The Protective Immune Response to Attenuated Enterotoxigenic Escherichia Coli Infection

Study Overview
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Brief Summary
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The existing diarrhoeagenic Escherichia coli (E. coli) challenge model is already suitable for dietary interventions in its current form, targeted to impact on the immediate clinical symptoms upon E. coli infection. In order to make the model also suitable for dietary interventions that are aimed at support of the protective response against reinfection, the immune response triggered by the primary infection should be suboptimal. The MIRRE pilot study is set up to determine how much the primary inoculation dose of diarrheagenic E. coli should be lowered in order to result in a reduced protective response upon a secondary infection.

Detailed Description
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The MIRRE pilot study is a randomized, double-blind dose-response, parallel 7 weeks human infection study. Healthy male subjects, 18-55 years of age who fulfil all of the inclusion criteria and none of the exclusion criteria, will be randomly assigned to one of five inoculation dosages of a live attenuated diarrheagenic E. coli strain (n=6 per group). Subjects will be instructed to maintain their usual pattern of physical activity and their habitual food intake, but to reduce and standardize their dietary calcium intake. After a standardized evening meal and an overnight fast, subjects will be orally infected on day 14 and 35 with a live, but attenuated, diarrheagenic E. coli (strain E1392/75-2A; collection NIZO food research). Five groups of 6 subjects will be provided one of the following dosages at day 14: 1E10 CFU (standard dose); 1E9 CFU; 1E8 CFU; 1E7 CFU or 1E6 CFU of the E.coli strain. At study day 35, all subjects will receive a second inoculation of 1E10 CFU of the E.coli strain. At various time points before and after diarrheagenic E. coli challenges an online diary will be kept to record information on stool consistency, frequency and severity of symptoms, and blood and stool samples are collected to quantify antibody levels and to quantify fecal infection parameters. The hypothesis of this pilot study is that the E. coli infection dose at primary inoculation determines the outcomes of the protective immune response and the extent of clinical symptoms at a secondary inoculation.Therefore the CFA/II-specific IgG antibody response, in relation to clinical symptoms, is the main outcome.

Official Title
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The Protective Immune Response to Attenuated Enterotoxigenic Escherichia Coli Infection in Healthy Human Subjects: A Pilot Study

Conditions
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Diarrhea

Intervention / Treatment
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* Biological: E. coli strain E1392-75-2A dose 1E10 CFU
* Biological: E. coli strain E1392-75-2A dose 1E9 CFU
* Biological: E. coli strain E1392-75-2A dose 1E8 CFU
* Biological: E. coli strain E1392-75-2A dose 1E7 CFU
* Biological: E. coli strain E1392-75-2A dose 1E6 CFU


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: In order to be eligible to participate in this study, a subject must meet all of the following criteria: Male Age between 18 and 55 years. BMI ≥18.5 and ≤30.0 kg/m2. Healthy as assessed by the NIZO health questionnaire. Ability to follow Dutch verbal and written instructions. Availability of internet connection. Signed informed consent. Willing to accept disclosure of the financial benefit of participation in the study to the authorities concerned. Willing to accept use of all encoded data, including publication, and the confidential use and storage of all data for at least 15 years. Willing to comply with study procedures, including collection of stool and blood samples. Willingness to abstain from high calcium containing products. Willingness to abstain from alcoholic beverages three days before, during and for 4 days after diarrheagenic E. coli challenge. Willingness to abstain from medications that contain acetaminophen, aspirin, ibuprofen, and other non-steroidal anti-inflammatory drugs, (OTC) antacids and antimotility agents (eg, loperamide) on the three days before, during and for 4 days after diarrheagenic E. coli challenge. Willingness to abstain from probiotics and prebiotic/fibers starting from runin and during the whole study. Willingness to give up blood donation starting at run-in and during the entire study. Exclusion Criteria: A potential subject who meets any of the following criteria will be excluded from participation in this study: Acute gastroenteritis in the 2 months prior to inclusion Any confirmed or suspected immunosuppressive or immunodeficient condition including human immunodeficiency virus infection (HIV). Disease of the GI tract, liver, gall bladder, kidney, thyroid gland (self-reported), except for appendicitis. History of microbiologically confirmed ETEC or cholera infection within 3 years prior to inclusion. Symptoms consistent with Travelers' Diarrhea concurrent with travel to countries where ETEC infection is endemic (most of the developing world) within 3 years prior to inclusion, OR planned travel to endemic countries during the length of the study. Vaccination for, or ingestion of cholera within 3 years prior to inclusion, including studies at NIZO. Occupation involving handling of ETEC or Vibrio cholerae currently, or within 3 years prior to inclusion. Vaccination for, or ingestion of ETEC or E coli heat labile toxin, including E. coli challenge studies at NIZO. Evidence of current excessive alcohol consumption (>4 consumptions/day or >20 consumptions/week) or drug (ab)use, and not willing/able to stop this during the study. Known allergy to the following antibiotics: ciprofloxacin, trimethoprim, sulfamethoxazole, and penicillins. Reported average stool frequency of >3 per day or <1 per 2 days. Use of antibiotics, (during 6 months prior to inclusion), norit, laxatives, cholestyramine, antacids H2 receptor antagonists or proton pump inhibitors (during 3 months prior to inclusion). Use of immunosuppressive drugs (e.g. cyclosporine, azathioprine, systemic corticosteroids, antibodies). Vegans. Mental status that is incompatible with the proper conduct of the study. Not having a general practitioner, not allowing disclosure of participation to the general practitioner or not allowing to inform the general practitioner about abnormal results. Participation in any clinical trial including blood sampling and/or administration of substances starting 1 month prior to study start and during the entire study. Personnel that is part of the study team at NIZO, their partner and their first and second degree relatives.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 55 Years

