic Escherichia coli (ETEC) and Shigella in Childhood Diarrhea in the Peri-Urban Area of Ouagadougou, Burkina Faso

by Alimatou Héma, Samuel S. Sermé, Jean Sawadogo, Amidou Diarra, Aissata Barry, Amidou Z. Ouédraogo, Issa Nébié, Alfred B. Tiono, Sophie Houard, Subhra Chakraborty, Alphonse Ouédraogo and Sodiomon B. Sirima

Microorganisms 2023, 11(11), 2809; https://doi.org/10.3390/microorganisms11112809 - 19 Nov 2023

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Abstract

The estimates of enterotoxigenic Escherichia coli (ETEC) and Shigella burden in developing countries are limited by the lack of rapid, accessible, and sensitive diagnostics and surveillance tools. We used a “Rapid LAMP based Diagnostic Test (RLDT)” to detect ETEC and Shigella in diarrheal [...] Read more.

The estimates of enterotoxigenic Escherichia coli (ETEC) and Shigella burden in developing countries are limited by the lack of rapid, accessible, and sensitive diagnostics and surveillance tools. We used a “Rapid LAMP based Diagnostic Test (RLDT)” to detect ETEC and Shigella in diarrheal and non-diarrheal stool samples from a 12-month longitudinal cohort of children under five years of age in a peri-urban area of Ouagadougou in Burkina Faso (West Africa). To allow comparison with the RLDT-Shigella results, conventional culture methods were used to identify Shigella strains in the stool samples. As conventional culture alone cannot detect ETEC cases, a subset of E. coli-like colonies was tested using conventional PCR to detect ETEC toxins genes. Of the 165 stool samples analyzed for ETEC, 24.9% were positive when using RLDT against 4.2% when using culture followed by PCR. ETEC toxin distribution when using RLDT was STp 17.6% (29/165), LT 11.5% (19/165), and STh 8.5% (14/165). Of the 263 specimens tested for Shigella, 44.8% were positive when using RLDT against 23.2% when using culture. The sensitivity and specificity of the RLDT compared to culture (followed by PCR for ETEC) were 93.44% and 69.8% for Shigella and 83.7% and 77.9% for ETEC, respectively. This study indicates that both Shigella and ETEC are substantially underdiagnosed when using conventional culture and highlights the potential contribution of the new RLDT method to improve enteric disease burden estimation and to guide future efforts to prevent and control bacterial enteric infection and disease. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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13 pages, 932 KiB

Open AccessArticle

Prevalence of Diarrhoeagenic Escherichia coli among Children Aged between 0–36 Months in Peri-Urban Areas of Lusaka

by Kapambwe Mwape, Samuel Bosomprah, Kennedy Chibesa, Suwilanji Silwamba, Charlie Chaluma Luchen, Nsofwa Sukwa, Cynthia Mubanga, Bernard Phiri, Mwelwa Chibuye, Fraser Liswaniso, Paul Somwe, Obvious Chilyabanyama, Caroline Cleopatra Chisenga, Monde Muyoyeta, Michelo Simuyandi, Tobias George Barnard and Roma Chilengi

Microorganisms 2023, 11(11), 2790; https://doi.org/10.3390/microorganisms11112790 - 17 Nov 2023

Cited by 1 | Viewed by 1228

Abstract

Diarrhoea is a major contributor to childhood morbidity and mortality in developing countries, with diarrhoeagenic Escherichia coli being among the top aetiological agents. We sought to investigate the burden and describe the diarrhoeagenic E. coli pathotypes causing diarrhoea among children in peri-urban areas [...] Read more.

