Restoring Gut Health With INF108F in Infants With Food Protein Induced Allergic Proctocolitis
Phase: PHASE4
Conditions: Infant Development, Gut Microbiome, Food Protein Induced Allergic Proctocolitis
Sponsor: Massachusetts General Hospital

 
Version 2023.01.06 
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Institutional Review Board 
Intervention/Interaction Detailed Protocol 
 
 
Principal Investigator: 
Qian Yuan, MD, PhD 
 
Project Title: 
The RESTORE Study: Restoring gut health with B. infantis INF108F in Infants with Food 
Protein Induced Allergic Proctocolitis  
 
Version Date: 
08/01/2023 
 
 
Version Name/Number: 
Version 0.1 
 
 
 
IND Sponsor: N/A 
PRINCIPAL INVESTIGATOR 
CO-INVESTIGATOR(S) 
Qian Yuan, MD, PhD 
Wayne Shreffler, MD, PhD 
BIOSTATISTICIAN 
INDEPENDENT SAFETY MONITOR 
Summit Analytical 
Jennifer J. Bragg, MD 
Confidentiality Statement 
 
The information contained within this document is not to be disclosed in any way without the prior permission of the Principal Investigator 
and Infinant Health. 
 
 
INVESTIGATOR SIGNATURE PAGE 
Protocol 
Version/Date: 
pending 
0.1 / June 01, 2023 

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IND Number 
N/A  
Principal Investigator: 
Qian Yuan, MD, PhD 
Title: The RESTORE Study: Restoring gut health with B. infantis INF108F in infants with Food 
Protein Induced Allergic Proctocolitis 
 Study Sponsor: Massachusetts General Hospital and Infinant Health 
INSTRUCTIONS: The Principal Investigator will print, sign, and date at the indicated location 
below. The original signature page will be kept by the PI and a copy will be sent to Infinant 
Health. After signature, the original will be kept by: 
Jannat Gill 
MGH 
55 Fruit Street, CPZS 553 
Boston, MA 02114 
I confirm that I have read the above protocol in the latest version. I understand it, and I will 
work according to the principles of good clinical practice (GCP) as described in the US Code 
of Federal Regulations (CFR) 45 CFR part 46 and 21 CFR parts 50, 56, and 312, and in the 
International Conference on Harmonization (ICH) document Guidance for Industry: E6 Good 
Clinical Practice: Consolidated Guidance dated April 1996. Further, I will conduct the study 
in keeping with local legal and regulatory requirements. 
As the principal investigator, I agree to carry out the study by the criteria written in the 
protocol and understand that changes to this protocol will only be made by mutual 
consensus with Infinant Health. 
Qian Yuan, MD, PhD 
 
Principal Investigator (Print) 
 
Principal Investigator (Signature)                                                                  Date 

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SYNOPSIS 
Title 
The RESTORE Study: Restoring gut health with B. infantis INF108F in Infants 
with Food Protein Induced Allergic Proctocolitis 
Short Title 
The RESTORE Study 
Rationale 
Food Protein Induced Allergic Protocolitis (FPIAP) is an early form of food 
allergy in infancy. It is thought be related to the dysbiosis of the infant gut 
microbiome. Bifidobacterium longum subspecies (subsp.) infantis (B. 
infantis) is one of the special bacterial strains which is adapted to 
metabolize human milk oligosaccharides (HMOs) from breast milk. Lack of 
bifidobacteria, and in particular depletion of genes required for HMO 
utilization from the metagenome, is associated with systemic 
inflammation and immune dysregulation early in life. In breastfed infants, 
supplementation with B. infantis has been shown to alter the gut 
microbiome composition and correct the dysbiosis associated with 
immune dysfunction. 
Clinical Phase 
Post-market, Food for Special Dietary Use with GRAS status 
Mechanistic Study 
Yes 
 
Study Sponsor 
Massachusetts General Hospital and Infinant Health 
Principal 
Investigator 
Qian Yuan, MD, PhD 
Participating Site(s) 
Food Allergy Center @ Massachusetts General Hospital 
Accrual Objective 
50 randomized 
Investigational 
Product 
/ 
Intervention 
B. infantis INF108F versus placebo 
Study Objectives 
To evaluate the ability of B. infantis INF108F to promote a change in the 
gut microbiome composition of infants with FPIAP over a 4-week period. 
Secondary objectives are to evaluate changes to clinical symptoms of FPIAP 
and additional stool biomarkers. 
Study Design 
This is a single-center, randomized, double-blind, placebo-controlled trial 
with two arms evaluating B. infantis INF108F (Food for Special Dietary Use) 
in breastfed infants with FPIAP. The particular sub-species of 
Bifidobacterium used in this study has historically dominated the breastfed 
infant gut, however, it is currently missing from the gastrointestinal tract 
of most infants in the industrialized world. There is evidence to support 
that B. infantis INF108F supplementation may ameliorate symptoms 
associated with FPIAP by establishing and maintaining the necessary gut 

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microbial composition to promote proper barrier and immune function in 
infants. 
Study Duration 
4 weeks 
 
SYNOPSIS CONTINUED 
Primary Endpoint 
Changes to the gut microbiome composition (including B. infantis 
colonization) in infants with FPIAP over a 4-week period (B. infantis 
INF108F vs placebo) 
Secondary 
Endpoints 
and 
Biomarkers 
/ 
Exploratory 
Endpoints 
Secondary Endpoints: 
• The percentage of patients testing negative for blood in stool by 
Study Day 7, 14 and 28 by hemoccult testing 
• The percentage of patients with no gross/visible blood in stool by 
Study Day 7, 14 and 28 by parental report 
• Changes to stool frequency by Study Day 7, 14 and 28 
• Changes to clinical symptoms including GER, feeding difficulties, 
sleep disturbance and poor growth 
• The occurrence of supplement-related adverse events 
Biomarkers / Exploratory Endpoints: 
• Fecal calprotectin levels 
• Fecal pH levels 
• Fecal metabolomics 
• Markers of inflammation and enteric cytokines including, but not 
limited to, IL-1, IL-8 and TNF-alpha in stool 
• Levels of eosinophil-related granular proteins in stool 
• Intestinal permeability/integrity markers including, but not limited 
to, lipocalin (NGAL) 

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Inclusion Criteria 
• Infants, male or female, of all ethnic/racial groups, with a gestational 
period of 37 to 42 weeks 
• Infants aged 1-90 days old at the time of enrollment with 
documented FPIAP with either gross blood or microscopic blood in 
stools, without other possible causes 
• Infants must be exclusively breastfed or at least half of oral intake is 
from breastfeeding or from expressed breast milk 
• A willing parent or legal guardian will sign the consent form either 
electronically or with a wet ink signature 
 
SYNOPSIS CONTINUED 
Exclusion Criteria 
• Infants born earlier than 37 weeks of gestation 
• Infants who are exclusively-formula fed or less than half of oral intake 
is from breastfeeding or from expressed breast milk at the time of 
enrollment 
• Infants born with medical complications (i.e., neurological, cerebral 
palsy, confirmed food allergies) 
• Diagnosis of other severe or complicating medical problems, 
including autoimmune or chronic immune inflammatory conditions, 
gastrointestinal inflammatory conditions, or renal insufficiency 
• History of abdominal surgery or congenital abnormalities of the GI 
tract, the cardiovascular system, the pulmonary system or the renal 
system 
• Antibiotic use (oral or systemic) within 7 days prior to enrollment 
• Parents’ intent to feed non-study probiotics or solid food to their 
infant at any time during the study 
• Infants who have consumed any B. infantis-containing probiotics 
since birth 
• Primary Immune Deficiency 
• Maternal use of probiotics containing B. infantis after the baby’s birth 
and/or intent to use probiotics containing B. infantis at any time 
throughout the study 
• Past or current medical problems or findings from physical 
examination or laboratory testing that are not listed above, which, in 
the opinion of the investigator, may pose additional risks from 
participation in the study, may interfere with the participant’s ability 

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to comply with study requirements or that may impact the quality or 
interpretation of the data obtained from the study 
Study Procedures 
Physical examination, stool collection, questionnaire administration 
Statistical 
Considerations 
20 FPIAP infants in each group, B. infantis INF108F versus placebo. 
Approximately 100 infants will be recruited and screened for the study to 
obtain a sample size of 50 participants for randomization, assuming a 50 
percent screen fail rate. Forty (40) participants are expected to complete 
the study assuming a 20 percent drop-out rate. This sample size is based 
on a literature review of randomized clinical trials and the anticipated 
changes to the microbiome based on previous studies of B. infantis. 
Analysis on primary and secondary objectives will be completed based on 
the mITT population and the PP population; analysis on the exploratory 
objectives will be completed based on the mITT population. 
 
 
 
 

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Contents 
SYNOPSIS …………………………………………………………………………………………………………………………………..   3 
Glossary of Abbreviations/Definitions ………………………………………………………………………………………. 10 
1 
BACKGROUND AND SINGIFICANCE ……………………………………………………………………………….. 12 
1.1 
Background ……………………………………………………………………………………………………….. 12 
1.2 
Rationale for Selection of Study Population ………………………………………………………………….. 13 
1.3 
Investigational Product(s) / Intervention(s) …………………………………………………………………… 13 
1.4 
Rationale for Selection of Investigational Product(s) / Intervention(s) and Regimen …….. 13 
1.5 
Preclinical and Clinical Experience ……………………………………………………………………… 14 
1.5.1 Preclinical Studies and Regulatory Status ……………………………………………….. 14 
1.5.2 Clinical Studies Relevant to FPIAP …………………………………………………………… 15 
2 
SPECIFIC AIMS AND OBJECTIVES ……………………………………………………………………………………. 15 
2.1 
Primary Objectives …………………………………………………………………………………………….. 15 
2.2 
Secondary and Exploratory Objective(s) …………………………………………………………….. 15 
3 
GENERAL DESCRIPTION OF STUDY DESIGN …………………………………………………………………….. 15 
3.1 
Study Endpoints …………………………………………………………………………………………………………….. 16 
3.1.1 Primary Endpoint ………………………………………………………………………………………………. 16 
3.1.2 Secondary Endpoints ………………………………………………………………………………………… 16 
3.1.3 Biomarkers / Exploratory Endpoints …………………………………………………………………. 16 
3.2 
Study Completion …………………………………………………………………………………………………………. 17 
4 
SUBJECT SELECTION ………………………………………………………………………………………………………. 17 
4.1 
Inclusion Criteria …………………………………………………………………………………………………………… 17 
4.2 
Exclusion Criteria ………………………………………………………………………………………………………….. 17 
4.3 
Participant Withdrawal Criteria …………………………………………………………………………………….. 18 
5 
SUBJECT ENROLLMENT …………………………………………………………………………………………………………… 18 
5.1 
Recruitment and Pre-Screening …………………………………………………………………………………….. 18 
5.2 
Informed Consent Process …………………………………………………………………………………………….. 19 
5.3 
Screening/Baseline ……………………………………………………………………………………………………….. 19 
5.4 
Randomization and Study Supply Shipment ………………………………………………………………….. 19 
6 
   STUDY PROCEDURES ……………………………………………………………………………………………………………….. 20 
6.1 
Investigational product(s)/intervention material(s),  
other study products (controls/placebos) …………………………………………………………………….. 20 
6.1.1 
Investigational Product(s)/Intervention(s) ……………………………………………………….. 20 
6.1.2 
Study Product Handling, Storage and Compliance ……………………………………………. 21 
6.1.3 
Preparation and Administration ………………………………………………………………………. 21 
6.1.4 
Accountability of Investigational Product(s) / Intervention(s) ………………… ……….. 22 
6.1.5 
Assessment of Compliance with Investigational  
Product(s)/Intervention Material(s) …………………………………………………………………. 22 
6.1.6 
Modification or Discontinuation of Investigational  
Product(s) / Intervention Material(s) ……………………………………………………………….. 22 
6.2 
Other Medications …………………………………………………………………………………………….. 22 
6.2.1 
Concomitant Medications ………………………………………………………………………………… 22 
6.2.2 
Prohibited Medications and Dietary Ingredients ………………………………………………. 22 
6.3 
Study assessments and Procedures …………………………………………………………………… 23 