Sexes Eligible for Study
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Male

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: The MIRRE pilot study is a randomized, double-blind dose-response, parallel human infection study in 30 healthy adults
Masking: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: 1E10 CFU Escherichia coli (E. coli)<br>At day 14, after a standardized evening meal and overnight fast, subjects receive a single oral dose of 1E10 CFU of the attenuated diarrhoeagenic E. coli strain E1392-75-2A. At day 35, after a standardized evening meal and overnight fast, all subjects receive a second inoculation 1E10 CFU of the diarrhoeagenic E. coli. | Biological: E. coli strain E1392-75-2A dose 1E10 CFU<br>* Diarrhoeagenic E. coli strain E1392/75-2A serotype O6:H16 belongs to Pathogen class 2 . The strain has a deletion of genes encoding the heat-labile (LT) and heat-stable (ST) toxins and can not produce any toxins. However, it continues to express Colonization Factor Antigen II (CFA/II) and provides 75% protection against challenge with an LT, ST, CFA/II strain<br>|
| Experimental: 1E9 CFU Escherichia coli (E. coli)<br>At day 14, after a standardized evening meal and overnight fast, subjects receive a single oral dose of 1E9 CFU of the attenuated diarrhoeagenic E. coli strain E1392-75-2A. At day 35, after a standardized evening meal and overnight fast, all subjects receive a second inoculation 1E10 CFU of the diarrhoeagenic E. coli. | Biological: E. coli strain E1392-75-2A dose 1E9 CFU<br>* Diarrhoeagenic E. coli strain E1392/75-2A serotype O6:H16 belongs to Pathogen class 2 . The strain has a deletion of genes encoding the heat-labile (LT) and heat-stable (ST) toxins and can not produce any toxins. However, it continues to express Colonization Factor Antigen II (CFA/II) and provides 75% protection against challenge with an LT, ST, CFA/II strain<br>|
| Experimental: 1E8 CFU Escherichia coli (E. coli)<br>At day 14, after a standardized evening meal and overnight fast, subjects receive a single oral dose of 1E8 CFU of the attenuated diarrhoeagenic E. coli strain E1392-75-2A. At day 35, after a standardized evening meal and overnight fast, all subjects receive a second inoculation 1E10 CFU of the diarrhoeagenic E. coli. | Biological: E. coli strain E1392-75-2A dose 1E8 CFU<br>* Diarrhoeagenic E. coli strain E1392/75-2A serotype O6:H16 belongs to Pathogen class 2 . The strain has a deletion of genes encoding the heat-labile (LT) and heat-stable (ST) toxins and can not produce any toxins. However, it continues to express Colonization Factor Antigen II (CFA/II) and provides 75% protection against challenge with an LT, ST, CFA/II strain<br>|
| Experimental: 1E7 CFU Escherichia coli (E. coli)<br>At day 14, after a standardized evening meal and overnight fast, subjects receive a single oral dose of 1E7 CFU of the attenuated diarrhoeagenic E. coli strain E1392-75-2A. At day 35, after a standardized evening meal and overnight fast, all subjects receive a second inoculation 1E10 CFU of the diarrhoeagenic E. coli. | Biological: E. coli strain E1392-75-2A dose 1E7 CFU<br>* Diarrhoeagenic E. coli strain E1392/75-2A serotype O6:H16 belongs to Pathogen class 2 . The strain has a deletion of genes encoding the heat-labile (LT) and heat-stable (ST) toxins and can not produce any toxins. However, it continues to express Colonizat<br>|
| Experimental: 1E6 CFU Escherichia coli (E. coli)<br>At day 14, after a standardized evening meal and overnight fast, subjects receive a single oral dose of 1E6 CFU of the attenuated diarrhoeagenic E. coli strain E1392-75-2A. At day 35, after a standardized evening meal and overnight fast, all subjects receive a second inoculation 1E10 CFU of the diarrhoeagenic E. coli. | Biological: E. coli strain E1392-75-2A dose 1E6 CFU<br>* Diarrhoeagenic E. coli strain E1392/75-2A serotype O6:H16 belongs to Pathogen class 2 . The strain has a deletion of genes encoding the heat-labile (LT) and heat-stable (ST) toxins and can not produce any toxins. However, it continues to express Colonizat<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in specific antibody titer | Serum IgG-CFA/II antibody levels in relation to clinical symptoms at second E.coli inoculation | Day 14,21,28 and Day 35,42,49 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in percentage of faecal wet weight from baseline | % of faecal wet weight determined by freeze-drying | Day 11,12 and Day 15-17. Day 33,34 and 36-38 | 
| Change in Stool consistency (Bristol stool scale) from baseline | Stool consistency for all stools reported by the subjects | Day 11-17 and Day 32-38 | 
| Change in Stool frequency from baseline | Stools per day reported by the subjects | Day 11-17 and Day 32-38 | 
| Change in incidence, duration and severity of Gastro-intestinal symptoms from baseline | Gastro-intestinal Symptom Rating Scale reported by the subjects in the online diary | Day 11-17 and Day 32-38 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Diarrhea, Gastroenteritis, Bacterial Infections, Escherichia Coli Infections, E.coli