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.

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.

The 2022 Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference: Summary of abstract-based presentations.

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.

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.

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.

Induction of mucosal and systemic immune responses against the common O78 antigen of an oral inactivated ETEC vaccine in Bangladeshi children and infants.

We tested an oral enterotoxigenic Escherichia coli (ETEC) vaccine, ETVAX, consisting of inactivated E. coli overexpressing the most prevalent ETEC colonization factors (CFs) and a toxoid (LCTBA), in Bangladeshi children for capacity to induce mucosal and plasma immune responses against O78 lipopolysaccharide (LPS) expressed on the vaccine strains. The vaccine was given ± double-mutant heat-labile toxin (dmLT) adjuvant. We evaluated the impact of dmLT on anti-O78 LPS immune responses and whether such responses can predict responses against the CFs as a marker for vaccine "take". Two fractionated doses of ETVAX ± different amounts of dmLT were administered biweekly to groups of children 24-59 (n = 125), 12-23 (n = 97) and 6-11 (n = 158) months of age. Immune responses were evaluated in antibody in lymphocyte supernatants (ALS), fecal extracts and plasma. ALS IgA responses against O78 LPS were induced in 44-49% of the children aged 12-59 months. The magnitudes of the ALS responses were significantly higher in children receiving a half-dose (5 × 1010 bacteria) of ETVAX ± dmLT than in placebo recipients. <10% of the vaccinees aged 6-11 months mounted ALS responses against O78 LPS. However, 49% of the infants developed fecal secretory IgA responses which were significantly more frequent in those receiving a quarter-dose (2.5 × 1010 bacteria) of vaccine + dmLT (62%) compared to a quarter-dose alone (36%). Plasma IgA antibody responses were induced in 80% of older children and 36% of infants. The frequencies of O78 LPS responses in plasma and feces were comparable or higher than against the vaccine CFs in infants. Our findings show that ETVAX induced mucosal and systemic immune responses against O78 LPS in all age groups and that dmLT improved intestinal immune responses among infants. These observations may have implications for more successful use of other oral vaccines based on O antigens in children.

A site assessment tool for inpatient controlled human infection models for enteric disease pathogens.

The use of the controlled human infection model to facilitate product development and to advance understanding of host-pathogen interactions is of increasing interest. While administering a virulent (or infective) organism to a susceptible host necessitates an ongoing evaluation of safety and ethical considerations, a central theme in conducting these studies in a safe and ethical manner that yields actionable data is their conduct in facilities well-suited to address their unique attributes. To that end, we have developed a framework for evaluating potential sites in which to conduct inpatient enteric controlled human infection model to ensure consistency and increase the likelihood of success.

A disease severity scale for the evaluation of vaccine and other preventive or therapeutic interventions for travellers' diarrhoea.

BACKGROUND: Travellers' diarrhoea (TD) is the most common travel-related illness with an estimated 10 million people afflicted annually. Outcome measures to assess the efficacy of primary and secondary TD interventions were historically based on diarrhoea frequency with ≥1 associated gastrointestinal symptom. Furthermore, efficacy determination is often made on the presence or absence of TD, rather than on TD illness severity. Current severity classifications are based on subjective consideration of impact of illness on activity. We sought to develop a standardized scoring system to characterize TD severity to potentially apply as a secondary outcome in future field studies. METHODS: Data on multiple signs and symptoms were obtained from a previously published multisite TD treatment trial conducted by the US Department of Defense (TrEAT TD). Correlation, regression and multiple correspondence analyses were performed to assess impact on activity and a TD severity score was established. RESULTS: Numerous signs and symptoms were associated with impaired function, with malaise and nausea most strongly associated [odds ratio (OR) 5.9-44.3, P < 0.0001 and OR 2.8-37.1, P < 0.0001, respectively). Based on co-varying symptomatology, a TD severity score accounting for diarrhoea frequency in addition to several signs and symptoms was a better predictor of negative impact on function than any single sign/symptom (X2 = 127.16, P < 0.001). Additionally, there was a significant difference (P < 0.0001) in the mean TD severity score between those with acute watery diarrhoea (3.9 ± 1.9) and those with dysentery or acute febrile illness (6.2 ± 2.0). CONCLUSIONS: The newly developed disease severity score better predicted a negative impact on activity due to TD than did any single sign or symptom. Incorporating multiple parameters into the TD severity score better captures illness severity and moves the field towards current recommendations for TD management by considering symptoms with high functional impact. Further validation of this score is needed in non-military travellers and other settings.