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6.3.1 
Efficacy Assessments ……………………………………………………………………………………….. 23 
6.3.2 
Safety and Other Exploratory Assessments ……………………………………………………… 24 
6.3.3 
Stool Sample Documentation, Training, Storage, and Handling ………………………. 24 
6.4 
Study Procedures ………………………………………………………………………………………………………… 
25 
6.4.1 
Study Product Supplementation Period ………………………………………………………….. 26 
6.4.2 
AE Follow-up …………………………………………………………………………………………………… 26 
6.4.3 
Compliance …………………………………………………………………………………………………….. 27 
6.4.4 
Double-Blind Placebo-Controlled Administration of INF108F and Placebo ……….. 27 
6.5 
Participant Completion and Withdrawal ………………………………………………………………………. 27 
6.5.1 
Participant Completion ……………………………………………………………………………………. 27 
6.5.2 
Participant Withdrawal ……………………………………………………………………………………. 27 
6.6 
Data Management ……………………………………………………………………………………………………….. 27 
6.6.1 
Data Collection ………………………………………………………………………………………………… 27 
6.6.2 
Access to Data …………………………………………………………………………………………………. 27 
6.6.3 
Stool Collection ……………………………………………………………………………………………….. 27 
6.6.4 
Physical Examination ……………………………………………………………………………………….. 28 
7 
RISKS AND DISCOMFORTS ……………………………………………………………………………………………….. 28 
7.1 
Risks of Investigational Product(s)/Intervention(s) ……………………………………………………….. 28 
7.2 
Risk of Study Procedures ……………………………………………………………………………………..……….. 30 
7.2.1 Double-Blind, Randomized, Placebo-Controlled clinical trial ……………………………. 30 
7.2.2 Administration Procedure ……………………………………………………………………….………. 30 
7.2.3 Stool collection ……………………………………………………………………………………………….. 30 
8 
BENEFITS ……………………………………………………………………………………………………………………… 31 
8.1 
Benefits of Investigational Product(s) / Intervention(s) ……………………………………………….. 31 
8.2 
Benefits of Study Procedure(s) …………………………………………………………………………………….. 31 
9 
STATISTICAL ANALYSIS ………………………………………………………………………………………..……….. 31 
9.1 
Sample Size Calculations and Statistical Analysis Plan …………………………..………….. 31 
9.1.1 Determination of Sample Size ………………………………………………………………..……..… 31 
9.2 
Data Analysis …………………………………………………………………………………………………..………..…. 31 
9.2.1 General Considerations ………………………………………………………………..…………………. 31 
9.2.2 Study Participant Populations …………………………………………………………………………. 32 
9.2.3 Efficacy Analysis ……………………………………………………………………………………………… 
32 
9.2.4 Study Participant Baseline Characteristics and Demographics ……………………...…. 33 
9.2.5 Study Completion ……………………………………………………………………………………………. 33 
9.3 
Interim Analyses ………………………………………………………………………………………………………….. 33 
9.4 
Deviations from Statistical Plan ………………………………………………………………….……………….. 33 
9.5 
Documenting an Unblinding ………………………………………………………………………………………… 33 
10 
MONITORING AND QUALITY ASSURANCE …………………………………………………………………………... 34 
10.1 
Safety Procedures …………………………………………………………………………………………….. 34 
10.1.1 Stopping Rules ………………………………………………………………………………………………… 34 
10.1.2 Early Discontinuation of Investigational Product(s)/Intervention(s)  
with continued study participation/follow-up …………………………………..…………….. 34 
10.1.3 Follow-up after early study termination ………………………………………………………….. 35 
10.1.4 Participant Replacement …………………………………………………………………………………. 35 
10.2 
Adverse Events ………………………………………………………………………………………………………….…. 35 

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10.2.1 Definitions ……………………………………………………………………………………………............ 35 
10.2.2 Collecting, Recording and Managing Adverse Events ……………………………........... 
36 
10.2.3 Grading and Attribution …………………………………………………………………………………. 
38 
10.2.4 SAE Reporting Criteria and Procedures ………………………………………………….. ……… 
40 
10.2.5 Non-Serious Adverse Events (NSAES) Reporting …………………………………………….. 
41 
10.3 
Protocol Deviations …………………………………………………………………………………………………….. 
42 
10.3.1 Protocol Deviation Definitions ………………………………………………………………………… 42 
10.3.2 Reporting Protocol Deviations ………………………………………………………………………… 43 
10.4 
Quality Control and Quality Assurance …………………………………………………………….. 43 
10.5 
Ethical Considerations and Compliance with Good Clinical Practice …………………. 44 
10.5.1 Statement of Compliance ……………………………………………………………………………….. 44 
10.6 
Informed Consent ……………………………………………………………………………………………………….. 44 
11 
PRIVACY AND CONFIDENTIALITY ……………………………………………………………………………………..….. 
45 
12 
REFERENCES …………………………………………………………………………………………………………………………… 45 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Glossary of Abbreviations / Definitions 
AE 
Adverse Event 
AR 
Adverse Reaction 
CFR 
Code of Federal Regulations 
CFU 
Colony Forming Units 
CMO 
Contract Manufacturing Organization 
CRA 
Clinical Research Associate 
CRC 
Clinical Research Center 
CRF 
Case Report Form 
CTCAE 
Common Terminology Criteria for Adverse Events 
DNA 
Deoxyribonucleic Acid 
EC 
Ethics Committee 
EGID 
Eosinophilic Gastrointestinal Disorder 
eIC 
Electronic Informed Consent 
EoE 
Eosinophilic esophagitis 
FDA 
Food and Drug Administration 
FPIAP 
Food Protein Induced Allergic Proctocolitis 
FPIES 
Food Protein Induced Enterocolitis Syndrome 
FSDU 
Foods for Special Dietary Use 
cGCP 
Current Good Clinical Practice 
GCP 
Good Clinical Practice 
GERD 
Gastroesophageal Reflux Disease 
GI 
Gastrointestinal 
GLP 
Good Laboratory Practices 
GMAP 
Gastrointestinal Microbiome Allergic Proctocolitis 
GMP 
Good Manufacturing Practices 
GRAS 
Generally Regarded as Safe 
HIPAA 
Health Insurance Portability and Accountability Act 
HMO 
Human Milk Oligosaccharide 
ICH 
International Conference on Harmonization 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ITT 
Intention to Treat 
NIH 
National Institutes of Health 
PCR 
Polymerase Chain Reaction 

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PHI 
Protected Health Information 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
RNA 
Ribonucleic Acid 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SAR 
Serious Adverse Reaction 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
US 
United States 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. 
 BACKGROUND AND SIGNIFICANCE 
 
1.1 
Background 
Oral tolerance is one of the fundamental mechanisms in preventing food allergies in humans. Breaching of oral 
tolerance may potentially lead to the development of food allergies (FA). Allergic food reactions can be divided 
into 2 categories: immunoglobulin E (IgE)-mediated and non-IgE-mediated FA. The hallmark of IgE-mediated FA is 
the potential of life-threatening anaphylaxis. The frequency of IgE-mediated FA in US children is about 6-8%. 
 
The non-IgE-mediated GI FA includes several clinical conditions, food protein-induced allergic protocolitis (FPIAP), 
food protein induced enterocolitis syndrome (FPIES), eosinophilic esophagitis (EoE) and eosinophilic 
gastrointestinal disorder (EGID). FPIAP is likely the earliest manifestation of non-IgE-mediated FA in childhood, 
and perhaps the very first condition of breaching oral tolerance, as it occurs in early infancy (2 weeks to 2 months) 
(1). In a prospective birth cohort study (GMAP), we have demonstrated that a prevalence of 17% of infants with 
FPIAP typically present with mucusy bloody stools, GER, irritability and feeding intolerances (1), and have 
increased risk of developing IgE-mediated FA later in life (2). FPIAP is also a risk factor for developing EoE (3). 
Children with FPIAP often have eczema (1). These suggest a possible common mechanism in non-IgE-mediated 
and IgE-mediated conditions. 
 
The human intestinal microbiome is found to play a key role in health and disease. Changes to the gut microbiota 
have been associated with a wide variety of diseases, including allergic diseases in human (4-9). It is known that 
the human gut microbiota undergoes rapid and dynamic changes during the first year of life (10-13). Many factors 
are known to influence the initial acquisition and subsequent composition of the gut microbiome in infants (11). 
These factors include mode of delivery, antibiotic exposure, presence of mother’s milk and breastfeeding, social 
economic status and household living conditions (11). Additionally, prenatal factors and in-utero exposures to 
certain environments likely play a role in the acquisition and composition of the infant gut microbiota (11). 
 
The gut microbiota plays a key role in the development of functional immune responses at all stages of 
development. The hygiene hypothesis suggests that changes in our microbial environment, including increased 
antibiotic usage and decreased environmental microbe exposure, are responsible for the rise of allergic and 
inflammatory diseases in the industrialized world. The human intestine is densely colonized by trillions of microbes 
that exist within an ecological network, interacting constantly with the human host to provide nutrient digestion 
and production of secondary metabolites, competitive exclusion of pathogenic species, and immune modulation. 
Dysbiosis refers to abnormal changes of the composition of the gut microbiota. The etiology of dysbiosis of the 
infant human gut microbiome is likely caused, at least in part, by epi-genetic factors over several generations (14). 
Intestinal dysbiosis has been suggested to contribute to a variety of inflammatory diseases, both within the gut 
and systemically (15). While food allergy patients do not share the severe bacterial imbalances observed in the 
intestines of inflammatory bowel disease or Clostridium difficile patients, studies suggest that they possess a 
microbiota that differs from that of healthy individuals. In a recent study, higher abundance of Ruminococcus 
gnavus and Faecalibacteerium prausnitzii and a depletion of Bifidobacterium longum, Bactereioudes dorei, B. 
vulgatus and fiber-degrading taxa were found in the gut microbiome of allergic children (16). The phylum 
Firmicutes, and specifically, bacteria from Clostridial Clusters IV and XIVa have been strongly associated with 
immunoregulatory responses in the intestine and have protective effects in models of food allergy and colitis (17-
20). Numerous animal studies have established that bacteria can modulate susceptibility to food allergy, and that 
beneficial Clostridia and their metabolites are protective in these models (18, 21-23). Thus, modulation of the 
microbiome shows promise as a therapeutic intervention for food allergies. 
 

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1.2 
Rationale for Selection of Study Population 
In a recent study, Henrick and colleagues elegantly demonstrated that a lack of bifidobacteria, specifically 
depletion of genes required for human milk oligosaccharide (HMO) utilization from the metagenome, is associated 
with systemic inflammation and immune dysregulation early in life (24). In our GMAP birth cohort, dysbiosis of 
the gut microbiome has been shown in both FPIAP (44) and FPIES (Su et al, submitted). 
 
Correction of the dysbiosis and restoration of the healthy gut microbiome in early infancy have great potential to 
alter abnormal immune responses, to prevent and treat non-IgE-mediated FA in infancy, and also to possibly 
prevent or alter the development of subsequent other non-IgE-mediated and IgE-mediated FA and allergic 
conditions. 
 
