A Phase 3, Randomized, Double-blind, Placebo-controlled Study to Evaluate the Effect of Bi-26 (Strain of Bifidobacterium Longum, B. Infantis) Supplementation Versus Placebo on Weight Gain in Underweight Infants
Phase: PHASE3
Conditions: Underweight, Paediatrics
Sponsor: Gates Medical Research Institute

 
 
 
 
 
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Title Page 
Protocol title: 
A Phase 3, randomized, double-blind, placebo-controlled study to evaluate the effect of Bi-26 (strain of 
Bifidobacterium longum, B. infantis) supplementation versus placebo on weight gain in underweight 
infants 
Protocol Number: Gates MRI-MNK01-301 
Version: 4.0 
Compound: Bifidobacterium longum subspecies infantis (B. infantis) strain Bi-26TM, product 
code MNK01 
Study Phase: Phase 3 
Short Title: Impact of Bi-26 supplementation on weight gain in underweight infants 
Sponsor Name: Bill & Melinda Gates Medical Research Institute 
Legal Registered Address: 
One Kendall Square 
Building 600, Suite 6-301 
Cambridge, MA USA 02139 
 
 
Medical Monitor and other important study personnel information will be provided in a separate 
document. 
 
Approval Date: 12 Sept 2022 
 
 

 
 
 
 
 
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Sponsor Signature Page 
See appended electronic signature page 
___________________________________  
____________________ 
Sponsor Signatory 
 
 
 
 
Date 
, M.D. 
Clinical Development Leader 
Bill & Melinda Gates Medical Research Institute 
___________________________________  
____________________ 
Biostatistician  
 
 
 
 
Date 
, DrPH 
Head of Biometrics & Data Management 
Bill & Melinda Gates Medical Research Institute 
___________________________________  
____________________ 
, M.D.  
 
 
 
Date 
Chief Medical Officer 
Bill & Melinda Gates Medical Research Institute 
 

 
 
 
 
 
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Principal Investigator Signature Page 
I have read the protocol, appendices, and accessory materials related to Gates MRI-MNK01-301 
entitled “A Phase 3, randomized, double-blind, placebo-controlled study to evaluate the effect of 
Bi-26 (strain of Bifidobacterium longum, B. infantis) supplementation versus placebo on weight 
gain in underweight infants”, and agree to the following: 
• To conduct this study as described by the protocol and any accessory materials 
• To protect the rights, safety, and welfare of the participants under my care 
• To provide oversight to all personnel to whom study activities have been delegated 
• To control all investigational products provided by the sponsor and maintain records of 
the disposition of those products 
• To conduct the study in accordance with: 
o consensus ethical principles derived from international guidelines including the 
Declaration of Helsinki and Council for International Organizations of Medical 
Sciences (CIOMS) International Ethical Guidelines 
o current International Council for Harmonization Guideline for Good Clinical 
Practice (GCP)  
o applicable laws and regulations 
• To obtain approval for the protocol and all written materials provided to participants prior 
to initiating the study at my site 
• To obtain informed consent – and updated consent in the event of new information or 
amendments – from all participants enrolled at my study site prior to initiating any 
study-specific procedures or administering investigational products to those participants 
• To maintain study records of each participant and all data required by the protocol. 
___________________________________  
____________________ 
Principal Investigator Signatory 
 
 
 
Date 
Name: 
Title: 
Affiliation: 
City and Country: 
 

 
 
 
 
 
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Protocol Version History and Summary of Changes 
DOCUMENT HISTORY 
Document 
Date 
Version 4.0 
12 Sept 2022 
Version 3.0 
15 Aug 2022 
Version 2.0  
27 June 2022 
Original Protocol Version 1.0  
28 April 2022 
Overall Rationale for changes in Version 4.0: 
The purpose of this substantial protocol amendment was to add an exclusion criterion as 
described in the table below. 
Section # and 
Name 
Description of Change 
Brief Rationale 
Section 5.2 
Exclusion Criteria 
Added new criterion (8. Ongoing infant 
antibiotic (e.g., as prophylaxis in sickle cell 
disease) and/or probiotic usage), and updated 
subsequent criteria numbering 
 
Included to prevent 
confounding of study results. 