Functional Antibodies and Innate Immune Responses to WRSS1, a Live Oral Shigella sonnei Vaccine Candidate, in Bangladeshi Adults and Children.

BACKGROUND: We demonstrated in a randomized placebo-controlled trial that WRSS1, a live oral Shigella sonnei vaccine candidate, is safe in Bangladeshi adults and children, and elicits antigen-specific antibodies. Here, we describe functional antibody and innate immune responses to WRSS1. METHODS: Adults (18-39 years) and children (5-9 years) received 3 doses of 3 × 105 or 3 × 106 colony forming units (CFU) of WRSS1 or placebo, 4 weeks apart; children additionally received 3 × 104 CFU. Blood and stool were collected at baseline and 7 days after each dose. Functional antibodies were measured using serum bactericidal antibody (SBA) assay. Cytokine/chemokine concentrations were measured in lymphocyte cultures. Host defense peptides LL-37, HBD-1, and HD-5 were analyzed in plasma and stool. RESULTS: Children showed increased SBA titers over baseline after the third dose of 3 × 106 CFU (P = .048). Significant increases of Th-17 and proinflammatory cytokines (TNF-α, G-CSF, MIP-1ß), and reduction of anti-inflammatory and Th2 cytokines (IL-10, IL-13, GM-CSF) were observed in children. Plasma HBD-1 and LL-37 decreased in children after vaccination but were increased/unchanged in adults. CONCLUSIONS: Functional antibodies and Th1/Th17 cytokine responses in children may serve as important indicators of immunogenicity and protective potential of WRSS1. Clinical Trials Registration: NCT01813071.

Safety and immunogenicity of intramuscularly administered CS6 subunit vaccine with a modified heat-labile enterotoxin from enterotoxigenic Escherichia coli.

INTRODUCTION: Enterotoxigenic Escherichia coli (ETEC) is a common cause of infectious diarrhoea and a leading cause of morbidity and mortality in children living in resource-limited settings. It is also the leading cause of travellers' diarrhoea among civilian and military travellers. Its dual importance in global public health and travel medicine highlights the need for an effective vaccine. ETEC express colonization factors (CFs) that mediate adherence to the small intestine. An epidemiologically prevalent CF is coli surface antigen 6 (CS6). We assessed the safety and immunogenicity of a CS6-targeted candidate vaccine, CssBA, co-administered intramuscularly with the double-mutant heat-labile enterotoxin, dmLT [LT(R192G/L211A)]. METHODS: This was an open-label trial. Fifty subjects received three intramuscular injections (Days 1, 22 and 43) of CssBA alone (5 µg), dmLT alone (0.1 µg) or CssBA (5, 15, 45 µg) + dmLT (0.1 and 0.5 µg). Subjects were actively monitored for adverse events for 28 days following the third vaccination. Antibody responses (IgG and IgA) were characterized in the serum and from lymphocyte supernatants (ALS) to CS6 and the native ETEC heat labile enterotoxin, LT. RESULTS: Across all dose cohorts, the vaccine was safe and well-tolerated with no vaccine-related severe or serious adverse events. Among vaccine-related adverse events, a majority (98%) were mild with 79% being short-lived vaccine site reactions. Robust antibody responses were induced in a dose-dependent manner with a clear dmLT adjuvant effect. Response rates in subjects receiving 45 µg CssBA and 0.5 µg dmLT ranged from 50 to 100% across assays. CONCLUSION: This is the first study to demonstrate the safety and immunogenicity of CssBA and/or dmLT administered intramuscularly. Co-administration of the two components induced robust immune responses to CS6 and LT, paving the way for future studies to evaluate the efficacy of this vaccine target and development of a multivalent, subunit ETEC vaccine.

Establishment of a Controlled Human Infection Model with a Lyophilized Strain of Shigella sonnei 53G.