A birth cohort study from Finland has demonstrated that loss of Bifidobacterium early in life has been associated 
with increased risk of developing autoimmunity (25). This has also been shown in atopic wheezing in another 
cohort in rural Ecuador (26). Studies by Henrick and colleagues (27) and Rhoads and colleagues (28) have identified 
an association between the loss of Bifidobacterium in infants and enteric inflammation early in life. 
 
Bifidobacterium longum subspecies infantis (B. infantis) is a specialized bacterium adapted to metabolize human 
milk oligosaccharides (HMOs). Human breast milk contains abundant HMOs that are not digestible by humans, as 
we lack the necessary glucosidases (29). Instead, the maternal energy spent to create such complex sugars is 
justified by providing a selective nutritional advantage to “beneficial” microbes specialized in metabolizing HMOs 
with evolutionarily important functions in newborns (24). 
 
Introducing B. infantis has been successfully accomplished in strains such as INF108F (Infinant Health), which is 
able to stably and persistently colonize and remodel the intestinal microbiome of breastfed infants (30), leading 
to reduced fecal calprotectin, a marker of intestinal inflammation (27). 
 
In breastfed infants given Bifidobacterium infantis INF108F, which expresses all HMO-utilization genes, intestinal 
T helper 2 (Th2) and Th17 cytokines were silenced and interferon  (INF-) was induced (24). Fecal water from 
INF108F -supplemented infants contains abundant indolelactate and B. infantis-derived indole-3-lactic acid (ILA) 
upregulated immunoregulatory galectin-1 in Th2 and Th17 cells during polarization. These data provide a 
functional link between beneficial microbes and immunoregulation during the first months of life. 
 
This protocol is designed to investigate the role of B. infantis in modulating the gut microbiome in infants with 
FPIAP and the changes from B. infantis on symptoms of FPIAP and stool biomarkers. 
1.3 
Investigational Product(s) / Intervention(s) 
The active product that will be used in the proposed clinical trial is currently available commercially as a food 
supplement (Food for Special Dietary Use).  
 
1.4      Rationale for Selection of Investigational Product(s) / Intervention(s) and Regimen 
B. infantis INF108F has been used in other trials and has been shown to be safe and well-tolerated in infants (term 
and preterm) when supplemented daily for variable durations. Studies of this strain in breastfed infants 

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demonstrate a significant change to the gut microbiome composition within days of initiating the supplement. 
This change generally persists beyond the supplementation period if infants continue to receive breast milk as all 
or part of their diet. 
1.5 
Preclinical and Clinical Experience 
1.5.1        Preclinical Studies and Regulatory Status 
The investigational study product is a probiotic, B. infantis INF108F. B. longum subsp. infantis INF108F (Infinant 
Health) was selected for this study due to its demonstrated ability to utilize many types of oligosaccharides found 
in breast milk (i.e., HMOs), including lacto-N-tetraose, lacto-N-neo-tetraose, 2’-fucosyllactose, 3’-fucosyllactose, 
3’-sialyllactose, and 6’-sialyllactose. 
 
B. infantis INF108F was isolated from a healthy term breastfed infant. Its identity was confirmed by genetic 
sequencing of the organism by Infinant Health. Master stocks and working stocks were created and the strain is 
routinely tested for purity after fermentation. According to cGMP, the strain was re-isolated before creating the 
inoculum for the fermentation run. 
 
B. infantis INF108F was produced by fermentation under cGMP conditions at a contract manufacturing 
organization (CMO). Following fermentation, the strain was centrifuged, freeze-dried, milled, and blended with 
lactose sourced from a Safe Quality Food (SQF) level 3 facility. The product was packaged by a second CMO into 
FDA-compliant single-serving nitrogen-flushed polyester-faced laminated sachets with an oxygen and moisture 
barrier following cGMP. Each sachet contains approximately 600 mg of the blended powder containing at least 8 
x 109 CFU B. infantis INF108F. 
 
By way of its unique combination with human breast milk and utilization of HMOs, B. infantis INF108F is designated 
as a “Foods for Special Dietary Use” (FSDU). The term “special dietary uses”, as applied to food for man, is 
described in Chapter 1, Subchapter B, Part 105 of the Code of Federal Regulations, 21 CFR and defined in Part 
105.3. It means foods supplying particular dietary needs which exist by reason of a physical, physiological, 
pathological or other condition, such as conditions of diseases, convalescence, pregnancy, lactation, allergic 
hypersensitivity to food, underweight and overweight. Included in the definition are uses for supplying particular 
dietary needs which exist by reason of age, including but not limited to the ages of infancy (not more than 12 
months old) and childhood (more than 12 months but less than 12 years). 
 
Within the FDA, the Office of Food Additive Safety oversees a process for evaluating the safety of food ingredients 
that have a history of use and can, by scientific consensus, be generally regarded as safe (GRAS). These food 
ingredients must complete a rigorous process of scientific and data review by independent qualified experts, who 
assess whether the product is safe under the conditions of its intended use. B. infantis INF108F gained GRAS status 
after successfully completing this process, with unanimous consensus by all independent qualified experts that 
this product is generally recognized as safe based upon the application of generally available and accepted 
scientific data, information, or methods (i.e., peer-reviewed scientific literature), and scientific principles for use 
in term infants. 
 
GRAS status for B. infantis INF108F was obtained for a use level of up to 2.8 x 1010 colony forming units (CFU) per 
day. The current commercial serving size of B. infantis INF108F is 8.0 x 109 CFU/day. The study product control is 
a lactose placebo. The lactose ingredient was sourced from a SQF level 3 facility and packaged into FDA-compliant 

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single-serving nitrogen-flushed polyester-faced laminated sachets with an oxygen and moisture barrier following 
cGMP. Each sachet contains approximately 600 mg of lactose powder. 
 
1.5.2 Clinical Studies Relevant for FPIAP 
As mentioned earlier in Background section 1.1, Food Protein-induced Allergic Proctocolitis (FPIAP) is likely the 
very first manifestation of breaching oral tolerance in infancy (1, 31, 32, 33). It is one of the non-IgE mediated food 
allergies in infancy and early childhood (34, 35). Children with FPIAP have increased risk to develop IgE-mediated 
food allergies later in life (2, 33). Although the precise mechanism of FPIAP is still unclear, multifactorial causes 
including dysbiosis likely plays a role (36, 44). 
 
Probiotic-based strategies have been used to treat allergic/atopic diseases in infancy and childhood (36, 37, 38, 
39). Probiotics are now added to infant formulas by several formula makers with the intention to reduce atopic 
diseases in infancy; however, the specificity and efficacy vary.  
 
B. infantis INF108F uses HMO-utilization genes to metabolize HMO in breastfed infants, suppresses Th2 and Th17 
cytokines and promotes INF-g production, proving a functional link between beneficial microbes and 
immunoregulation during the first months of life (24). 
 
 
 
2. 
SPECIFIC AIMS AND OBJECTIVES  
 
2.1 
Primary Objective 
Assess the effect of modulating the gut microbiome by B. infantis INF108F in infants with FPIAP in early infancy. 
2.2    
Secondary and Exploratory Objective(s) 
• 
Evaluate changes in FPIAP symptoms (blood in stool, stool frequency, GER, feeding difficulties, and sleep 
disturbance) from feeding B. infantis INF108F. 
• 
Evaluate changes in enteric markers of inflammation and biochemistry from feeding B. infantis INF108F 
 
3. 
GENERAL DESCRIPTION OF STUDY DESIGN 
 
This single-center, double-blind, randomized and placebo-controlled clinical study will evaluate modulation of the 
intestinal microbiome in breastfed infants consuming a probiotic, B. infantis INF108F. The specific sub-species of 
bifidobacteria used in this study has historically dominated the breastfed infant gut; however, it is currently 
missing from the gastrointestinal tract of most infants in the industrialized world (40). There is evidence to support 
that B. infantis INF108F supplementation may ameliorate symptoms associated with FPIAP by establishing and 
maintaining the necessary gut microbial composition to promote proper barrier and immune function in infants 

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(41). We intend to enroll 100 infants for the study. The parents of each newborn will complete questionnaires and 
study logs and collect stool samples for the study. . Of the enrolled infants, we expect to randomize 50 in a 1:1 
allocation to the following study arms: 
 
• 
8.0 x 109 CFU daily serving of B. infantis INF108F (25 infants) 
• 
Lactose placebo (25 infants) 
 
Study participants and staff will be blinded to the treatment assignments. Infants in the active cohort will receive 
a once-daily oral feeding of B. infantis INF108F (8.0 x 109 CFU) and infants in the control cohort will receive a 
lactose placebo (both supplements mixed with breast milk) for 14 consecutive days. Study product feeds will be 
initiated on Day 1 (after the baseline period) and will continue for 14 days. The B. infantis INF108F and placebo 
will both be provided as a powder in sachets and will be prepared by mixing with a small amount of breast milk. 
The mixture will be fed by syringe to ensure the entire serving is consumed. Placebo sachets will contain lactose, 
the carrier used in the active product. The total duration of the family’s participation in the study will be 
approximately 4 weeks. 
3.1 
Study Endpoints 
3.1.1.    Primary Endpoint 
Changes to the gut microbiome composition (including B. infantis colonization) in infants with FPIAP over a 4-week 
period (B. infantis INF108F vs placebo) 
3.1.2.    Secondary Endpoints 
• The percentage of patients testing negative for blood in stool by Study Day 7, 14 and 28 by hemoccult testing 
• The percentage of patients with no gross/visible blood in stool by Study Day 7, 14 and 28 by parental report 
• Changes to stool frequency by Study Day 7, 14 and 28 
• 
Changes to clinical symptoms including GER, feeding difficulties, sleep disturbance and poor growth 
• 
The occurrence of supplement-related adverse events 
 
3.1.3 
Biomarkers / Exploratory Endpoints: 
• 
Fecal calprotectin levels 
• 
Fecal pH levels 
• 
Fecal metabolomics 
• 
Markers of inflammation and enteric cytokines including, but not limited to, IL-1, IL-8 and TNF-alpha in stool 
• 
Levels of eosinophil-related granular proteins in stool 
• 
Intestinal permeability/integrity markers including, but not limited to, lipocalin (NGAL) 

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3.2 
Study Completion 
This study will be considered “completed” when the primary and secondary objectives have been met. This 
includes the analysis of all the data required to meet the chosen objectives. 
 
After the study is completed, the Principal Investigator and the Data Center will compile a final study report as per 
ICH E6 and 21CFR312. The study report will be submitted to the local IRB. 
 