Diarrhoea is a major contributor to childhood morbidity and mortality in developing countries, with diarrhoeagenic Escherichia coli being among the top aetiological agents. We sought to investigate the burden and describe the diarrhoeagenic E. coli pathotypes causing diarrhoea among children in peri-urban areas of Lusaka, Zambia. This was a facility-based surveillance study conducted over an 8-month period from 2020 to 2021. Stool samples were collected from children aged 0–3 years presenting with diarrhoea at five peri-urban health facilities in Lusaka. Stool samples were tested for diarrhoeagenic E. coli using the Novodiag bacterial GE+^® panel, a platform utilising real-time PCR and microarray technology to detect bacterial pathogens. Of the 590 samples tested, diarrhoeagenic E. coli were detected in 471 (76.1%). The top three pathogens were enteropathogenic E. coli 45.4% (n = 268), enteroaggregative E. coli 39.5% (n = 233), and enterotoxigenic E. coli 29.7% (n = 176). Our results revealed that 50.1% of the diarrhoeagenic E. coli positive samples comprised multiple pathotypes of varying virulence gene combinations. Our study demonstrates a high prevalence of diarrhoeagenic E. coli in childhood diarrhoea and the early exposure (<12 months) of children to enteric pathogens. This calls for the early implementation of preventive interventions for paediatric diarrhoea. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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14 pages, 4809 KiB

Open AccessArticle

Anti-Toxin Responses to Natural Enterotoxigenic Escherichia coli (ETEC) Infection in Adults and Children in Bangladesh

by Petra Girardi, Taufiqur Rahman Bhuiyan, Samuel B. Lundin, Shushan Harutyunyan, Irene Neuhauser, Farhana Khanam, Gábor Nagy, Valéria Szijártó, Tamás Henics, Eszter Nagy, Ali M. Harandi and Firdausi Qadri

Microorganisms 2023, 11(10), 2524; https://doi.org/10.3390/microorganisms11102524 - 09 Oct 2023

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Abstract

A sero-epidemiology study was conducted in Dhaka, Bangladesh between January 2020 and February 2021 to assess the immune responses to ETEC infection in adults and children. (1) Background: Enterotoxigenic Escherichia coli infection is a main cause of diarrheal disease in endemic countries. The [...] Read more.

A sero-epidemiology study was conducted in Dhaka, Bangladesh between January 2020 and February 2021 to assess the immune responses to ETEC infection in adults and children. (1) Background: Enterotoxigenic Escherichia coli infection is a main cause of diarrheal disease in endemic countries. The characterization of the immune responses evoked by natural infection can guide vaccine development efforts. (2) Methods: A total of 617 adult and 480 pediatric diarrheal patients were screened, and 43 adults and 46 children (below 5 years of age) with an acute ETEC infection completed the study. The plasma samples were analyzed for antibody responses against the ETEC toxins. (3) Results: Heat-stable toxin (ST)-positive ETEC is the main cause of ETEC infection in adults, unlike in children in an endemic setting. We detected very low levels of anti-ST antibodies, and no ST-neutralizing activity. However, infection with ETEC strains expressing the heat-labile toxin (LT) induced systemic antibody responses in less than 25% of subjects. The antibody levels against LTA and LTB, as well as cholera toxin (CT), correlated well. The anti-LT antibodies were shown to have LT- and CT- neutralizing activity. The antibody reactivity against linear LT epitopes did not correlate with toxin-neutralizing activity. (4) Conclusions: Unlike LT, ST is a poor antigen and even adults have low anti-ST antibody levels that do not allow for the detection of toxin-neutralizing activity. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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13 pages, 1864 KiB

Open AccessArticle

A Polyvalent Adhesin–Toxoid Multiepitope-Fusion-Antigen-Induced Functional Antibodies against Five Enterotoxigenic Escherichia coli Adhesins (CS7, CS12, CS14, CS17, and CS21) but Not Enterotoxins (LT and STa)

by Siqi Li, Hyesuk Seo, Ipshita Upadhyay and Weiping Zhang

Microorganisms 2023, 11(10), 2473; https://doi.org/10.3390/microorganisms11102473 - 01 Oct 2023

Cited by 2 | Viewed by 1020

Abstract

The increasing prevalence and association with moderate-to-severe diarrhea make enterotoxigenic Escherichia coli (ETEC) adhesins CS7, CS12, CS14, CS17, and CS21 potential targets of ETEC vaccines. Currently, there are no vaccines licensed to protect against ETEC, a top cause of children’s diarrhea and travelers’ [...] Read more.