 
 
 
 
 
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Table of Contents 
1. 
Protocol Summary ..........................................................................................................9 
1.1. 
Synopsis ............................................................................................................................9 
2. 
Introduction ...................................................................................................................16 
2.1. 
Background and Study Rationale ...................................................................................17 
2.2. 
Benefit/Risk Assessment ................................................................................................18 
3. 
Objectives and Endpoints ............................................................................................19 
4. 
Study Design ..................................................................................................................19 
4.1. 
Overall Design ................................................................................................................19 
4.2. 
Scientific Rationale for Study Design ............................................................................20 
4.3. 
Justification for Dose ......................................................................................................21 
4.4. 
End of Study Definition ..................................................................................................21 
4.5. 
Optional Sub-study .........................................................................................................21 
5. 
Study Population and Eligibility Criteria ...................................................................22 
5.1. 
Inclusion Criteria ............................................................................................................22 
5.2. 
Exclusion Criteria ...........................................................................................................23 
5.3. 
Screen Failures ................................................................................................................23 
6. 
Study Intervention ........................................................................................................24 
6.1. 
Handling, Storage, and Accountability ...........................................................................25 
6.2. 
Measures to Minimize Bias: Randomization and Blinding ............................................26 
6.3. 
Study Intervention Compliance ......................................................................................27 
6.4. 
Feeding History...............................................................................................................27 
6.5. 
Concomitant Therapy .....................................................................................................27 
6.6. 
Dose Modification ..........................................................................................................28 
7. 
Discontinuation of Study Intervention and Participant 
Discontinuation/Withdrawal........................................................................................28 
7.1. 
Discontinuation of Study Intervention ............................................................................28 
7.2. 
Participant Discontinuation/Withdrawal from the Study ................................................28 
7.3. 
Replacement of Participants ...........................................................................................29 
7.4. 
Lost to Follow-up............................................................................................................29 
8. 
Study Assessments and Procedures.............................................................................29 
8.1. 
Demographic and Baseline Characteristics ....................................................................30 
8.2. 
Efficacy Assessments .....................................................................................................30 
8.3. 
Safety Assessments .........................................................................................................31 
8.3.1. 
Medical History and Full Physical Examination ............................................................31 
8.3.2. 
Interim History and Focused Physical Examination .......................................................31 
8.3.3. 
Vital Signs .......................................................................................................................31 
8.4. 
Adverse Events, Serious Adverse Events .......................................................................32 
8.4.1. 
Time Period and Frequency for Collecting AE and SAE 
Information .....................................................................................................................32 
8.4.2. 
Method of Detecting AEs and SAEs ..............................................................................32 
8.4.3. 
Follow-up of AEs and SAEs ...........................................................................................33 

 
 
 
 
 
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8.4.4. 
Regulatory Reporting Requirements for SAEs ...............................................................33 
8.4.5. 
Independent Data Monitoring Committee ......................................................................34 
8.5. 
Genetics ..........................................................................................................................34 
8.6. 
Biomarkers ......................................................................................................................34 
9. 
Statistical Considerations .............................................................................................35 
9.1. 
General ............................................................................................................................35 
9.2. 
Subject Disposition, Demographics and Baseline Data, 
Concomitant Medication, and Compliance ....................................................................36 
9.2.1. 
Subject Disposition .........................................................................................................36 
9.2.2. 
Demographic and Baseline Data .....................................................................................36 
9.2.3. 
Concomitant Medication .................................................................................................36 
9.2.4. 
Compliance .....................................................................................................................36 
9.3. 
Randomization ................................................................................................................36 
9.4. 
Primary Statistical Hypotheses and Sample Size Determination ...................................36 
9.5. 
Populations for Analyses ................................................................................................37 
9.6. 
Statistical Analyses .........................................................................................................38 
9.7. 
Primary and Secondary Estimands .................................................................................38 
9.7.1. 
Other Secondary Analyses ..............................................................................................40 
9.7.2. 
Exploratory Analyses ......................................................................................................40 
10. 
Supporting Documentation and Operational Considerations ..................................41 
10.1. 
Regulatory, Ethical, and Study Oversight Considerations .............................................41 
10.1.1. Regulatory and Ethical Considerations ...........................................................................41 
10.1.2. Financial Disclosure........................................................................................................41 
10.1.3. Informed Consent Process ..............................................................................................42 
10.1.4. Data Protection................................................................................................................43 
10.1.5. Dissemination of Clinical Study Data.............................................................................43 
10.1.6. Data Quality Assurance ..................................................................................................43 
10.1.7. Record Retention ............................................................................................................44 
10.1.8. Source Documents ..........................................................................................................44 
10.1.9. Study and Site Closure ....................................................................................................44 
10.1.10. Publication Policy ...........................................................................................................45 
10.1.11. Financing and Insurance .................................................................................................45 
10.2. 
Adverse Events and Serious Adverse Events: Definitions and 
Procedures for Recording, Evaluating, Follow-up, and Reporting.................................45 
10.2.1. Definition of AE .............................................................................................................45 
10.2.2. Definition of SAE ...........................................................................................................46 
10.2.3. Recording and Following Up SAEs ................................................................................47 
10.2.4. Reporting SAEs ..............................................................................................................50 
11. 
References ......................................................................................................................51 
12. 
Protocol Amendments ..................................................................................................54 
 