4. 
SUBJECT SELECTION 
 
4.1 
Inclusion Criteria 
Individuals who meet all of the following criteria are eligible for enrollment as study participants. Potential 
candidates will be identified at the Pediatrics at Newton Wellesley (PNW) and MGH Pediatric Gastroenterology 
Clinic. The recruitment will occur at Newton Wellesley Hospital or Massachusetts General Hospital. 
• 
Infants, male or female, of all ethnic/racial groups, with a gestational period of 37 to 42 weeks 
• 
Infants aged 1-90 days old with a documented FPIAP with either gross blood or microscopic blood in stools, 
without other possible causes 
• 
Infants must be exclusively breastfed or at least half of oral intake is from breast feeding or from expressed 
breast milk 
• 
A willing parent or legal guardian will sign the consent form either electronically or with a wet ink signature 
4.2 
Exclusion Criteria 
Individuals who meet any of these criteria are not eligible for enrollment as study participants: 
• 
Infants born earlier than 37 weeks of gestation 
• 
Infants who are exclusively formula-fed or less than half of oral intake is from breastfeeding or from 
expressed breast milk at the time of enrollment 
• 
Infants born with medical complications (i.e., neurological, cerebral palsy, confirmed food allergies) 
• 
Diagnosis of other severe or complicating medical problems, including autoimmune or chronic immune 
inflammatory conditions, gastrointestinal inflammatory conditions, or renal insufficiency 
• 
History of abdominal surgery or congenital abnormalities of the GI track, the cardiovascular system, the 
pulmonary system or the renal system 
• 
Antibiotic use (oral or systemic) within 7 days prior to enrollment 
• 
Parents’ intent to feed non-study probiotics or solid food to their infant at any time during the study 
• 
Mothers with substance use disorder (SUD) or on Nicotine replacement therapy (NRT) 
• 
Infants who have consumed any B. infantis-containing probiotics since birth 

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• 
Primary Immune Deficiency 
• 
Maternal use of probiotics containing B. infantis during pregnancy, after the baby’s birth and/or intent to 
use probiotics containing B. infantis at any time throughout the study 
• 
Past or current medical problems or findings from physical examination or laboratory testing that are not 
listed above, which, in the opinion of the investigator, may pose additional risks from participation in the 
study, may interfere with the participant’s ability to comply with study requirements, or that may impact 
the quality or interpretation of the data obtained from the study.  
 
4.3 
Participant Withdrawal Criteria 
Participants may be terminated early from the study for the following reasons: 
• 
The participant elects to withdraw consent from all future study activities, including follow-up. 
• 
The participant is “lost to follow-up” (i.e., no further follow-up is possible because attempts to reestablish 
contact with the participant have failed). 
• 
The participant dies. 
• 
The participant develops a medical condition or is started on new medication(s) not previously mentioned 
in the list of prohibited medications that, in the opinion of the investigator, may pose additional risks from 
participation in the study, may interfere with the participant’s ability to comply with study requirements or 
that may impact the quality of the data obtained from the study. 
• 
The participant meets any of the individual stopping rules as delineated in Section 10.1.1. 
Participants with early termination from this study will not be replaced. 
 
5. 
SUBJECT ENROLLMENT 
 
5.1      Recruitment and Pre-Screening 
Breastfed infants with FPIAP, with either grossly visible bloody stool or microscopic bloody stools, excluding other 
causes, will be recruited at PNW or MGH Pediatric GI clinic. These individuals will be provided basic information 
about the study and ask potential participants to provide basic contact information and answer some study-
specific questions as a pre-qualifier. Individuals who are interested to learn more will receive more details about 
the study, will be presented with an informed consent (IC), and will receive instructions as to how they are able 
to communicate with the study PI and other study team members to address any questions or concerns during 
the IC process and study duration.  
 

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5.2.      Informed Consent Process 
The parent of each infant participant will be required to sign an IRB-approved IC. Only participants for whom there 
is a signed and approved IC will be enrolled in the study. The IC will explain the study, and fully disclose its known 
risks and benefits by using simple, nontechnical terms wherever possible. The IC will include the elements required 
by the FDA in 21 CFR 50 and International Conference on Harmonization (ICH) guidelines. Parents will also be 
required to consent to the use of their protected health information (PHI) according to all applicable health 
information privacy regulations in the US. This authorization can be part of the IC or can be a separate document. 
 
The investigator of the study is responsible for ensuring that a signed IC and authorization for use of PHI is 
collected from each participant that they enroll into the study at the time of that participant’s enrollment. No 
study-related activities may be performed with, or by, any participant, and no data may be collected from any 
participant for study purposes prior to obtaining these consents and authorizations. 
 
Participation in this study is voluntary and parents will be allowed to withdraw consent for their infant at any time 
for any reason with no impact on their eligibility to be treated for any adverse events or on their eligibility to 
receive other therapy. 
5.3.      Screening/Baseline 
After signing the IC, parents will complete a screening questionnaire, demographics, medical history, and 
ConMeds to assess alignment with the eligibility criteria. Once eligibility at this point is confirmed, the study site 
will provide stool collection kits and the Study Product feeding kit. Parents will be provided with a binder, including 
logs and questionnaires that they will be expected to complete throughout the study.  
Prior to start oral administration of B. infantis INF108F or placebo, the participant will have a physical exam with 
vital signs and parents will be expected to collect a baseline stool sample. 
5.4.      Randomization and Study Supply Shipment 
Randomization lists will be maintained in a secured area by the study team. If the randomization key is shared 
such that blinding is compromised, the Principal Investigator will be notified. 
 
Randomization will occur immediately after all screening and baseline tasks have been confirmed to be completed 
and baseline data has been deemed acceptable. Baseline tasks include:  
 
• Confirming infant/mother eligibility  
• Obtaining informed consent  
• Confirming baseline data collection  
• Confirming baseline stool specimen collection.  
 
Only infants for whom acceptable baseline data and stool samples have been collected will be randomized into 
the supplementation phase of the study. Using the randomization key generated by an unblinded statistician and 
held by an unblinded study site team member, eligible infants will be assigned a randomization code and 
randomized to a study product arm. The unblinded team member will provide the enrolled patient’s parents with 

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the appropriate Study Product Kit in accordance with the randomization key. Infants will be randomized in a 1:1 
ratio to the following arms: 
 
• 8.0 x 109 CFU B. infantis INF108F 
• Lactose placebo 
 
After randomization, the study team will provide the assigned feeding kit to the participant. Study supplies that 
will be provided include: 
 
• Feeding kits to include 14 sachets of study product (B. infantis INF108F or lactose placebo depending on 
randomization), oral syringes, and mixing cups 
• Stool collection kits  
 
 
 
6. 
STUDY PROCEDURES 
 
6.1 INVESTIGATIONAL PRODUCT(S)/INTERVENTION MATERIAL(S), OTHER STUDY PRODUCTS 
(CONTROLS/PLACEBOS) 
6.1.1.   Investigational Product(s) / Intervention(s) 
INF108F 
INF108F is formulated as water-soluble powder.  The investigational study product is a probiotic, B. infantis 
INF108F. B. longum subsp. infantis INF108F (Infinant Health) was selected for this study due to its demonstrated 
ability to utilize many types of oligosaccharides found in breast milk (i.e., HMOs), including lacto-N-tetraose, lacto-
N-neo-tetraose, 2’-fucosyllactose, 3’-fucosyllactose, 3’-sialyllactose, and 6’-sialyllactose. 
 
The B. infantis INF108F was isolated from a healthy term breastfed infant. Its identity was confirmed by genetic 
sequencing of the organism by Infinant Health. Master stocks and working stocks were created and the strain is 
routinely tested for purity after fermentation. According to cGMP, the strain was re-isolated before creating the 
inoculum for the fermentation run. 
 
The INF108F strain of B. infantis was produced by fermentation under cGMP conditions at a contract 
manufacturing organization (CMO). Following fermentation, the strain was centrifuged, freeze-dried, milled, and 
blended with lactose sourced from a Safe Quality Food (SQF) level 3 facility. The product was packaged by a second 
CMO into FDA compliant single-serving nitrogen-flushed polyester-faced laminated sachets with an oxygen and 
moisture barrier following cGMP. Each sachet contains approximately 600 mg of the blended powder containing 
at least 8 x 109 CFU B. infantis INF108F. 
 
By way of its unique combination with human breast milk and utilization of HMOs, B. infantis INF108F is designated 
as a “Foods for Special Dietary Use” (FSDU). The term “special dietary uses”, as applied to food for man, is 
described in Chapter 1, Subchapter B, Part 105 of the Code of Federal Regulations, 21 CFR and defined in Part 
105.3. It means foods supplying particular dietary needs which exist by reason of a physical, physiological, 

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pathological or other condition, such as conditions of diseases, convalescence, pregnancy, lactation, allergic 
hypersensitivity to food, underweight and overweight. Included in the definition are uses for supplying particular 
dietary needs which exist by reason of age, including but not limited to the ages of infancy (not more than 12 
months old) and childhood (more than 12 months but less than 12 years). 
 
Within the FDA, the Office of Food Additive Safety oversees a process for evaluating the safety of food ingredients 
that have a history of use, and can, by scientific consensus, be generally regarded as safe (GRAS). These food 
ingredients must complete a rigorous process of scientific and data review by independent qualified experts, who 
assess whether the product is safe under the conditions of its intended use. B. infantis INF108F gained GRAS status 
after successfully completing this process, with unanimous consensus by all independent qualified experts that 
this product is generally recognized as safe based upon the application of generally available and accepted 
scientific data, information, or methods (i.e., peer-reviewed scientific literature), and scientific principles for use 
in term infants. 
 
GRAS status for B. infantis INF108F was obtained for a use level of up to 2.8 x 1010 colony forming units (CFU) per 
day. The current commercial serving size of B. infantis INF108F is 8.0 x 109 CFU/day. 
 
INF108F Placebo: 
INF108F Placebo is lactose water soluble powder 
The study product control is a lactose placebo. The lactose ingredient was sourced from a SQF level 3 facility and 
packaged into FDA compliant single-serving nitrogen-flushed polyester-faced laminated sachets with an oxygen 
and moisture barrier following cGMP. Each sachet contains 600 mg of lactose powder. 
6.1.2 
Study Product Handling, Storage and Compliance 
Once a participant has been randomized, a feeding kit containing 14 sachets of B. infantis INF108F or placebo, oral 
syringes, and mixing cups will be provided. Parents will be instructed to store the supplement immediately upon 
receipt in their freezer until use.  
 
Parents will be expected to record missed study product feedings in their daily feeding log. Compliance will be 
evaluated based on these data. Parents may also be requested at the end of the supplementation period to count 
the number of sachets remaining. Unused sachets may be kept by participants or disposed of. 
6.1.3           Preparation and Administration 
Parents will be trained on how to prepare and feed the study product to their infants. Infants enrolled into the 
control cohort will receive a lactose placebo and infants enrolled into the active cohort will receive B. infantis 
INF108F in lactose carrier. Both placebo and B. infantis INF108F feedings will be provided in powder form to be 
mixed in 3-5 mL (approximately 1 teaspoon) of breast milk each day for 14 consecutive days.  
 
Study product cannot be initiated until 1) an infant has been deemed eligible for this study by the Investigator, 2) 
informed consent has been obtained, 3) the baseline stool sample has been collected and frozen by the parents, 
and 4) baseline study data has been obtained. 

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6.1.4 
Accountability of Investigational Product(s) / Intervention(s) 
Under Title 21 of the Code of Federal Regulations (21CFR §312.62) the investigator will maintain adequate records 
of the disposition of the investigational product(s) / intervention material(s), including the date and quantity of 
the product received, to whom the product was dispensed (participant-by-participant accounting), and a detailed 
accounting of any investigational product(s) / intervention material(s) accidentally or deliberately destroyed. 
 
Records for receipt, storage, use, and disposition will be maintained by the study site. A Master Dispensation Log 
will be kept current. This log will contain the identification of each participant and the date and quantity of 
investigational product(s) / intervention material(s) dispensed. Any product that remains unused at the end of 
study will be destroyed. 
 
All records regarding the disposition of the investigational product(s) / intervention material(s) will be available 
for inspection by the Clinical Research Associate (CRA). 
6.1.5 
Assessment of Compliance with Investigational Product(s) / Intervention Material(s) 
Every study subject will be asked to record consumption of their feedings of INF108F/placebo in a daily feeding 
log. Investigational product compliance will be recorded as a percentage, calculated as the number of days of 
reported consumption divided by the number of days of supplementation expected. 
 