The increasing prevalence and association with moderate-to-severe diarrhea make enterotoxigenic Escherichia coli (ETEC) adhesins CS7, CS12, CS14, CS17, and CS21 potential targets of ETEC vaccines. Currently, there are no vaccines licensed to protect against ETEC, a top cause of children’s diarrhea and travelers’ diarrhea. Recently, a polyvalent adhesin protein (adhesin MEFA-II) was demonstrated to induce antibodies that inhibited adherence from these five ETEC adhesins and reduced the enterotoxicity of ETEC heat-stable toxin (STa), which plays a key role in causing ETEC-associated diarrhea. To improve adhesin MEFA-II for functional antibodies against STa toxin and the other ETEC toxin, heat-labile toxin (LT), we modified adhesin MEFA-II by adding another STa toxoid and an LT epitope; we examined the new antigen immunogenicity (to five adhesins and two toxins) and more importantly antibody functions against ETEC adherence and STa and LT enterotoxicity. Data show that mice intramuscularly immunized with the new antigen (adhesin MEFA-IIb) developed robust IgG responses to the targeted adhesins (CS7, CS12, CS14, CS17, and CS21) and toxins (STa and LT). Mouse antibodies inhibited the adherence of ETEC strains expressing any of these five adhesins but failed to neutralize STa or LT enterotoxicity. In further studies, rabbits intramuscularly immunized with adhesin MEFA-IIb developed robust antigen-specific antibodies; when challenged with an ETEC isolate expressing CS21 adhesin (JF2101, CS21, and STa), the immunized rabbits showed a significant reduction in intestinal colonization by ETEC bacteria. These data indicate that adhesin MEFA-IIb is broadly immunogenic and induces functional antibodies against the targeted ETEC adhesins but not the toxins. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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16 pages, 724 KiB

Open AccessArticle

Efficacy of an Enterotoxigenic Escherichia coli (ETEC) Vaccine on the Incidence and Severity of Traveler’s Diarrhea (TD): Evaluation of Alternative Endpoints and a TD Severity Score

by Nicole Maier, Shannon L. Grahek, Jane Halpern, Suzanne Restrepo, Felipe Troncoso, Janet Shimko, Olga Torres, Jaime Belkind-Gerson, David A. Sack, Ann-Mari Svennerholm, Björn Gustafsson, Björn Sjöstrand, Nils Carlin, A. Louis Bourgeois and Chad K. Porter

Microorganisms 2023, 11(10), 2414; https://doi.org/10.3390/microorganisms11102414 - 27 Sep 2023

Cited by 1 | Viewed by 1180

Abstract

The efficacy of an Oral Whole Cell ETEC Vaccine (OEV) against Travelers’ Diarrhea (TD) was reexamined using novel outcome and immunologic measures. More specifically, a recently developed disease severity score and alternative clinical endpoints were evaluated as part of an initial validation effort [...] Read more.