 
 
 
 
 
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LIST OF TABLES 
Table 1: Objectives and Endpoints ............................................................................................... 12 
Table 2: Schedule of Activities (SoA) .......................................................................................... 14 
Table 3: Study Intervention .......................................................................................................... 25 
Table 4: Primary Estimands .......................................................................................................... 38 
Table 5: Secondary Estimands ...................................................................................................... 39 
 
 
LIST OF FIGURES 
Figure 1: Main Study Schema....................................................................................................... 11 
Figure 2: Sample size sensitivity .................................................................................................. 37 

 
 
 
 
 
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List of Abbreviations 
AE 
 
Adverse event 
CFU 
 
Colony forming units 
CRF 
 
Case Report Form, Electronic Case Report Form 
CRO 
 
Contract research organization 
EDC 
 
Electronic data capture 
EoS 
 
End of study 
GCP 
 
Good Clinical Practice 
GRAS  
Generally Recognized as Safe 
HMO  
Human milk oligosaccharides 
ICF 
 
Informed consent form 
ICH 
International Council for Harmonization of Technical Requirements for Pharmaceuticals 
for Human Use 
IDMC  
Independent Data Monitoring Committee 
IRB 
 
Institutional Review Board (ethics committee) 
ITT 
 
Intention-to-treat 
IWRS  
Interactive Web Response System 
LMIC  
Low- and middle-income countries 
mITT   
modified intention-to-treat 
MUAC  
Mid-upper arm circumference 
NEC 
 
Necrotizing enterocolitis 
PP 
 
Per-protocol 
SAE 
 
Serious adverse event 
SAM 
 
Severe acute malnutrition 
SAP 
 
Statistical analysis plan 
SoA 
 
Schedule of activities 
US 
 
United States 
WAZ  
Weight-for-age Z score 
WHO  
World Health Organization 
WLZ 
 
Weight-for-length Z score 

 
 
 
 
 
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1. 
Protocol Summary 
1.1. 
Synopsis 
Protocol Title: A Phase 3, randomized, double-blind, placebo-controlled study to evaluate the 
effect of Bi-26 (strain of Bifidobacterium longum, B. infantis) supplementation versus placebo on 
weight gain in underweight infants 
Short Title: Impact of Bi-26 supplementation on weight gain in underweight infants 
Rationale: Bi-26 is a nutritional probiotic intervention that is being evaluated for its impact on 
weight gain and other health outcomes in underweight infants who have been hospitalized with 
acute illness. 
The burden of disease experienced by severely malnourished infants is significant in low-income 
countries. Gut dysbiosis, an imbalance in microbial composition, is thought to play a role in 
nutrient malabsorption leading to underweight infants and failure to thrive. B. infantis is thought 
to play an important role in the breakdown and adsorption of human milk oligosaccharides 
(HMOs) [Duar 2020; Casaburi 2021]. 
Supplementation with B. infantis strain EVC001 has been shown to colonize the gut and is 
associated with significant changes to fecal microbiome composition in healthy breast-fed 
infants [Frese 2017; Henrick 2019]. Supplementation of B. infantis has also been associated with 
a reduction in pro-inflammatory cytokines [Henrick 2019]. In a pilot trial evaluating B. infantis 
supplementation with strain EVC001 in hospitalized 2- to 6-month-old infants with severe acute 
malnutrition (SAM) in Bangladesh, daily supplementation of B. infantis for 4 weeks was 
associated with B. infantis colonization and an increase in weight-for-age Z score (WAZ) 
[Barratt 2022]. 
If confirmed in a larger clinical trial, B. infantis strains, such as Bi-26 used in this study, could 
potentially be used as a dietary supplement in underweight infants in low-income countries. 
Overall Design 
Disclosure statement: This is a Phase 3, randomized, double-blind, placebo-controlled study to 
demonstrate increased weight gain with Bi-26 supplementation versus placebo in underweight 
infants (WAZ<-2) discharged from hospital following acute illness and to evaluate the safety of 
Bi-26 supplementation in this population. 
Underweight infants (WAZ<-2), who are between 1 and 4 months of age at enrollment and are 
currently hospitalized for acute non-surgical illness, will be enrolled to receive daily 
supplementation of Bi-26 or placebo for 28 days with a total study duration of approximately 90 
days. 