For participants who are withdrawn prior to the end of the supplementation period, percent adherence will be 
based on the number of days active in the study prior to withdrawal. 
6.1.6 
Modification or Discontinuation of Investigational Product(s) / Intervention Material(s) 
The parents may withdraw their infant from the study at any time by notifying the study team. Participants may 
be withdrawn from the study by the investigator if study procedures have not been followed or if the investigator 
determines the participant is not able to safely continue with the study due to an adverse event or other health-
related issue. If greater or equal to 20 percent drop-out occurs, infants may be replaced to assure adequate 
statistical power. 
6.2.       OTHER MEDICATIONS 
6.2.1 
Concomitant Medications 
Concomitant medications may be used as necessary throughout the study unless noted in section 6.2. 
6.2.2 Prohibited Medications and Dietary Ingredients 
Medications and dietary ingredients prohibited during the study include: 
 

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For Infant: 
• 
Any probiotics (including infant formulas containing probiotics) 
• 
Antibiotics (oral or systemic)  
• 
Infant formula greater than half of the infant’s daily nutrition intake 
• 
Solid food 
For Mother: 
• 
Antibiotics 
• 
Probiotics containing B. infantis 
6.3.       STUDY ASSESSMENTS AND PROCEDURES 
6.3.1 
Efficacy Assessments 
6.3.1.1      Primary objective  
The primary objective of the study is to assess changes to the gut microbiome composition (including B. infantis 
colonization) in infants with FPIAP over a 4-week period (B. infantis INF108F vs placebo) 
6.3.1.2     Secondary Objectives  
The secondary objectives of this study are to evaluate: clinical symptoms including blood in stool, stool frequency, 
GER, feeding difficulties, sleep disturbance and poor growth 
 
6.3.1.2.1    Blood in stool  
Blood in stool will be assessed by daily hemoccult stool test on Days 0, 1-14, 21 
(+/- 1 day), 28 (+/- 1 day) 
6.3.1.2.2    Stooling  
 
Infant stooling will be assessed by daily log capturing frequency, consistency, 
night stooling, and presence of mucus or gross blood  
6.3.1.2.3    Infant GER   
Infant GER will be assessed by standard Reflux Questionnaire on Day 7, 14, 21, 
and 28 
6.3.1.2.4    Infant Sleep   
Infant sleep duration/quality will be assessed by Questionnaire on Day 7, 14, 21, 
and 28 
6.3.1.2.5    Infant Feeding  
Infant feeding difficulties will be assessed by Questionnaire on Day 7, 14, 21, and 
28 
6.3.1.2.6    Infant Growth  
Infant growth will be monitored at weekly visits by measuring weight, length, and 
head circumference  
 
6.3.1.2.7    Adverse Events  
Supplement-related adverse events will be recorded by parents in a health log, in 
consultation with the study team and PI.  

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6.3.2 
Safety and Other Exploratory Assessments 
Safety assessments will include parent-reported adverse events as described in Section 10.2. 
 
6.3.2.1      Stool Sample Collection  
In addition to being used for hemoccult testing, infant stool specimens will be analyzed as an exploratory objective 
of the study and will be collected once during the Baseline period and daily (when available) for Days 1-14, 21, 
and 28, and assessments may include the following markers: 
 
• Fecal calprotectin levels 
• Fecal pH levels 
• Fecal metabolomics 
• Markers of inflammation and enteric cytokines including, but not limited to, IL-1, IL-8 and TNF-alpha 
• Levels of eosinophil-related granular proteins 
• Intestinal permeability/integrity markers including, but not limited to, lipocalin (NGAL) 
6.3.3 
Stool Sample Documentation, Training, Storage, and Handling 
6.3.3.1       Baseline Samples 
Baseline stool swabs and chunks of stool must be collected before the first study supplementation is consumed. 
After collection, the parents will store the swab tubes (white top tube) at room temperature and will store the 
chunk tubes (brown top tube)  in their home freezer in a bag provided to retain those samples. At the Day 7 in-
person Study Visit, parents will bring all Days 0-7 stool samples (swab, chunk, and card) with them to the visit. At 
the end of the study, parents will bring all remaining stool samples to the study site at the Day 28 End of Study in-
person Visit. All efforts should be made to collect stool samples within the target collection windows. Parents will 
be trained on how to collect the samples at enrollment and as needed throughout the study.  
 
6.3.3.2      Stool Swabs 
Starting with a freshly soiled diaper, the parent will first collect the stool swab sample using the supplies provided 
in the stool collection kit (green-topped tube containing a buffer/bead mixture and packaged swab). Parents will 
swab their infant’s soiled diaper, collecting enough sample to entirely coat the head of the swab. They will place 
the stool-covered swab into a white-topped collection tube containing stabilization buffer and will snap the swab 
head off at the breakpoint, leaving the swab head in the tube. They will seal the tube and shake vigorously to 
ensure distribution of the stool within the stabilization buffer. Swab-containing tubes (white cap tube) will be 
stored at room temperature. . 

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6.3.3.3 Stool Chunks 
Using the supplies provided in the stool collection kit (empty snap-cap tubes and white stool collection spatulas), 
mothers will collect as much loose stool sample from their infant’s soiled diaper as possible before snapping the 
lid tightly closed for storage. They will likely need to take multiple scoops to fill the tube as much as possible. If 
little stool is available for collection in a particular diaper, mothers will be instructed to use subsequent soiled 
diapers that fall within the same stool collection window to add to the already frozen snap-cap tube. 
 
For example: A mother can only collect the swab tube and a very small spatula-full of stool for the stool ‘chunk’ 
sample. If baby stools later within the same stool collection window, the mother will remove the previously frozen 
partial ‘chunk’ sample tube(s) from the freezer, add as much stool as possible and re-freeze the entire sample. 
She will not need to prepare a second swab sample and should not try to add more material to the green-topped 
tube. 
6.3.3.4 
Stool Sample Storage and Transport 
Immediately after collecting their infant’s stool swab and chunk samples, parents will store the whole chunk 
sample (tube with brown cap) in their home freezer within the study-provided freezer bag. Parents will bring the 
freezer bag containing the collected stool chunk samples on a provided frozen ice pack to the in-person Study Visit 
on Day 7 and the in-person Study Visit on Day 28.  
Stool swab samples (tubes with white cap) should be stored at room temperature. Parents will bring the collected 
stool swab samples with them to the in-person Study Visit on Day 7 and the in-person Study Visit on Day 28. 
Hemoccult cards should be stored at room temperature. Parents will bring the completed hemoccult cards with 
them to the Study Visits on day 7 and day 28. 
6.3.3.5 
Stool Sample Analysis 
DNA and RNA will be extracted from stool swab samples and will be used for shotgun sequencing, next generation 
sequencing, and/or quantitative PCR to determine levels of B. infantis, total Bifidobacterium, microbial signatures, 
and relative abundance of the most abundant bacterial taxa. Biochemical analyses may be performed on stool 
chunk samples to evaluate markers as described in Section 6.3.2. 
6.3.3.6 
Stool Sample Retention by the Sponsor 
Infant stool samples collected during the conduct of the study may be kept by the Sponsor for up to 10 years for 
use in future research. No personally identifiable information will be recorded on stool specimen tubes. 
 
6.4 
Study Procedures 
All study procedures are listed in Table 1: Schedule of Events below. 

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Table 1: Schedule of Events 
 
Screening 
Day 
0 
(Baseline) 
Days  
1-6 
Day 7 
Days  
8-13 
Day 14 Days  
15-27 
Day 28 
(End of 
Study) 
Diagnosis 
x 
 
 
 
 
 
 
 
Consent 
x 
 
 
 
 
 
 
 
Screening 
x 
 
 
 
 
 
 
 
Study Visit 
x 
x 
 
x 
 
x 
 
x 
Physical exam 
 
x 
 
x 
 
x 
 
x 
Supplementation  
 
x 
x 
x 
x 
 
 
Questionnaire 
 
x 
x 
x 
x 
x 
x 
 
Feeding log 
 
x 
x 
x 
x 
x 
x 
 
Stooling log 
 
x 
x 
x 
x 
x 
x 
 
Clinician 
symptom tool 
 
x 
 
x 
 
x 
 
x 
Randomization 
 
x 
 
 
 
 
 
 
Stool collection 
 
x 
X Day 3  
 
x 
 
x 
Hemoccult test 
 
x 
x 
x 
x 
x 
x 
x 
Stool to site 
 
 
 
x 
 
 
 
x 
AE reporting 
 
 
x 
x 
x 
x 
x 
x 
 
 
 
6.4.1 
Study Product Supplementation Period Days 1-14  
• Daily oral administration of B. infantis INF108F or placebo 

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• Daily stool collection 
• Questionnaires for Sleep, Feeding and Stool frequency and consistency 
 
On or after Day 28, study staff will check-in with parents to make sure that the final stool samples have been 
collected and all remaining Questionnaires have been completed. Parents will bring all remaining frozen stool 
samples to the Day 28 in-person study visit.  
6.4.2 
AE Follow-up 
Days 15-28  
Parents will report any post-supplementation period adverse events experienced by their infant during this time.  
6.4.3 
Compliance 
Compliance will be determined by treatment consumption, as reported in the Daily Log. Treatment compliance 
will be recorded as a percentage, calculated as the number of days of reported consumption divided by the 
number of days of treatment expected. Missed assessments will be recorded. For participants who are withdrawn 
prior to the end of the treatment period, percent adherence will be based on the number of days active in the 
study prior to withdrawal. 
6.4.4      Double-Blind Placebo-Controlled Administration of INF108F and Placebo 
Prior to start of oral administration of B. infantis INF108F or placebo, the participant will have a physical exam 
with vital signs. 
6.5 
Participant Completion and Withdrawal 
6.5.1 
Participant Completion 
Participants will have completed the study upon completion of Day 28 AE follow-up period. 
6.5.2 
Participant Withdrawal 
The parents may withdraw their infant from the study at any time by notifying the study team. Participants may 
be withdrawn from the study by the investigator if study procedures have not been followed or if the investigator 
determines the participant is not able to safely continue with the study due to an adverse event or other health-
related issue. If 20 percent drop-out occurs, infants may be replaced to ensure adequate statistical power. 
 

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6.6 
Data Management 
6.6.1 
Data Collection 
Study data will be collected on paper Case Report Forms and entered by study staff into a RedCap database. 
6.6.2 
Access to Data 
The PI, study coordinators, and CRAs (Study Monitors) will only have access to the blinded study data. 
6.6.3 
Stool Collection 
Stool samples will be collected from the participants at the following time points: 
• At Screening / Baseline 
• Day 3 (within a window of Study Day 3-5) 
• Day 14 (+/- 1 day) 
• Day 28 (+/- 1 day) 
The stool samples will be banked and used for 16s rRNA or metagenomic sequencing for microbiome 
evaluation and biologic studies. 
6.6.4        Physical Examination 
This will include measurement of vital signs, including weight, temperature, heart rate, and blood pressure, 
and physical examination including the skin, ears, nose and throat, abdomen, lungs, and heart. 
 
7. 
RISKS AND DISCOMFORTS 
 
7.1     Risks of Investigational Product(s) / Intervention(s) 
Previous studies have shown that B. infantis INF108F is safe and well-tolerated by infants (both term and preterm) 
with no known side effects. 
 
7.2 
Risk of Study Procedures 
7.2.1       Double-Blind, Randomized, Placebo-Controlled clinical trial Possible adverse events during the clinical 
trial include mainly GI symptoms (gas, abdominal distension, vomiting, diarrhea, constipation or abdominal pain). 