The efficacy of an Oral Whole Cell ETEC Vaccine (OEV) against Travelers’ Diarrhea (TD) was reexamined using novel outcome and immunologic measures. More specifically, a recently developed disease severity score and alternative clinical endpoints were evaluated as part of an initial validation effort to access the efficacy of a vaccine intervention for the first time in travelers to an ETEC endemic area. A randomized, double-blind, placebo-controlled trial followed travelers to Guatemala or Mexico up to 28 days after arrival in the country following vaccination (two doses two weeks apart) with an ETEC vaccine. Fecal samples were collected upon arrival, departure, and during TD for pathogen identification. Serum was collected in a subset of subjects to determine IgA cholera toxin B subunit (CTB) antibody titers upon their arrival in the country. The ETEC vaccine’s efficacy, utilizing a TD severity score and other alternative endpoints, including the relationship between antibody levels and TD risk, was assessed and compared to the per-protocol primary efficacy endpoint. A total of 1435 subjects completed 7–28 days of follow-up and had available data. Vaccine efficacy was higher against more severe (≥5 unformed stools/24 h) ETEC-attributable TD and when accounting for immunologic take (PE ≥ 50%; p < 0.05). The vaccine protected against less severe (3 and 4 unformed stools/24 h) ETEC-attributable TD when accounting for symptom severity or change in activity (PE = 76.3%, p = 0.01). Immunologic take of the vaccine was associated with a reduced risk of infection with ETEC and other enteric pathogens, and with lower TD severity. Clear efficacy was observed among vaccinees with a TD score of ≥4 or ≥5, regardless of immunologic take (PE = 72.0% and 79.0%, respectively, p ≤ 0.03). The vaccine reduced the incidence and severity of ETEC, and this warrants accelerated evaluation of the improved formulation (designated ETVAX), currently undergoing advanced field testing. Subjects with serum IgA titers to CTB had a lower risk of infection with ETEC and Campylobacter jejuni/coli. Furthermore, the TD severity score provided a more robust descriptor of disease severity and should be included as an endpoint in future studies. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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13 pages, 811 KiB

Open AccessArticle

Seroprevalence Study of Conserved Enterotoxigenic Escherichia coli Antigens in Globally Diverse Populations

by Frederick Matthew Kuhlmann, Vadim Grigura, Timothy J. Vickers, Michael G. Prouty, Lora L. Iannotti, Sherlie Jean Louis Dulience and James M. Fleckenstein

Microorganisms 2023, 11(9), 2221; https://doi.org/10.3390/microorganisms11092221 - 31 Aug 2023

Cited by 1 | Viewed by 945

Abstract

Enterotoxigenic Escherichia coli (ETEC) are common causes of infectious diarrhea among young children of low-and middle-income countries (LMICs) and travelers to these regions. Despite their significant contributions to the morbidity and mortality associated with childhood and traveler’s diarrhea, no licensed vaccines are available. [...] Read more.

Enterotoxigenic Escherichia coli (ETEC) are common causes of infectious diarrhea among young children of low-and middle-income countries (LMICs) and travelers to these regions. Despite their significant contributions to the morbidity and mortality associated with childhood and traveler’s diarrhea, no licensed vaccines are available. Current vaccine strategies may benefit from the inclusion of additional conserved antigens, which may contribute to broader coverage and enhanced efficacy, given their key roles in facilitating intestinal colonization and effective enterotoxin delivery. EatA and EtpA are widely conserved in diverse populations of ETEC, but their immunogenicity has only been studied in controlled human infection models and a population of children in Bangladesh. Here, we compared serologic responses to EatA, EtpA and heat-labile toxin in populations from endemic regions including Haitian children and subjects residing in Egypt, Cameroon, and Peru to US children and adults where ETEC infections are sporadic. We observed elevated IgG and IgA responses in individuals from endemic regions to each of the antigens studied. In a cohort of Haitian children, we observed increased immune responses following exposure to each of the profiled antigens. These findings reflect the wide distribution of ETEC infections across multiple endemic regions and support further evaluation of EatA and EtpA as candidate ETEC vaccine antigens. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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14 pages, 1894 KiB

Open AccessArticle

Heat-Labile Enterotoxin Decreases Macrophage Phagocytosis of Enterotoxigenic Escherichia coli

by Ian E. Hollifield, Natalya I. Motyka, Kaylynn A. Fernando and Jacob P. Bitoun

Microorganisms 2023, 11(8), 2121; https://doi.org/10.3390/microorganisms11082121 - 21 Aug 2023

Cited by 1 | Viewed by 1174

Abstract

Enterotoxigenic E. coli (ETEC) are endemic in low-resource settings and cause robust secretory diarrheal disease in children less than five years of age. ETEC cause secretory diarrhea by producing the heat-stable (ST) and/or heat-labile (LT) enterotoxins. Recent studies have shown that ETEC can [...] Read more.

