The Protective Immune Response to Attenuated Enterotoxigenic Escherichia Coli Infection
The Protective Immune Response to Attenuated Enterotoxigenic Escherichia Coli Infection
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.
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.
Inclusion Criteria:

 In order to be eligible to participate in this study, a subject must meet all of the
 following criteria:

 1. Male

 2. Age between 18 and 55 years.

 3. BMI ≥18.5 and ≤30.0 kg/m2.

 4. Healthy as assessed by the NIZO health questionnaire.

 5. Ability to follow Dutch verbal and written instructions.

 6. Availability of internet connection.

 7. Signed informed consent.

 8. Willing to accept disclosure of the financial benefit of participation in the study to
 the authorities concerned.

 9. Willing to accept use of all encoded data, including publication, and the confidential
 use and storage of all data for at least 15 years.

 10. Willing to comply with study procedures, including collection of stool and blood
 samples.

 11. Willingness to abstain from high calcium containing products.

 12. Willingness to abstain from alcoholic beverages three days before, during and for 4
 days after diarrheagenic E. coli challenge.

 13. 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.

 14. Willingness to abstain from probiotics and prebiotic/fibers starting from runin and
 during the whole study.

 15. 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:

 1. Acute gastroenteritis in the 2 months prior to inclusion

 2. Any confirmed or suspected immunosuppressive or immunodeficient condition including
 human immunodeficiency virus infection (HIV).

 3. Disease of the GI tract, liver, gall bladder, kidney, thyroid gland (self-reported),
 except for appendicitis.

 4. History of microbiologically confirmed ETEC or cholera infection within 3 years prior
 to inclusion.

 5. 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.

 6. Vaccination for, or ingestion of cholera within 3 years prior to inclusion, including
 studies at NIZO.

 7. Occupation involving handling of ETEC or Vibrio cholerae currently, or within 3 years
 prior to inclusion.

 8. Vaccination for, or ingestion of ETEC or E coli heat labile toxin, including E. coli
 challenge studies at NIZO.

 9. 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.

 10. Known allergy to the following antibiotics: ciprofloxacin, trimethoprim,
 sulfamethoxazole, and penicillins.

 11. Reported average stool frequency of >3 per day or <1 per 2 days.

 12. 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).

 13. Use of immunosuppressive drugs (e.g. cyclosporine, azathioprine, systemic
 corticosteroids, antibodies).

 14. Vegans.

 15. Mental status that is incompatible with the proper conduct of the study.

 16. 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.

 17. 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.

 18. Personnel that is part of the study team at NIZO, their partner and their first and
 second degree relatives.