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7.2.2         Administration Procedure Minimal risk is associated with administration of B. infantis INF108F, including 
choking,  spit up, and vomiting. 
7.2.3       Stool collection All stool collection will be from fresh diapers, which does not impose any potential risk 
to the infants. 
8. 
BENEFITS 
 
8.1.    Benefits of Investigational Product(s) / Intervention(s) 
The risks associated with participation in this study are minimal in relation to the potential benefits derived by 
both the subject and society. Participants may derive two major benefits from the study. First, participants may 
have faster resolution of FPIAP. Second, participants may have modified gut microbiota which could potentially 
lead to reduced risk, or even prevention of, other allergic diseases in life. 
 
Potential benefits to society include a better understanding of modulation of the infant gut microbiome and the 
associated clinical outcomes in infants with FPIAP. 
8.2     Benefits of Study Procedure(s) 
With the treatment of B infantis INF108F, we anticipate that the infant’s gut microbiome will likely be modified in 
a positive manner to correct the dysbiosis which leads to the development of FPIAP. 
 
 
9. 
STATISTICAL ANALYSIS 
 
9.1    SAMPLE SIZE CALCULATIONS AND STATISTICAL ANALYSIS PLAN 
9.1.1  Determination of Sample Size 
Approximately 100 infants and 100 mothers will be recruited and screened for the study to obtain a sample size 
of 50 participants for randomization, assuming a 50 percent screen fail rate. Forty (40) participants are expected 
to complete the study assuming a 20 percent drop-out rate. This sample size is based on a literature review of 
randomized clinical trials and the anticipated changes to the microbiome based on previous studies of B. infantis. 
 9.2 
Data Analysis 
9.2.1     General Considerations 
Data will be collected and curated in a REDCap database [43] with support from the Human participants Core and 
exported into a suitable analysis format (e.g., STATA, SPSS, R analysis) for analysis. For continuous variables that 

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are normally distributed we will summarize them using mean with standard derivations and for those that are 
non-normally distributed, we will summarize with medians and inter-quartile ranges. For dichotomous data we 
will provide proportions. In addition to the baseline and outcome data, we will also summarize the recruitment 
numbers, those participants lost to follow-up, protocol violations and other relevant data. 
 
9.2.2     Study Participant Populations 
Randomization Approximately fifty (50) infants will be randomized in a 1:1 ratio to treatment probiotic or placebo. 
Randomization will occur immediately after all baseline period tasks have been confirmed to be completed and 
provide acceptable data. 
 
Populations for Analyses The modified Intent to Treat (mITT) population will include all infants who are 
randomized and receive at least one supplementation. The Per Protocol (PP) population will include all 
randomized participants who do not have major protocol deviations. Major protocol deviations will be defined 
prior to unblinding of the data. Analysis on primary and secondary objectives will be completed based on the mITT 
population and the PP population; analysis on the exploratory objectives (perception of treatment) will be 
completed based on the mITT population. 
 
Descriptive Statistics Descriptive statistics (mean, median, and standard deviation, minimum and maximum) of 
the quantitative variables will be summarized using number of subjects (n). The qualitative variables will be 
summarized using counts and percentages. All summaries will be presented by treatment group and time point 
(where relevant). 
 
All CRF and laboratory data will be analyzed. Comparisons between treatment groups will be performed using 
Fisher’s exact test or Pearson’s Chi-Square test for categorical data and one-way ANOVA with Tukey’s post hoc 
comparisons for individual timepoints or two-way repeated measures ANOVA for all timepoints for continuous 
data. Mean and standard deviations for certain groups and timepoints will be used as estimates in power 
calculations for future studies. Missing data will not be imputed in this study. 
 
9.2.3 
Efficacy Analysis  
Primary Endpoint(s) The primary objective of the study is to assess the effectiveness of the probiotic, B. infantis 
INF108F, on modulating the gut microbiome composition of breastfed infants diagnosed with FPIAP from Baseline 
to Day 28, including colonization of INF108F. This will be measured using stool 16S analysis and quantitative PCR. 
Treatments will be compared using a Wilcoxon Rank Sum test. 
 
Secondary Endpoint(s) The secondary objectives of this study as measured:  
• Evaluating the reduction in stool frequency and improvement of stool consistency by Day 7 using 
Questionnaire 
• Evaluating the improvement of infant GER symptoms by Day 14 using Questionnaire. Reduction by 50 
percent from Baseline to Day 14 Infants with a reduction in the number of crying/fussing episodes of greater 
than or equal to 50 percent on Day 14 will be counted as a success. The number of successes per treatment 
group will be compared using a Chi-square or Fisher’s Exact test, as appropriate. 
• Evaluating the improvement of feeding difficulties by Day 14, using Questionnaire. The number of successes 
per treatment group will be compared using a Chi-square or Fisher’s Exact test, as appropriate. 

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• Evaluating the improvement of infant’s sleep by Day 14, using Questionnaire.  
• Changes of stool cytokine profile by Day 28. The changes from baseline to Day 28 will be compared for the 
two groups using a repeated measures ANOVA. 
Safety Analysis Fisher’s Exact Test will also be used to compare treatment groups for SAEs and AEs resulting in 
withholding or discontinuing the study products. Adverse events will be summarized. Frequencies and 
percentages of subjects with treatment-emergent adverse events (TEAEs), serious TEAEs and TEAEs causing 
premature discontinuation will be provided by treatment group. An AE is treatment emergent if it 1) occurs after 
the first dose of randomized study treatment or 2) if it is present prior to receipt of randomized study treatment 
but worsens in severity or increases in frequency after the first dose of randomized study treatment. 
9.2.4     Study Participant Baseline Characteristics and Demographics 
Summary of descriptive statistics for baseline and demographic characteristics will be provided for all enrolled 
participants. Demographic data will include age, race, sex, body weight, and height; these data will be presented 
in summary tables. 
 
9.2.5     Study Completion 
Premature Termination of Study: Should it prove necessary to discontinue the study permanently prior to 
completion, the Sponsor will notify Infinant Health and the IRB of the rationale. Participants will be informed by 
the study site. All relevant study documents and data will then be returned to the Sponsor, and the study product 
will be destroyed or returned.  
Termination of Study: After the completion or termination of the study, all relevant study documents and data 
will then be sent to the Sponsor and the study product will be destroyed. The Investigator will inform the IRB of 
the end of the study and a certificate of study closure will be issued. 
9.3 
Interim Analyses 
One Interim Analysis will occur when the first 10 subjects (at least) complete the study. Interim Analysis data will 
be analyzed and reported by an unblinded statistician. 
9.4       Deviations from Statistical Plan 
The principal features of the study design and of the plan for statistical analysis of the data are outlined in this 
protocol. Any changes in these principal features will require a protocol amendment and will be described in the 
final report. Any such changes will be subject to review by the IRB. 

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9.5        Documenting an Unblinding 
Any unblinding will require a full written account of the event(s) that necessitated the unblinding of the treatment 
assignment for an individual participant(s). This account will be made in the participant(s) study file and in the 
final study report and will include the reason(s) for the unblinding, the name of the Independent Study Monitor 
who was notified and approved the unblinding, the names of the individuals unblinded, and the date and time the 
unblinding occurred. 
 
 
 
10  MONITORING AND QUALITY ASSURANCE 
 
10.1        SAFETY PROCEDURES 
10.1.1     Stopping Rules 
10.1.1.1   Study Stopping Rules 
Study enrollment and study procedures will be suspended pending expedited review of all pertinent data by the 
Partners institutional review board (IRB), the ISM, and the Infinant Medical Officer, if a participant at any time or 
in any group develops a severe or life-threatening adverse event such that he or she requires hospitalization, or 
an unexpected (non-allergy-related) hospitalization or death. 
 
Study enrollment will be suspended pending expedited review of all pertinent data by the ISM if any of the 
following occur: 
– Any death related to B. infantis INF108F 
– Any serious adverse reaction to B. infantis INF108F 
All above events will be reported immediately to the Partners IRB, the ISM, and Infinant’s Medical Officer. The 
study will not resume until approval is given by the IRB, the ISM, and Infinant’s Medical Officer. 
 
10.1.1.2   Individual Stopping Rules 
Safety of the participant will remain of primary importance. Any participant who develops documented 
hypotension (systolic BP>30% fall from baseline systolic BP), neurological compromise (confusion, loss of 
consciousness), and/or significant hypotension during any stage of the during any administration of the B. infantis 
product will be withdrawn from the study. 
 
10.1.2    Early Discontinuation of Investigational Product(s) / Intervention(s) with continued study participation 
/ follow-up 
10.1.2.1 Unscheduled Termination In accordance with the Declaration of Helsinki, patients have the right to 
withdraw from the study at any time for any reason. Participants may withdraw with or without medical advice. 
The investigator also has the right to withdraw participants from the study. Participants will be removed from the 

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study for the following reasons: adverse experience, intercurrent illness or medication that in the judgment of the 
investigator may place the participant at risk, request of the investigator or participant for administrative or other 
reasons, protocol violation, determination that the participant is non-compliant or has unreliable behavior. 
Withdrawal from this study will have no impact on the future care of the participant at the Massachusetts General 
Hospital or any of its affiliated hospitals or health clinics. 
10.1.3    Follow-up after early study termination 
Participants who are prematurely terminated from the study will be followed to monitor safety for a minimum of 
30 days or until resolution of the disqualifying event whichever is longer or until the Independent Study Monitor 
and the Principal Investigator determine that the follow-up is complete. 
10.1.4    Participant Replacement 
Participants who are permanently discontinued from the study will not be replaced. 
 
 
10.2     ADVERSE EVENTS 
This section defines the types of adverse events and outlines the procedures for appropriately collecting, grading, 
recording, and reporting them. Information in this section complies with ICH Guideline E2A: Clinical Safety Data 
Management: Definitions and Standards for Expedited Reporting and ICH E6: Guideline for Good Clinical Practice, 
and applies the standards set forth in the National Cancer Institute (NCI), Common Terminology Criteria for 
Adverse Events version 4.0. These criteria have been reviewed by the study investigators and have been 
determined appropriate for this study population. 
10.2.1   Definitions 
10.2.1.1    Adverse Events An adverse event (AE) is any occurrence or worsening of an undesirable or unintended 
sign, symptom, laboratory finding, or disease that is experienced during participation in the trial. An AE can 
therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or 
disease temporarily associated with the use of a medicinal (investigational) Study Agent(s) whether related to the 
medicinal (investigational) Study Agent(s) or not. Any medical condition that is present at the time that the 
participant is screened will be considered as baseline and not recorded as an AE. However, if the condition 
deteriorates or changes in severity at any time during the study it will be recorded and reported as an AE. 
10.2.1.2  Suspected Adverse Reaction and Adverse Reaction Suspected adverse reaction (SAR) means any 
adverse event for which there is a reasonable possibility that the drug caused the adverse event. For the purposes 
of safety reporting, “reasonable possibility” means there is evidence to suggest a causal relationship between the 
drug and the adverse event. 
 
An adverse reaction (AR) means any adverse event caused by a drug. Adverse reactions are a subset of all 
suspected adverse reactions for which there is reason to conclude that the drug caused the event. A suspected 

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adverse reaction implies a lesser degree of certainty about causality than adverse reaction, which means any 
adverse event caused by a drug. 
10.2.1.3   Adverse Events Associated with Study Procedures The following clinical situations, when associated 
with study procedures, are defined as adverse events and will be recorded on the AE CRF. These situations do not 
limit the principal investigator from recording and reporting any other events as AEs, associated or not with these 
procedures. 
10.2.1.4  Serious Adverse Event (SAE) An AE or SAR (including AR) is considered serious if, in the view of the 
investigator, it results in any of the following outcomes (21 CFR 312.32): 
– 
Death. A death that occurs during the study or that comes to the attention of the investigator during the 
protocol-defined follow-up. 
 