Enterotoxigenic E. coli (ETEC) are endemic in low-resource settings and cause robust secretory diarrheal disease in children less than five years of age. ETEC cause secretory diarrhea by producing the heat-stable (ST) and/or heat-labile (LT) enterotoxins. Recent studies have shown that ETEC can be carried asymptomatically in children and adults, but how ETEC subvert mucosal immunity to establish intestinal residency remains unclear. Macrophages are innate immune cells that can be exploited by enteric pathogens to evade mucosal immunity, so we interrogated the ability of ETEC and other E. coli pathovars to survive within macrophages. Using gentamicin protection assays, we show that ETEC H10407 is phagocytosed more readily than other ETEC and non-ETEC isolates. Furthermore, we demonstrate that ETEC H10407, at high bacterial burdens, causes nitrite accumulation in macrophages, which is indicative of a proinflammatory macrophage nitric oxide killing response. However, at low bacterial burdens, ETEC H10407 remains viable within macrophages for an extended period without nitrite accumulation. We demonstrate that LT, but not ST, intoxication decreases the number of ETEC phagocytosed by macrophages. Furthermore, we now show that macrophages exposed simultaneously to LPS and LT produce IL-33, which is a cytokine implicated in promoting macrophage alternative activation, iron recycling, and intestinal repair. Lastly, iron restriction using deferoxamine induces IL-33 receptor (IL-33R) expression and allows ETEC to escape macrophages. Altogether, these data demonstrate that LT provides ETEC with the ability to decrease the perceived ETEC burden and suppresses the initiation of inflammation. Furthermore, these data suggest that host IL-33/IL-33R signaling may augment pathways that promote iron restriction to facilitate ETEC escape from macrophages. These data could help explain novel mechanisms of immune subversion that may contribute to asymptomatic ETEC carriage. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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19 pages, 3900 KiB

Open AccessArticle

The Immunogenicity and Properties of a Whole-Cell ETEC Vaccine Inactivated with Psoralen and UVA Light in Comparison to Formalin

by Marlena M. Westcott, Maria Blevins, Thomas F. Wierzba, Alexis E. Morse, Kinnede R. White, Leigh Ann Sanders and John W. Sanders

Microorganisms 2023, 11(8), 2040; https://doi.org/10.3390/microorganisms11082040 - 09 Aug 2023

Cited by 1 | Viewed by 1629

Abstract

Inactivated whole-cell vaccines present a full repertoire of antigens to the immune system. Formalin treatment, a standard method for microbial inactivation, can modify or destroy protein antigenic epitopes. We tested the hypothesis that photochemical inactivation with psoralen and UVA light (PUVA), which targets [...] Read more.

Inactivated whole-cell vaccines present a full repertoire of antigens to the immune system. Formalin treatment, a standard method for microbial inactivation, can modify or destroy protein antigenic epitopes. We tested the hypothesis that photochemical inactivation with psoralen and UVA light (PUVA), which targets nucleic acid, would improve the immunogenicity of an Enterotoxigenic E. coli (ETEC) vaccine relative to a formalin-inactivated counterpart. Exposure of ETEC H10407 to PUVA using the psoralen drug 4′-Aminomethyltrioxsalen hydrochloride (AMT) yielded replication-incompetent bacteria that retained their metabolic activity. CFA/I-mediated mannose-resistant hemagglutination (MRHA) was equivalent for PUVA-inactivated and live ETEC, but was severely reduced for formalin–ETEC, indicating that PUVA preserved fimbrial protein functional integrity. The immunogenicity of PUVA–ETEC and formalin–ETEC was compared in mice ± double mutant heat-labile enterotoxin (dmLT) adjuvant. Two weeks after an intramuscular prime/boost, serum anti-ETEC IgG titers were similar for the two vaccines and were increased by dmLT. However, the IgG responses raised against several conserved ETEC proteins were greater after vaccination with PUVA–ETEC. In addition, PUVA–ETEC generated IgG specific for heat-labile toxin (LT) in the absence of dmLT, which was not a property of formalin–ETEC. These data are consistent with PUVA preserving ETEC protein antigens in their native-like form and justify the further testing of PUVA as a vaccine platform for ETEC using murine challenge models. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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12 pages, 2063 KiB