Controlled human infection models (CHIMs) are useful for vaccine development. To improve on existing models, we developed a CHIM using a lyophilized preparation of Shigella sonnei strain 53G produced using current good manufacturing practice (cGMP). Healthy adults were enrolled in an open-label dose-ranging study. Following administration of a dose of rehydrated S. sonnei strain 53G, subjects were monitored for development of disease. The first cohort received 500 CFU of 53G, and dosing of subsequent cohorts was based on results from the previous cohort. Subjects were administered ciprofloxacin on day 5 and discharged home on day 8. Subjects returned as outpatients for clinical checks and sample collection. Attack rates increased as the dose of S. sonnei was increased. Among those receiving the highest dose (1,760 CFU), 70% developed moderate to severe diarrhea, 50% had dysentery, and 40% had fever. Antilipopolysaccharide responses were observed across all cohorts. An S. sonnei CHIM using a lyophilized lot of strain 53G was established. A dose in the range of 1,500 to 2,000 CFU of 53G was selected as the dose for future challenge studies using this product. This model will enable direct comparison of study results between institutions and ensure better consistency over time in the challenge inoculum.IMPORTANCE Controlled human infection models (CHIMs) are invaluable tools utilized to understand the human response to infection, potentially leading to protective immune mechanisms and allowing efficacy testing of enteric countermeasures, including vaccines, antibiotics, and other products. The development of an improved Shigella CHIM for both Shigella sonnei and Shigella flexneri is consistent with international efforts, supported by international donors and the World Health Organization, focused on standardizing Shigella CHIMs and using them to accelerate Shigella vaccine development. The use of lyophilized Shigella challenge strains rather than plate-grown inoculum preparations is considered an important step forward in the standardization process. Furthermore, the results of studies such as this justify the development of lyophilized preparations for additional epidemiologically important S. flexneri serotypes, including S. flexneri 3a and S. flexneri 6.

Safety and immunogenicity of the oral, inactivated, enterotoxigenic Escherichia coli vaccine ETVAX in Bangladeshi children and infants: a double-blind, randomised, placebo-controlled phase 1/2 trial.

BACKGROUND: Enterotoxigenic Escherichia coli causes diarrhoea, leading to substantial mortality and morbidity in children, but no specific vaccine exists. This trial tested an oral, inactivated, enterotoxigenic E coli vaccine (ETVAX), which has been previously shown to be safe and highly immuongenic in Swedish and Bangladeshi adults. We tested the safety and immunogenicity of ETVAX, consisting of four E coli strains overexpressing the most prevalent colonisation factors (CFA/I, CS3, CS5, and CS6) and a toxoid (LCTBA) administered with or without a double-mutant heat-labile enterotoxin (dmLT) as an adjuvant, in Bangladeshi children. METHODS: We did a randomised, double-blind, placebo-controlled, dose-escalation, age-descending, phase 1/2 trial in Dhaka, Bangladesh. Healthy children in one of three age groups (24-59 months, 12-23 months, and 6-11 months) were eligible. Children were randomly assigned with block randomisation to receive either ETVAX, with or without dmLT, or placebo. ETVAX (half [5·5 × 1010 cells], quarter [2·5 × 1010 cells], or eighth [1·25 × 1010 cells] adult dose), with or without dmLT adjuvant (2·5 µg, 5·0 µg, or 10·0 µg), or placebo were administered orally in two doses 2 weeks apart. Investigators and participants were masked to treatment allocation. The primary endpoint was safety and tolerability, assessed in all children who received at least one dose of vaccine. Antibody responses to vaccine antigens, defined as at least a two-times increase in antibody levels between baseline and post-immunisation, were assessed as secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02531802. FINDINGS: Between Dec 7, 2015, and Jan 10, 2017, we screened 1500 children across the three age groups, of whom 430 were enrolled and randomly assigned to the different treatment groups (130 aged 24-59 months, 100 aged 12-23 months, and 200 aged 6-11 months). All participants received at least one dose of vaccine. No solicited adverse events occurred that were greater than moderate in severity, and most were mild. The most common solicited event was vomiting (ten [8%] of 130 patients aged 24-59 months, 13 [13%] of 100 aged 12-23 months, and 29 [15%] of 200 aged 6-11 months; mostly of mild severity), which appeared related to dose and age. The addition of dmLT did not modify the safety profile. Three serious adverse events occurred but they were not considered related to the study drug. Mucosal IgA antibody responses in lymphocyte secretions were detected against all primary vaccine antigens (CFA/I, CS3, CS5, CS6, and the LCTBA toxoid) in most participants in the two older age groups, whereas such responses to four of the five antigens were less frequent and of lower magnitude in infants aged 6-11 months than in older children. Faecal secretory IgA immune responses were recorded against all vaccine antigens in infants aged 6-11 months. 78 (56%) of 139 infants aged 6-11 months who were vaccinated developed mucosal responses against at least three of the vaccine antigens versus 14 (29%) of 49 of the infants given placebo. Addition of the adjuvant dmLT enhanced the magnitude, breadth, and kinetics (based on number of responders after the first dose of vaccine) of immune responses in infants. INTERPRETATION: The encouraging safety and immunogenicity of ETVAX and benefit of dmLT adjuvant in young children support its further assessment for protective efficacy in children in enterotoxigenic E coli-endemic areas. FUNDING: PATH (Bill & Melinda Gates Foundation and the UK's Department for International Development), the Swedish Research Council, and The Swedish Foundation for Strategic Research.