– 
A life-threatening event. A life-threatening event is any adverse experience that, in the view of the 
investigator, places the study participant at immediate risk of death from the reaction as it occurred. It 
does not include an adverse event or suspected adverse reaction that, had it occurred in a more severe 
form, might have caused death. 
 
– 
An inpatient hospitalization or prolongation of existing hospitalization. 
 
– 
Persistent or significant disability/incapacity or substantial disruption of the ability to conduct normal life 
functions. 
– 
An important medical event that may not result in death, be life threatening, or require hospitalization 
may be considered an SAE when, based on appropriate medical judgment, it may jeopardize the 
participant and may require medical or surgical intervention to prevent one of the outcomes listed above. 
 
– 
Congenital anomaly or birth defect. 
 
Regardless of the relationship of the adverse event to the study, the event will be reported per Section 8.2.2.3 as 
an SAE if it meets any of the above definitions. 
10.2.1.5   Unexpected Adverse Event An AE or SAR (including AR) is considered “unexpected” if it is not consistent 
with the risk information described in the study protocol. 
10.2.2       Collecting, Recording and Managing Adverse Events 
10.2.2.1    Identifying Adverse Events Any adverse event that occurs from the moment the participant has signed 
the consent form will be recorded and is reportable. Adverse events will be reported until the participant has 
completed the long-term follow-up phase. 
 

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Adverse events may be discovered through any of these methods: 
– 
Observing the participant. 
 
– 
Questioning the participant with standardized questions/procedures. 
 
– 
Receiving an unsolicited complaint from the participant. 
 
– 
An abnormal value or result from a clinical or laboratory evaluation (e.g., a radiograph, an ultrasound, or 
an electrocardiogram) can also indicate an adverse event. 
Adverse events will be captured as follows: (1) every occurrence as per the NCI-CTCAE criteria, (2) for protocol-
specific adverse events (Section 10.2.1.3) or (3) when determined to be clinically significant by the Principal 
Investigator. 
 
All adverse events occurring during or within 24 hours of the study procedures will be reported as an adverse 
event. 
 
10.2.2.2 Recording AEs Throughout the study, all identified adverse events (serious and non-serious) will be 
recorded on all appropriate source document and adverse event case report forms regardless of their severity or 
relation to the study. 
 
A complete description of all adverse events will include event description, time of onset, investigator assessment 
of severity, relationship to study agent(s) or procedures/intervention(s), time of resolution/stabilization of the 
event, expectedness, determination of whether the AE qualifies as a SAE, and action taken. A change in the 
severity of the AE will also be documented. The PI will document assessment of severity and relationship on the 
source documents or the on the CRF. 
10.2.2.3  Recording SAEs Serious adverse events will be recorded on the serious adverse event case report form 
and will include a narrative of the event signed and dated by the Principal Investigator and the Independent Safety 
Monitor. 
10.2.2.4  Managing Adverse Events The site investigator must apply his or her clinical judgment as to whether an 
AE is of sufficient severity to require that the participant immediately be removed from further treatment under 
the protocol. The investigator must institute any necessary medical therapy to protect a participant from any 
immediate risk. 
 
There is no risk for possible anaphylaxis with our study agent INF108F and placebo. Epinephrine auto injectors 
and diphenhydramine will not be needed. Potential gastrointestinal adverse effects might occur, including 
abdominal discomfort and changes of stool frequency and consistency. We will stop administering the study agent 
if any of adverse events occur and we will treat the symptoms accordingly. 
 
 
The same protocol will be recommended when signs/symptoms of acute gastrointestinal infection occur. An 
adverse event will be followed until any of the following takes place: a) it is resolved, b) participant is stable, c) a 
minimum of 30 days after participant is terminated from the study and the Independent Safety Monitor and the 

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Principal Investigator determine that follow-up is complete. If an abnormal value or result is determined by the 
investigator to be clinically significant, it must be recorded as an adverse event on the appropriate laboratory 
evaluation form(s). 
 
10.2.3      Grading and Attribution 
10.2.3.1   Grading criteria In addition to determining whether an adverse event fulfills criteria for a serious adverse 
event or not, the severity of adverse events experienced by study participants will be graded according to the 
criteria set forth in the National Cancer Institute’s Common Terminology Criteria for Adverse Events Version 4.0. 
This document (referred to herein as the NCI-CTCAE manual) provides a common language to describe levels of 
severity, to analyze and interpret data, and to articulate the clinical significance of all adverse events. The grading 
criteria listed below will supersede any in the NCI-CTCAE manual. 
 
All adverse events whether or not listed in the NCI-CTCAE will be graded on a scale from 1 to 5 according to the 
following standards in the NCI-CTCAE manual (A semi-colon indicates “or” within the description of the grade.): 
– 
Grade 1 = Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not 
indicated. 
 
– 
Grade 2 = Moderate; minimal, local, or noninvasive intervention indicated; limiting age-appropriate 
instrumental ADL (preparing meals, shopping for groceries or clothes, using the telephone, money, etc.). 
 
– 
Grade 3 = Severe or medically significant but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling; limiting self-care ADL (bathing, dressing and 
undressing, feeding self, using he toilet, taking medications, and not bedridden). 
 
– 
Grade 4 = Life-threatening consequences; or urgent intervention indicated. 
 
– 
Grade 5 = Death related to AE. 
Adverse events not included in the NCI-CTCAE listing or which have relative specificity for this protocol will be 
recorded and graded 1 to 5 according to the grade definition provided below: 
 
ADVERSE EVENT: CARDIAC ARRHYTHMIA (CONDUCTION DISORDER) 
Definition: A disorder characterized by pathological irregularities in the cardiac conduction system. 
Grade 1 = Mild symptoms; intervention not indicated 
Grade 2 = Moderate symptoms 
Grade 3 = Severe symptoms; intervention indicated 
Grade 4 = Life-threatening consequences; urgent intervention indicated 
Grade 5 = Death 
ADVERSE EVENT: BRONCHOSPASM 
Definition: A disorder characterized by a sudden contraction of the smooth muscles of the bronchial wall. 
Grade 1 = Mild symptoms; intervention not indicated 
Grade 2 = Symptomatic; medical intervention indicated; limiting instrumental ADL 
Grade 3 = Limiting self care ADL; oxygen saturation decreased 
Grade 4 = Life-threatening respiratory or hemodynamic compromise; intubation or urgent intervention indicated Grade 5 = Death 

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ADVERSE EVENT: COUGH 
Definition: A disorder characterized by sudden, often repetitive, spasmodic contraction of the thoracic cavity, resulting in violent release of air 
from the lungs and usually accompanied by a distinctive sound. 
Grade 1 = Mild symptoms; nonprescription intervention indicated 
Grade 2 = Moderate symptoms, medical intervention indicated; limiting instrumental ADL 
Grade 3 = Severe symptoms; limiting self care ADL 
Grade 4 = N/A 
Grade 5 = N/A 
ADVERSE EVENT: FEVER 
Definition: A disorder characterized by elevation of the body’s temperature above the upper limit of normal. 
Grade 1= 38.0 – 39.0 degrees C (100.4- 102.2 degrees F) despite use of acetaminophen 
Grade 2= >39.0 – 40.0 degrees C (102.3- 104.0 degrees F) despite use of acetaminophen Grade 3= >40.0 degrees C (>104.0degrees F) for <24 hrs 
despite use of acetaminophen 
Grade 4= >40.0 degrees C (>104.0degrees F) for >24 hrs despite use of acetaminophen Grade 5= Death 
ADVERSE EVENT: NAUSEA 
Definition: A disorder characterized by a queasy sensation and/or the urge to vomit. 
Grade 1 = Loss of appetite without alteration in eating habits 
Grade 2 = Oral intake decreased without significant weight loss, dehydration or malnutrition 
Grade 3 = Inadequate oral caloric or fluid intake, likely associated with losses due to vomiting; hospitalization may be warranted Grade 4 = N/A 
Grade 5 = N/A 
ADVERSE EVENT: ABDOMINAL PAIN 
Definition: A disorder characterized by pain or discomfort localized to the abdominal cavity. 
Grade 1 = Mild: possibly associated with loss of appetite without alteration in eating habits, or normal activity 
Grade 2 = Moderate: Oral intake and normal activity decreased without significant weight loss, dehydration or malnutrition 
Grade 3 = Severe: Inadequate oral caloric or fluid intake; debilitating pain causing significant curtailment of normal activity Grade 4 = N/A 
Grade 5 = N/A 
10.2.3.2    Definition of Attribution The attribution of an adverse event to the study will initially be determined 
by the Principal Investigator or designated physician co/sub-investigator. The Principal Investigator or designee 
will record the determination of attribution on the appropriate adverse event or serious adverse event form. The 
attribution of an adverse event to the investigational drug(s) or other study drug (s) will be determined using the 
descriptors in the following table. 
 
For the purpose of this study, in addition to all study medications, the following interventions/procedures will be 
considered when determining attribution: 
10.2.3.2.1 Products 
– B. infantis INF108F powder 
– Placebo INF108F powder 
10.2.3.2.2   Interventions 
– Double-Blind Placebo-Controlled administration of B. infantis INF108F or Placebo 
 
Code 
Descriptor 
Definition (guidelines) 

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UNRELATED CATEGORIES 
1 
Unrelated 
The adverse event is clearly not related to study. The event is completely related to an 
etiology other than the study product or study intervention (the alternative etiology 
must be documented in the study participant’s medical record). 
2 
Unlikely 
The adverse event is doubtfully related to study and likely to be related to factors other 
than study product or study intervention. 
RELATED CATEGORIES 
3 
Possible 
The adverse event may be related to study. There is an association between the event 
and the administration of study product and there is a plausible mechanism for the event 
to be related to the study product; there may be also an alternative etiology, such as 
characteristics of the participant’s clinical status and/or underlying disease. 
4 
Probable 
The adverse event is likely related to study. There is (1) an association between the event 
and the administration of study product or study intervention, (2) a plausible mechanism 
for the event to be related to the study product, and (3) the event could not be 
reasonably explained by known characteristics of the participant’s clinical status and or 
an alternative etiology is not apparent. 
5 
Definite 
The adverse event is clearly related to study. There is (1) an association between the 
event and the administration of the study product or study intervention, (2) a plausible 
mechanism for the event to be related to the related to the study product, and (3) causes 
other than the study product have been ruled out and/or the event re-appeared on re-
exposure to the study product. 
(For additional information and a printable version of the NCI-CTCAE manual, consult the NCI-CTCAE 
website: http://ctep.cancer.gov/reporting/ctc.html) 
In a clinical trial, the study product/intervention will always be suspect when attributing an AE and the 
“unrelated” attribution will be used only when there is an indisputable or likely alternative explanation for 
the AE. 
10.2.4    SAE Reporting Criteria and Procedures 
The Principal Investigator will be notified by the study staff as soon as a staff member becomes aware of the SAE. 
In the absence of the Principal Investigator, a physician sub-investigator will be notified. 
10.2.4.1  Notifying Infinant’s Medical Officer Infinant’s Medical Officer and the Independent Safety Monitor will 
be notified by the Principal Investigator no later than 24 hours after the investigative site becomes aware of the 
SAE, regardless of the presumed relationship to the study product. Reporting to Infinant’s Medical Officer will 
utilize an initial SAE case report form in draft format. Contact information for Infinant’s Medical Officer is listed 
below: 
Karl Sylvester, MD 
Infinant Health 
2121 2nd Street 
Davis, CA 95616  
Tel 650-804-0597  
ksylvester@infinanthealth.com 
 
Within another 24 hours, Infinant’s Medical Officer, Independent Safety Monitor, and the Principal Investigator 
will discuss the impact of the SAE on the participant and on the study and Infinant’s Medical Officer will decide 

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whether standard or expedited reporting will be applied. A finalized, SAE case report form will be generated by 
the Principal Investigator and must be approved by Infinant’s Medical Officer. The finalized, Infinant Health-
approved case report form will be placed in the participant study chart and will be sent to the Independent Safety 
Monitor. As additional clinical information is obtained by the Principal Investigator regarding the SAE, the SAE 
case report will be revised and submitted to Infinant’s Medical Officer and the Independent Safety Monitor. 
10.2.4.2      Unexpected, Non-Serious Adverse Events An unexpected, non-serious adverse event that is of Grade 
2 severity or higher and study-related will be recorded and reported to the Independent Safety Monitor and 
Infinant’s Medical Officer under the serious adverse event reporting procedure outlined in the SAE Reporting and 
Criteria Section (Section 8) of the protocol (i.e., within 24 hours). 
10.2.4.3     Notifying the Data and Independent Safety Monitor (ISM) The Principal Investigator is responsible for 
submitting all expedited SAEs on an ongoing basis to the Independent Safety Monitor. Individual or clusters of 
SAEs may be reported expeditiously to the ISM either when specified by the ISM, or upon determination of 
Infinant’s Medical Officer. 
 
