Pre-Study of Wild Type Enterotoxigenic E. Coli (ETEC) Strain for Verification of a Planned Challenge Dose

This was an open-label, single-center pre-study designed to evaluate the safety, tolerability, and infectivity of wild-type Enterotoxigenic Escherichia coli (ETEC) strain E24377A in healthy adults. The goal was to estimate the incidence of moderate and severe diarrhea following challenge with approximately 4 × 10⁹ colony forming units (cfu) of the strain. This challenge model was intended to inform a subsequent controlled human infection model (CHIM) study assessing the efficacy of the oral inactivated tetravalent ETEC vaccine ETVAX® (containing LCTBA and dmLT).

Up to 30 healthy adult subjects were planned to be enrolled; 23 were ultimately challenged at the Center for Immunization Research (CIR), Johns Hopkins Bloomberg School of Public Health in Baltimore, Maryland, USA. Participants underwent screening, then entered an inpatient unit on Day -1. On Day 1, they received a single oral dose of strain E24377A (lot 0807) suspended in 30 mL sodium bicarbonate buffer, preceded by 120 mL of buffer to reduce gastric acidity.

During the inpatient period, subjects were monitored at least three times daily for vital signs, signs and symptoms of ETEC illness, and stool output. All stools were collected, weighed, graded, and cultured. Stools were graded on a 5-point scale: Grade 1 = firm/formed; Grade 2 = soft/formed; Grade 3 = thick liquid; Grade 4 = thin liquid; Grade 5 = clear/watery. Loose stools were defined as Grade 3-5.

Diarrhea was defined as ≥2 loose stools (Grade 3-5) within a 24-hour period. Severity was categorized as:

Mild: 2-3 Grade 3-5 stools and ≤400 g in 24 h

Moderate: 4-5 Grade 3-5 stools or >400-800 g

Severe: ≥6 Grade 3-5 stools or >800 g

An independent adjudication committee determined whether episodes were attributable to ETEC and whether participants met the primary endpoint. Participants received ciprofloxacin 500 mg twice daily for 3 days starting on Day 6, or earlier if needed. Discharge occurred after meeting microbiological and clinical criteria, with follow-up visits on Day 29 (clinic) and Day 180 (phone).

Secondary and exploratory endpoints included stool frequency and weight, symptom scores, microbiological shedding, and immunologic assessments (serum, fecal, and ALS IgA/IgG, memory B cells, transcriptomics, and biomarkers of intestinal/systemic inflammation). Safety endpoints included adverse events through Day 29 and serious adverse events through Day 180.

Inclusion criteria

• Healthy adults between 18 and 50 years of age, inclusive, at the time of signing the informed consent.
• General good health, without clinically significant medical history, physical examination findings or clinical laboratory abnormalities per clinical judgment of PI.
• Negative pregnancy test at screening and prior to challenge for people of childbearing potential. People of childbearing potential must agree to use an efficacious hormonal or barrier method of birth control during the study. Abstinence is acceptable. People of childbearing potential unable to bear children must have this documented (e.g. tubal ligation or hysterectomy) or must have negative pregnancy tests at screening and prior to challenge.
• Willingness to participate in the study after all aspects of the protocol have been explained and written informed consent obtained.
• Completion of a training session and demonstrated comprehension of the protocol procedures and knowledge of ETEC associated illness by passing a written examination (70% pass score).
• Availability for the study duration, including planned follow-up visit/contact.

Exclusion criteria

• Presence of a significant medical or psychiatric condition which in the opinion of the investigator precludes participation in the study, including gastrointestinal disease (gastritis, irritable bowel disease as suggested by Rome III criteria or medical diagnosis, inflammatory bowel disease). Some medical conditions which are adequately treated and stable would not preclude entry into the study. These conditions might include stable asthma controlled with inhalers or mild hypertension stably controlled.
• Significant abnormalities in screening haematology, or serum chemistry as determined by PI.
• Presence in the serum of HIV antibody, HBsAg, or HCV antibody with confirmation of infection (e.g. by HCV PCR).
• Evidence of IgA deficiency (serum IgA < 7 mg/dl or limit of detection of assay).
• Evidence of current alcohol or drug dependence.
• Subjects whose Body Mass Index (BMI) is less than 19.0 or greater than 37.0 (kg/m2).
• Recent vaccination or receipt of an investigational product (within 30 days before challenge) or intended vaccination or receipt of investigational products until 60 days after challenge, with the exception of licensed vaccine for influenza or SARS-CoV-2 vaccination that may be given up to 7 days prior to challenge.
• Positive test for SARS-CoV-2 at arrival to the unit on the day of in-patient admission.
• Intention to donate blood or blood products within one month following the completion of study participation (note: The Red Cross will not allow blood donations for 1 year following participation in an investigational research study).
• Any other criteria which, in the investigator's opinion, would compromise the ability of the subject to participate in the study, the safety of the study, or the results of the study.
• Abnormal stool pattern (fewer than 3 per week or more than 3 per day).
• Regular (≥ weekly) use of laxatives, antacids, or other agents to lower stomach acidity.
• Use of any medication known to affect the immune function (e.g., corticosteroids and others) within 30 days preceding the challenge or planned use during the active study period. Use of inhaled or topical steroids may be permitted per PI discretion.
• History of microbiologically confirmed ETEC infection in the last 3 years.
• Occupational handling of ETEC currently, or in the past 3 years.
• Travel to countries where ETEC infection is endemic (most of the developing world) within two years prior to dosing OR visit for > two months in ETEC endemic countries during the last 10 years, OR planned travel to endemic countries prior to study day 180.
• Vaccination for or ingestion of ETEC, cholera, or LT toxin within 5 years prior to dosing.
• Use of antibiotics during the 14 days before challenge dosing or proton pump inhibitors, H2 blockers or antacids within 48 hours prior to challenge dosing.
• History of diarrhea in the 7 days prior to challenge (outpatient diarrhea is defined as ≥ 3 unformed (grade 3 or greater) loose stools in 24 hours).
• Known allergy to two of the three following antibiotics: ciprofloxacin, amoxicillin, and/or azithromycin.