A phase I trial of WRSS1, a Shigella sonnei live oral vaccine in Bangladeshi adults and children.

Shigella sonnei live vaccine candidate, WRSS1, which was previously evaluated in US, Israeli and Thai volunteers, was administered orally to Bangladeshi adults and children to assess its safety, clinical tolerability and immunogenicity. In a randomized, placebo-controlled, dose-escalation, age-descending study, 39 adults (18-39 years) and 64 children (5-9 years) were enrolled. Each adult cohort (n = 13) received one dose of 3x104, or three doses of 3 × 105 or 3 × 106 colony forming unit (CFU) of WRSS1 (n = 10) or placebo (n = 3). Each child cohort (n = 16) received one dose of 3x103, or three doses of 3x104, 3x105, or 3 × 106 CFU WRSS1 (n = 12) or placebo (n = 4). WRSS1 elicited mostly mild and transient reactogenicity events in adults and children. In the 3 × 106 dose group, 50% of the adults shed the vaccine; no shedding was seen in children. At the highest dose, 100% of adults and 40% of children responded with a ≥ 4-fold increase of S. sonnei LPS-specific IgA antibody in lymphocyte supernatant (ALS). At the same dose, 63% of adults and 70% of children seroconverted with IgA to LPS, while in placebo, 33% of adults and 18% of children seroconverted. Both the vaccinees and placebos responded with fecal IgA to LPS, indicating persistent exposure to Shigella infections. In conclusion, WRSS1 was found safe up to 106 CFU dose and immunogenic in adults and children in Bangladesh. These data indicate that live, oral Shigella vaccine candidates, including WRSS1 can potentially be evaluated in toddlers and infants (<2 years of age), who comprise the target population in an endemic environment.

Live attenuated enterotoxigenic Escherichia coli (ETEC) vaccine with dmLT adjuvant protects human volunteers against virulent experimental ETEC challenge.