Open AccessArticle

Reduced Plasma Guanylin Levels Following Enterotoxigenic Escherichia coli-Induced Diarrhea

by Ingeborg Brønstad, Hilde Løland von Volkmann, Sunniva Todnem Sakkestad, Hans Steinsland and Kurt Hanevik

Microorganisms 2023, 11(8), 1997; https://doi.org/10.3390/microorganisms11081997 - 03 Aug 2023

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Abstract

The intestinal peptide hormones guanylin (GN) and uroguanylin (UGN) interact with the epithelial cell receptor guanylate cyclase C to regulate fluid homeostasis. Some enterotoxigenic Escherichia coli (ETEC) produce heat-stable enterotoxin (ST), which induces diarrhea by mimicking GN and UGN. Plasma concentrations of prohormones [...] Read more.

The intestinal peptide hormones guanylin (GN) and uroguanylin (UGN) interact with the epithelial cell receptor guanylate cyclase C to regulate fluid homeostasis. Some enterotoxigenic Escherichia coli (ETEC) produce heat-stable enterotoxin (ST), which induces diarrhea by mimicking GN and UGN. Plasma concentrations of prohormones of GN (proGN) and UGN (proUGN) are reportedly decreased during chronic diarrheal diseases. Here we investigate whether prohormone concentrations also drop during acute diarrhea caused by ST-producing ETEC strains TW10722 and TW11681. Twenty-one volunteers were experimentally infected with ETEC. Blood (n = 21) and urine (n = 9) specimens were obtained immediately before and 1, 2, 3, and 7 days after ETEC ingestion. Concentrations of proGN and proUGN were measured by ELISA. Urine electrolyte concentrations were measured by photometry and mass spectrometry. Ten volunteers developed diarrhea (D group), and eleven did not (ND group). In the D group, plasma proGN, but not proUGN, concentrations were substantially reduced on days 2 and 3, coinciding with one day after diarrhea onset. No changes were seen in the ND group. ETEC diarrhea also seemed to affect diuresis, the zinc/creatinine ratio, and sodium and chloride secretion levels in urine. ETEC-induced diarrhea causes a reduction in plasma proGN and could potentially be a useful marker for intestinal isotonic fluid loss. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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17 pages, 282 KiB

Open AccessPerspective

A Perspective on the Strategy for Advancing ETVAX^®, An Anti-ETEC Diarrheal Disease Vaccine, into a Field Efficacy Trial in Gambian Children: Rationale, Challenges, Lessons Learned, and Future Directions

by M. Jahangir Hossain, Ann-Mari Svennerholm, Nils Carlin, Umberto D’Alessandro and Thomas F. Wierzba

Microorganisms 2024, 12(1), 90; https://doi.org/10.3390/microorganisms12010090 - 31 Dec 2023

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Abstract

For the first time in over 20 years, an Enterotoxigenic Escherichia coli (ETEC) vaccine candidate, ETVAX^®, has advanced into a phase 2b field efficacy trial for children 6–18 months of age in a low-income country. ETVAX^® is an inactivated whole [...] Read more.