 
 
 
 
 
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Intervention: 
The Bi-26 supplement is presented as a lyophilized powder. A single dose of supplement will be 
resuspended and administered to the participant each day for 28 days. Once a day, the mother 
mixes the powder with approximately 3 mL to 5 mL of breastmilk and administers to the infant 
orally using a feeding syringe. In keeping with current World Health Organization (WHO) 
recommendation that children are exclusively breast-fed for the first 6 months of life [WHO 
2022], breastmilk is preferred for mixing the supplement. If the mother is unable to express 
breastmilk, the powder may be mixed in approximately 3 mL to 5 mL of water. 
While a total of 7 doses of Bi-26 supplement are to be administered per week, one each day, 9 
doses will be provided each week to allow for an additional 2 doses, if needed, for repeat dose 
administration in the event of vomiting, or unexpected events which may render a dose unusable 
(e.g., spillage or otherwise compromised). Any additional dose/s not administered will be 
collected by study staff at the following visit. 
Doses will either 1) be delivered to the mother by study staff, or 2) stored by staff, at the local 
health center to be picked up from the health center. 
Study activities are assigned to the mother of the infant participant because the preferred method 
of reconstitution of the study intervention is in breastmilk. However, other caretakers may 
perform certain study activities (e.g., picking up the study doses, assisting in completion of the 
feeding diary, etc.).  
Two treatment groups, shown below, will be enrolled in parallel. 
Intervention 
Duration of Study Intervention  Number of Participants Randomized 
Bi-26 
28 days 
198 
Placebo 
28 days 
198 
Number of Participants: A sufficient number of participants will be screened to randomize 
approximately 396 study participants. 
Screening (within a week before anticipated hospital discharge and 
enrollment/randomization): Informed consent will be obtained, and screening activities will 
take place during the infant’s hospitalization. If a participant is discharged to a nutrition 
rehabilitation unit, this would still be considered a hospital discharge. 
Enrollment and Randomization (day of hospital discharge): After the infant participant has 
completed the acute stabilization phase of treatment, including fluid rehydration and antibiotic 
course as needed, eligibility criteria will be checked and verified on study Day 1, the day of 
hospital discharge. Eligible participants will be enrolled and randomized at the time of hospital 
discharge. 
Note: Screening may be conducted on the same day as enrollment/randomization (Day 1). Study 
assessments should not be duplicated if screening and Day 1 occur on the same day.  

 
 
 
 
 
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To minimize differences between groups, randomization will be stratified by: 
• country 
• age at enrollment (30-59 days of age, 60-120 days of age) 
• discharge destination (home, nutritional rehabilitation unit) 
• WAZ (≤ -3, > -3) 
o Target randomization is approximately 2/3 of participants with WAZ between -2 
and -3 and approximately 1/3 with WAZ ≤ -3. 
Blinding: This is a double-blind study. The placebo doses will be prepared to be as similar as 
possible in appearance and odor to Bi-26. The final packaging material and the final target fill 
weight for Bi-26 and placebo will be identical. 
Total duration of study participation: The study duration for each participant is approximately 
3 months. 
Study sites: The study will be conducted at 4 or more sites in 2 or more low-income countries. 
Infant Feeding: Breast-feeding counseling as recommended by the WHO will be conducted by 
study staff. 
Independent Data Monitoring Committee: An IDMC will be used in this study for safety 
review. Methods for unblinding of IDMC members and support staff will be outlined in an 
IDMC charter. 
Figure 1: Main Study Schema 
Screening within a week before, up to and including the day of discharge from hospital
Hospitalized underweight infants (WAZ<-2)
1-4 months of age at enrollment
Enrollment and Randomization on day of discharge (Day 1,Visit 1)
2 groups:  4 weeks of Bi-26 vs placebo
Clinic
Visit 1 
•Anthropometric measurements and bilateral pedal edema
•Focused physical exam (PE) and interim medical history
•Vital signs, length, and weight
•Collect stool samples Visits 1, 2, 3, and 4
•Collect serum samples Visits 1 and 2 
•Collect dose usage and storage information through Visit 2
•*Collect AEs, SAEs, medications
•*Collect feeding information
Clinic 
Visit 2 
Day 28
Clinic 
Visit 3
Day 56
Clinic
Visit 4
Day 90
•Anthropometric measurements 
•Focused PE and interim medical history
•Bilateral pedal edema
•Vital signs, length, and weight
•Collect stool sample(engraftment)
•Collect AEs, SAEs, medications
•Collect feeding information
Home visits 
Weeks 5,6,& 7
Data collection*
Home visits 
Weeks 1,2,& 3 
Dose delivery 
Doses 
administered to 
infant 
Data collection*
 