BACKGROUND: There is no licensed vaccine against enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhea-associated morbidity and mortality among infants and children in low-income countries and travelers. The results of this vaccination/challenge study demonstrate strong protection by an attenuated ETEC vaccine candidate, ACE527, when co-administered with a mucosal adjuvant, the double-mutant heat-labile toxin (dmLT) of ETEC. METHODS: Sixty healthy adults participated in a randomized, placebo-controlled, double-blind study with three doses of lyophilized ACE527 (∼3 × 109 of each strain per dose) administered orally with or without dmLT adjuvant (25 µg/dose). Six months later, 36 of these volunteers and a control group of 21 unvaccinated volunteers were challenged with virulent ETEC strain H10407. The primary outcome was severe diarrhea, defined as passing >800 g of unformed stools during the inpatient period following challenge. FINDINGS: The vaccine was well tolerated and induced robust immune responses to key antigens. The protective efficacy (PE) against the primary outcome of severe diarrhea was 65.9% (95% confidence interval [CI] 5.4-87.7, p = 0.003). Among subjects receiving the adjuvanted vaccine, the attack rate of severe diarrhea was 23.1, while in unimmunized controls it was 67.7%. The PE against diarrhea of any severity was 58.5% (95% CI 3.8- 82.1, p = 0.016). There was a strong inverse correlation between shedding of the vaccine strain after either of the first two doses and absence of severe diarrhea upon challenge (RR = 0.29, 95% CI 0.08-1.05, p = 0.041). Challenge strain shedding was 10-fold lower in those receiving the adjuvant than in those receiving vaccine alone. The unadjuvanted vaccine was not protective (PE = 23.1%). INTERPRETATION: The results of this study support further development of ACE527 + dmLT as a vaccine for children in endemic countries and travelers. This is the first clinical demonstration that dmLT can contribute significantly to vaccine efficacy and may warrant testing with other oral vaccines. (ClinicalTrials.gov registration: NCT01739231).

Evaluation of the safety and immunogenicity of the oral inactivated multivalent enterotoxigenic Escherichia coli vaccine ETVAX in Bangladeshi adults in a double-blind, randomized, placebo-controlled Phase I trial using electrochemiluminescence and ELISA assays for immunogenicity analyses.

The safety and immunogenicity of the second generation oral enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, consisting of inactivated recombinant E. coli strains over-expressing the colonization factors (CFs) CFA/I, CS3, CS5 and CS6 and the heat labile toxoid LCTBA, were evaluated in Bangladeshi volunteers. To enable analysis of antibody responses against multiple vaccine antigens for subsequent use in small sample volumes from children, a sensitive electrochemiluminescence (ECL) assay for analysis of intestine-derived antibody-secreting cell responses using the antibodies in lymphocyte secretions (ALS) assay was established using Meso Scale Discovery technology. Three groups of Bangladeshi adults (n = 15 per group) received two oral doses of ETVAX with or without double mutant LT (dmLT) adjuvant or placebo in the initial part of a randomized, double-blind, placebo-controlled, age-descending, dose-escalation trial. CF- and LTB-specific ALS and plasma IgA responses were analyzed by ECL and/or ELISA. ETVAX was safe and well tolerated in the adults. Magnitudes of IgA ALS responses determined by ECL and ELISA correlated well (r = 0.85 to 0.98 for the five primary antigens, P < 0.001) and ECL was selected as the ALS readout method. ALS IgA responses against each of the primary antigens were detected in 87-100% of vaccinees after the first and in 100% after the second vaccine dose. Plasma IgA responses against different CFs and LTB were observed in 62-93% and 100% of vaccinees, respectively. No statistically significant adjuvant effect of dmLT on antibody responses to any antigen was detected, but the overall antigenic breadth of the plasma IgA response tended to favor the adjuvanted vaccine when responses to 4 or more or 5 vaccine antigens were considered. Responses in placebo recipients were infrequent and mainly detected against single antigens. The promising results in adults supported testing ETVAX in descending age groups of children. ClinicalTrials.gov Identifier: NCT02531802.

A Phase 1 dose escalating study of double mutant heat-labile toxin LTR192G/L211A (dmLT) from Enterotoxigenic Escherichia coli (ETEC) by sublingual or oral immunization.