For the first time in over 20 years, an Enterotoxigenic Escherichia coli (ETEC) vaccine candidate, ETVAX^®, has advanced into a phase 2b field efficacy trial for children 6–18 months of age in a low-income country. ETVAX^® is an inactivated whole cell vaccine that has gone through a series of clinical trials to provide a rationale for the design elements of the Phase 2b trial. This trial is now underway in The Gambia and will be a precursor to an upcoming pivotal phase 3 trial. To reach this point, numerous findings were brought together to define factors such as safe and immunogenic doses for children, and the possible benefit of a mucosal adjuvant, double mutant labile toxin (dmLT). Considering the promising but still underexplored potential of inactivated whole cells in oral vaccination, we present a perspective compiling key observations from past ETVAX^® trials that informed The Gambian trial design. This report will update the trial’s status and explore future directions for ETEC vaccine trials. Our aim is to provide not only an update on the most advanced ETEC vaccine candidate but also to offer insights beneficial for the development of other much-needed oral whole-cell vaccines against enteric and other pathogens. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

17 pages, 1213 KiB

Open AccessProject Report

A First in Human Clinical Trial Assessing the Safety and Immunogenicity of Two Intradermally Delivered Enterotoxigenic Escherichia coli CFA/I Fimbrial Tip Adhesin Antigens with and without Heat-Labile Enterotoxin with Mutation LT(R192G)

by Ramiro L. Gutiérrez, Mark S. Riddle, Chad K. Porter, Milton Maciel, Jr., Steven T. Poole, Renee M. Laird, Michelle Lane, George W. Turiansky, Abel Jarell and Stephen J. Savarino

Microorganisms 2023, 11(11), 2689; https://doi.org/10.3390/microorganisms11112689 - 02 Nov 2023

Cited by 1 | Viewed by 1082

Abstract

Introduction: Enterotoxigenic E. coli (ETEC) is a leading cause of diarrhea in travelers as well as for children living in low- to middle-income countries. ETEC adhere to intestinal epithelium via colonization factors (CFs). CFA/I, a common CF, is composed of a polymeric stalk [...] Read more.

Introduction: Enterotoxigenic E. coli (ETEC) is a leading cause of diarrhea in travelers as well as for children living in low- to middle-income countries. ETEC adhere to intestinal epithelium via colonization factors (CFs). CFA/I, a common CF, is composed of a polymeric stalk and a tip-localized minor adhesive subunit, CfaE. Vaccine delivery by the transcutaneous immunization of dscCfaE was safe but was poorly immunogenic in a phase 1 trial when administered to volunteers with LTR(192G) and mLT. To potentially enhance the immunogenicity of CfaE while still delivering via a cutaneous route, we evaluated the safety and immunogenicity of two CfaE constructs administered intradermally (ID) with or without mLT. Methods: CfaE was evaluated as a donor strand-complemented construct (dscCfaE) and as a chimeric construct (Chimera) in which dscCfaE replaces the A1 domain of the cholera toxin A subunit and assembles non-covalently with the pentamer of heat-labile toxin B (LTB). Subjects received three ID vaccinations three weeks apart with either dscCfaE (1, 5, and 25 µg) or Chimera (2.6 and 12.9 µg) with and without 0.1 µg of mLT. Subjects were monitored for local and systemic adverse events. Immunogenicity was evaluated by serum and antibody-secreting cell (ASC) responses. Results. The vaccine was well-tolerated with predominantly mild and moderate local vaccine site reactions characterized by erythema, induration and post-inflammatory hyperpigmentation. High rates of serologic and ASC responses were seen across study groups with the most robust responses observed in subjects receiving 25 µg of dscCfaE with 0.1 mcg of LT(R192G). Conclusion: Both ETEC adhesin vaccine prototypes were safe and immunogenic when co-administered with mLT by the ID route. The observed immune responses induced with the high dose of dscCfaE and mLT warrant further assessment in a controlled human infection model. Full article

(This article belongs to the Special Issue Enterotoxigenic Escherichia coli Infection and Vaccine-Mediated Immunity)

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