10.2.4.4     Notifying the Institutional Review Board The Principal Investigator will ensure the timely dissemination 
of SAE information, including expedited reports, to the IRB in accordance with IRB regulations and guidelines. 
 
 10.2.5      Non-Serious Adverse Events (NSAES) Reporting 
10.2.5.1   Notifying the Independent Safety Monitor The Principal Investigator will provide the Independent 
Safety Monitor with a listing of all AEs in a Vedanta Biosciences-provided standard format and timeline for review 
during planned protocol reviews. Individual or clusters of AEs may be reported expeditiously to the ISM either 
when specified by the ISM, or upon determination of Infinant’s Medical Officer. 
10.2.5.2     Notifying the Institutional Review Board The Principal Investigator will ensure the timely dissemination 
of AE information to the IRB in accordance with applicable regulations and guidelines. 
10.3        PROTOCOL DEVIATIONS 
The practices below are consistent with Good Clinical Practice (GCP ICH E6) Sections: 
– 
Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3 
– 
Quality Assurance and Quality Control, section 5.1.1 
– 
Noncompliance sections 5.20.1, and 5.20.2 
 
10.3.1      Protocol Deviation Definitions 
10.3.1.1  Protocol Deviation – Any change, divergence, or departure from the study design or procedures of a 
research protocol that affects the participant’s rights, safety, or well-being and/or the completeness, accuracy 
and reliability of the study data constitutes a protocol violation. Changes or alterations in the conduct of the trial 
which do not have a major impact on the participant’s rights, safety or well-being, or the completeness, accuracy 

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and reliability of the study data are considered minor protocol deviations. The Principal Investigator is responsible 
for reporting protocol deviations to the IRB using the standard reporting form. As a result of deviations, corrective 
actions are to be developed by the site and implemented promptly. 
10.3.1.2     Major Protocol Deviation – A protocol violation is a deviation from the IRB approved protocol that may 
affect the participant’s rights, safety, or well-being and/or the completeness, accuracy, and reliability of the study 
data. 
 
If the deviation meets any of the following criteria, it is considered a major protocol deviation (protocol violation). 
Example list is not exhaustive. 
1. The deviation has harmed or posed a significant or substantive risk of harm to the research 
participant. 
Examples: 
– A research participant received the wrong treatment or incorrect dose. 
– A research participant met withdrawal criteria during the study but was not withdrawn. 
2. The deviation compromises the scientific integrity of the data collected for the study. Examples: 
– A research participant was enrolled but does not meet the protocol’s eligibility criteria. 
– Failure to treat research participants per protocol procedures that specifically relate to primary 
efficacy outcomes (if it involves patient safety it meets the first category above) – Changing the 
protocol without prior IRB approval. 
– Inadvertent loss of samples or data. 
3. The deviation is a willful or knowing breach of human participant protection regulations, policies, or 
procedures on the part of the investigator(s). Examples: 
– Failure to obtain informed consent prior to initiation of study-related Procedures 
– Use of outdated or incorrect consent forms 
– Falsifying research or medical records 
– Performing tests or procedures beyond the individual’s professional scope or privilege status 
(credentialing) 
4. The deviation involves a serious or continuing noncompliance with federal, state, local or institutional 
human participant protection regulations, policies, or procedures. Examples: 
– Working under an expired professional license or certification 
– Failure to follow federal and/or local regulations, and intramural research – Repeated minor 
deviations. 
5. The deviation is inconsistent with the NIH Human Research Protection Program’s research, medical, 
and ethical principles. Examples: 

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– A breach of confidentiality. 
– Inadequate or improper informed consent procedure. 
10.3.1.3      Non-Major Protocol Deviation A non-major protocol deviation is any change, divergence, or departure 
from the study design or procedures of a research protocol that has not been approved by the IRB and which 
DOES NOT have a major impact on the participant’s rights, safety or well-being, or the completeness, accuracy, 
and reliability of the study data. 
10.3.2        Reporting Protocol Deviations 
Upon determination that a protocol deviation has occurred, the study staff will a) notify the Principal Investigator, 
b) notify the Infinant Health Project Manager (refer to investigator’s signature page for contact information) and 
c) will complete the Protocol Deviation form. Infinant Health may request discussion with the Principal Investigator 
and the Independent Safety Monitor to determine the effect of the protocol deviation on the study participant 
and his/her further study participation, the effect of the protocol deviation on the overall study and corrective 
actions. The Principal Investigator will complete and sign the Protocol Deviation form and submit it to Infinant’s 
Medical Officer and Project Manager, to the Independent Safety Monitor and to the site IRB, per IRB regulations. 
Major protocol deviations will be reported to the ISM and Infinant’s Medical Officer.  
 
All study staff be educated about the adverse event reporting policy and will be instructed to notify an investigator 
if an event occurs. 
10.4   QUALITY CONTROL AND QUALITY ASSURANCE 
The Principal Investigator will keep accurate records to ensure that the conduct of the study is fully documented. 
The investigator will ensure that all CRFs and participant study files are legible and complete for every participant. 
The Principal Investigator, through the use of a Clinical Research Associate (CRA), will be responsible for the 
regular review of the conduct of the trial, for verifying adherence to the protocol, and for confirming the 
completeness, consistency, and accuracy of all documented data and accuracy of source documentation 
verification. The reports of the CRA will be submitted to the Principal Investigator and the Infinant Health Project 
Manager. Infinant Health will independently review these reports. 
When the CRFs are complete, they will be reviewed and signed by the Principal Investigator. All discrepancies 
identified will be reviewed, and any resulting queries will be resolved with the Principal Investigator and the CRFs 
will be amended as needed. 
 

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10.5       ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL PRACTICE 
10.5.1   Statement of Compliance 
This study was designed to ensure the protection of participants according to the ethical principles of the 
Declaration of Helsinki and amendments concerning medical research in human participants. This clinical study 
will be conducted using current good clinical practice (cGCP), as delineated in Guidance for Industry: E6 Good 
Clinical Practice Consolidated Guidance 1, and according to the criteria specified in this study protocol. Before 
study initiation, the protocol and the informed consent documents will be reviewed and approved by the local 
IRB. Any amendments to the protocol or to the consent materials will also be approved by the local IRB prior to 
implementation. 
10.6   Informed Consent 
The informed consent form will provide information about the study to a prospective participant or participant’s 
legal representative to allow for an informed decision about participation in the study. All participants (or their 
legally acceptable representative) must read, sign (electronically or with wet ink), and date a consent form prior 
to study participation. The electronic consent process will be FDA 21 CFR Part 11 compliant.  
 
The informed consent form will be revised and receive IRB approval whenever important new safety information 
is available, whenever the protocol is amended, and/or whenever any new information becomes available that 
may affect participation in the trial. 
 
A copy of the appropriate informed consent form will be given to a prospective participant for review. The 
Principal Investigator or an approved designee, will discuss the consent with the prospective participant and 
answer questions, either in person or via institution-approved video conferencing platform. The prospective 
participant will be told that being in the trial is voluntary and that he or she may withdraw from the study at any 
time, for any reason. 
 
 
 
 
11.  Privacy and Confidentiality 
 
 
A participant’s privacy and confidentiality will be respected throughout the study. Each participant will be assigned 
a sequential identification number and these numbers rather than names will be used during collection, storage, 
and reporting of participant information. 
☒ Study procedures will be conducted in a private setting 
☒ Only data and/or specimens necessary for the conduct of the study will be collected 
☒ Data collected (paper and/or electronic) will be maintained in a secure location with appropriate 
protections such as password protection, encryption, physical security measures (locked files/areas) 

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☒ Specimens collected will be maintained in a secure location with appropriate protections (e.g. locked 
storage spaces, laboratory areas) 
☒ Data and specimens will only be shared with individuals who are members of the IRB-approved research 
team or approved for sharing as described in this IRB protocol 
☒  Data and/or specimens requiring transportation from one location or electronic space to another will be 
transported only in a secure manner (e.g. encrypted files, password protection, using chain-of-custody 
procedures, etc.) 
☒ All electronic communication with participants will comply with Mass General Brigham secure 
communication policies 
☒ Identifiers will be coded or removed as soon as feasible and access to files linking identifiers with coded 
data or specimens will be limited to the minimal necessary members of the research team required to 
conduct the research 
☒ All staff are trained on and will follow the Mass General Brigham policies and procedures for maintaining 
appropriate confidentiality of research data and specimens 
☒ The PI will ensure that all staff implement and follow any Research Information Service Office (RISO) 
requirements for this research 
☒ Additional privacy and/or confidentiality protections 
 
 
 
 
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APPENDIX A 
 
Data Monitoring Committee / Data and Safety Monitoring Board 
Appendix 
 
There is no separate Data Monitoring Committee (DMC) or Data and Safety Monitoring Board (DSMB) 
for this study. We have specific procedures to ensure the quality and monitor safety for the study as 
described in Section 10.  
 
 
 
• 
To be completed for studies monitored by Data Monitoring Committee (DMC) or Data and Safety Monitoring 
Board (DSMB) if a full DMC/DSMB charter is not available at the time of initial IRB review. 
• 
DMC/DSMB Charter and/or Roster can be submitted to the IRB later via Amendment, though these are not 
required.  
 
A Data Monitoring Committee (DMC) or Data and Safety Monitoring Board (DSMB) will be convened for safety 
monitoring of this research study.  The following characteristics describe the DMC/DSMB convened for this study 
(Check all that apply): 
 
☐ The DMC/DSMB is independent from the study team and study sponsor. 
 
☐ A process has been implemented to ensure absence of conflicts of interest by DMC/DSMB members. 
 
☐ The DMC/DSMB has the authority to intervene on study progress in the event of safety concerns, e.g., to 
suspend or terminate a study if new safety concerns have been identified or need to be investigated.   
 
☐ Describe number and types of (i.e., qualifications of) members: 
Click or tap here to enter text. 
 
☐  Describe planned frequency of meetings: 
Click or tap here to enter text. 
 

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☐ DMC/DSMB reports with no findings (i.e., “continue without modifications”) will be submitted to the IRB 
at the time of Continuing Review. 
 
☐ DMC/DSMB reports with findings/modifications required will be submitted promptly (within 5 business 
days/7 calendar days of becoming aware) to the IRB as an Other Event.