Aimmune Longitudinal Collaboration Study

Participants will be either selected from a pool of previously screened and identified candidates or who have communicated interest in participating in research through GoodNature Program, run by Aimmune. No participants who have made or have potential to make a lot of a finished product will be allowed to participate. Those who agree to participate will complete a health pre-screening survey over electronic form or phone call and complete the informed consent form (ICF). After completion of the ICF, participants will be assigned a unique, anonymous participant ID. Only a select few members of the Study Sponsor will have access to the identification codes; deidentification codes will not be shared with collaborators. Following this, participants will be given general information regarding diet practices, fiber benefits, and details regarding the study, including sample collection and labeling of samples, diet tracking via surveys, photos of meal submissions, etc.

Participants will meet at a study site and will be provided with chia seeds, the manual stool sampling tool, and pre-labeled specimen containers if they are collecting their samples at home. At this time, participants may also provide blood specimens.

Once the pre-intervention materials are completed, participants (60) will begin a 16-week intervention period; half of participants will add daily 2.5 tbsp chia seeds for the first 8-weeks of the study and return to their normal diet for the second 8-weeks, half of participants will maintain their normal diet for the first 8-weeks and add daily 2.5 tbsp chia seeds for the second 8-weeks as a crossover design. Multiple people in the household can participate, but because we want to understand transmissibility of SCFU and/or IgA levels.

Participants will be randomized into two groups, but members of the same household will be assigned into opposite groups.

All participants will be asked to complete a short, electronic daily stress survey, including providing daily photos of meals. The daily survey will include the Patient Health Questionnaire (PHQ-4), a brief validated screening questionnaire consisting of a 2-item depression scale (PHQ-2) and a 2-item anxiety scale (GAD-2)2. The PHQ-4 data will be used to track stress. Daily photos of meals will be used to track diet data throughout the study.

PHQ-4 total score ranges from 0 to 12, with categories of psychological distress being2:

• None 0-2
• Mild 3-5
• Moderate 6-8
• Severe 9-12

Anxiety subscale = sum of items 2 and 3 on Daily Survey Form (score range 0 to 6) Depression subscale = sum of items 4 and 5 on Daily Survey Form (score range 0 to 6) On each subscale, a score of 3 or greater is considered positive for screening purposes.

Participants will be also be asked to collect a minimum of 3 stool samples per week during the study. Participants who are collecting stool samples at home will store samples in their freezers in a provided biohazard storage container and samples will be picked up weekly by study staff or a courier. All participants will scan the QR code on the container to record the sample collection date and time and will write the collection date on the container.

On weeks 1, 8, and 16, participants will be asked to collect detailed diet data via electronic survey, using the NIH Automated Self-Administered 24-Hour Dietary Assessment Tool. Stool samples will be prepared and aliquoted by the Study Sponsor and sent to UCI and Cleveland Clinic for bacterial PCR, Metabolomic analysis, Shotgun metagenomic sequencing, bacterial spore titer evaluation, and IgA Testing. All samples, including blood samples, will be retained indefinitely in a -80C freezer at Aimmune Therapeutics for research per the existing Biobank Protocol #20234092. Mid- and post- intervention surveys will be sent to participants to evaluate experiences with the study and chia seeds, as well as to identify any side effects or complaints.

Deidentified surveys will be provided to and analyzed by the Study Sponsor.

IgA low subjects throughout the study. To discover the unknown IgA-degrading bacteria, researchers will spread fecal samples of IgA-Low subjects on CDC anaerobic blood agar plates to form single colonies. Next, researchers will subculture all the colonies on new CDC blood agar by streaking. Researchers will scrape each subculture colony into sterile PBS buffer and pool ~15 individual colonies for the in vitro incubation with IgA and Pigr. After overnight incubation at 37 °C, WB analysis will be used for evaluation of the degradation of IgA and Pigr. Colonies from the bacterial pool with IgA/Pigr-cleaving activity will be individually incubated with the substrates again and analyzed by immunoblotting.

Evaluate complexity of IgA degrading bacteria. Researchers will determine antibiotic sensitivities of the candidate bacteria in vitro. Because of the challenges with T. immunophilia (IgA degrader in mice), researchers will evaluate the complexity of colonies (are they easily grown in pure culture versus the presence of companion bacteria within the isolated colony). If the candidate IgA degrader grows slowly in culture, researchers will evaluate the bacteria for auxotrophy as for T. immunophila. Researchers will use genomic and metabolic tools in combination to test for auxotrophy.

IgA high subjects at the end of the study. Researchers will inoculate germ free mice with stool samples from these donors and evaluate IgA levels after two weeks in these mice (we will IgA low samples as a control). For mice that have high levels of IgA, researchers will isolate spores be various methods and test in germ free mice. Researchers will refine the IgA stimulating community as much as is possible and test if it is culturable. The goal is to identify human gut microbes that respond to fiber in the diet and alter IgA levels to all gut microbes.