BACKGROUND: The public health burden of Enterotoxigenic Escherichia coli (ETEC) is high but no vaccine is specifically approved to prevent ETEC infections. METHODS: We performed a Phase 1, dose escalation study (1-50 µg) evaluating the sublingual (SL) delivery of the double mutant heat-labile toxin LTR192G/L211A (dmLT) in 80 healthy adult volunteers. The primary objective was safety and the secondary was the immunogenicity of the dmLT. Subjects received 3 doses of dmLT at days 1, 15, and 29. Subjects receiving the first dose at each dosage level were observed overnight in a research facility. The second and third doses were administered on an outpatient basis. Data from cohorts 1-4 were used to select the cohort 5 dose (25 µg), comparing SL and oral routes. RESULTS: The vaccine appeared safe and well tolerated with only rare development of vomiting or diarrhea. The serum anti-dmLT IgA and IgG and neutralizing antibody responses were modest after any of the SL immunizations. Serum IgA and IgG titers were increased at the higher antigen doses (25 or 50 µg) but the percent with 4-fold increases was at best 38% for both IgA and IgG. The 4-fold increase among subjects receiving all 3 doses was 43% for both IgA and IgG. Antibody titers following oral administration were, in general, significantly higher than after SL. The frequency of IgA- or IgG-ASCs in circulation were somewhat vaccine dose dependent and were detected at a moderate level. However, antibodies in saliva or stool were rarely detected. Post-vaccination increases in T cells or cytokine production were also infrequent. CONCLUSION: The dmLT vaccine formulation evaluated here was safe but only moderately immunogenic at doses up to 50 µg when administered by the SL or oral route. Studies at higher doses with better formulations appear warranted.

Impact of lower challenge doses of enterotoxigenic Escherichia coli on clinical outcome, intestinal colonization and immune responses in adult volunteers.

A reliable and effective human challenge model is needed to help down-select the most promising ETEC vaccines currently under development. Such a model would need to reliably induce diarrhea in a high proportion of volunteers using the lowest possible inoculum to maximize safety and sensitivity. Previously we validated a challenge model that utilized a dose of 2x107 CFU of ETEC strain H10407 (LT+, ST+, CFA/I+ and O78+) to induce attack rates for moderate to severe diarrhea (MSD) of ~60-70%. Here we detail efforts to further refine the model in an attempt to determine if a lower challenge dose of H10407 can be used. Thirty subjects were randomized 1:1 to receive an oral administration of H10407 at doses of 106 or 105 CFU in bicarbonate buffer. After challenge, subjects were monitored for signs and symptoms of enteric illness and stool samples were collected to detect shedding of the challenge strain. Systemic and mucosal immune responses were measured using serum, antibody in lymphocyte supernatant and fecal samples. The attack rate was 13.3% (2/15) and 26.7% (4/15) for MSD in the 105 and 106 groups, respectively. Four MSD cases met criteria for early antibiotic treatment. All subjects but one shed the challenge strain in fecal samples. The frequency and magnitude of anti-LT toxin, CFA/I and LPS O78 immune responses were antigen, dose, severity of diarrhea and shedding levels dependent. Notably, although of lower magnitude, there were considerable immune responses in the subjects with no diarrhea. This may indicate that immune responses to asymptomatic infections of ETEC in children in the endemic countries may contribute to protection. Based on this and our prior studies, we conclude that a dose of 2x107 H10407 remains the lowest practical dose for use in future volunteer studies evaluating candidate vaccines and other preventive or therapeutic ETEC interventions. TRIAL REGISTRATION: ClinicalTrials.gov NCT00844493.

Developing and utilizing controlled human models of infection.

The controlled human infection model (CHIM) to assess the efficacy of vaccines against Shigella and enterotoxigenic Escherichia coli (ETEC) has several unique features that could significantly enhance the ability to test candidate vaccines. Despite increasing interest in these models, questions remain as to how to best incorporate them into vaccine development and how to maximize results. We designed a workshop focused on CHIM as part of the Vaccines Against Shigella and ETEC (VASE) Conference. The workshop, using the World Café method, focused on; clinical outcomes, nonclinical outcomes and model standardization. Researchers with a variety of expertise and experience rotated through each focus area and discussed relevant sub-topics. The results of these discussions were presented and questions posed to guide future workshops. Clinical endpoint discussions focused on the need for harmonized definitions; optimized attack rates; difficulties of sample collection and a need for non-stool based endpoints. Nonclinical discussions centered on evolving omics-based opportunities, host predictors of susceptibility and novel characterizations of the immune response. Model standardization focused on the value of shared procedures across institutions for clinical and non-clinical endpoints as well as for strain preparation and administration and subject selection. Participants agreed CHIMs for Shigella and ETEC vaccine development could accelerate vaccine development of a promising candidate; however, it was also appreciated that variability in the model and our limited understand of the host-pathogen interaction may yield results that could negatively impact a suitable candidate. Future workshops on CHIM are needed to ensure the optimal application of these models moving forward.