Note that screening may occur on the same day as enrollment and randomization (Day 1) 
An optional sub-study will be initiated at Day 35 to monitor refrigerator temperatures (summarized below). 

 
 
 
 
 
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Optional Sub-study 
Approximately 20% of the study population enrolled at each site will also be enrolled into a 
prospective, observational sub-study to gather information on the reliability of refrigeration in 
the participants’ homes over a 2-week period. The monitoring device will be placed in the 
refrigerator after the study intervention period has completed. 
At the Day 35 home visit, a box with a temperature data logger will be placed in the refrigerator 
used by the sub-study participant’s parent/legal guardian, and collected from the refrigerator at 
the Day 49 home visit.  
Table 1: Objectives and Endpoints 
Objectives 
Endpoints 
Primary 
To evaluate the change in weight 
(standardized for age) of infants receiving 
Bi-26 
WAZ change from baseline (Day 1) to the Day 56 visit in 
Bi-26 group  
Key Secondary 
To evaluate the change in weight of infants 
receiving Bi-26 
Change in weight (in grams) from baseline (Day 1) to the 
Day 56 visit in the Bi-26 group 
Other Secondary 
To estimate the treatment response over 
time associated with Bi-26 
WAZ change from baseline (Day 1) over time through 
the Day 90 visit (longitudinal assessment) by duration of 
dosing 
To assess the proportion of infants who 
achieve a specified change in WAZ from 
baseline to Day 56 
Proportion of infants with a ≥ 0.3, ≥ 0.4, and ≥ 0.5 
change in WAZ from baseline (Day 1) to Day 56 visit 
To assess the proportion of infants who 
achieve a specified WAZ at Day 56 
Proportion of infants who achieve a WAZ > -2 at Day 56 
visit 
To assess the re-hospitalization rate  
Number of re-hospitalizations for acute non-surgical 
illness through the Day 56 visit 
To assess the safety of Bi-26 
supplementation through End of Study 
(EoS, Day 90 visit) 
Number of adverse events (AEs) and serious adverse 
events (SAEs) through Day 90 
To measure engraftment of B. infantis in 
participants  
Presence of B. infantis in stool on Days 1, 28, 56, and 90  

 
 
 
 
 
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Objectives 
Endpoints 
Exploratory 
To describe stool biomarkers in response 
to Bi-26 supplementation in terms of stool 
composition (e.g., microbiome, pH) 
Stool biomarkers on Days 1, 28, and 56 
To describe the blood biomarkers (e.g., 
inflammation, metabolomics), in response 
to Bi-26 supplementation 
Blood biomarkers on Days 1 and 28 
 

 
 
 
 
 
 
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Table 2: Schedule of Activities (SoA) 
Activities 
Screen Clinic 
Visit 1 
Home 
Visits 
Clinic 
Visit 2 
Home 
Visit  
Home 
Visit 
Home 
Visit 
Clinic 
Visit 3 
Clinic 
Visit 4 
Discon 
Visitb 
Visit window Days (D) 
≤7 days 
before 
Day 1 
Day 1a 
 
Wk 1
 D 7  
Wk 2 
D 14 
Wk 3 
D 21  
M 1 
D 28 
Wk 5 
D 35 
Wk 6 
D 42 
Wk 7 
D 49 
D 56 
M 2 
D 90 
M 3 
 
 
±0 
±1 
±1 
±1 
±1 
±2 
±2  
±2 
±2 
±2 
 
 Informed consent (while in hospital) c 
X 
 
 
 
 
 
 
 
 
 
 
 
Check inclusion and exclusion criteria /verify eligibility d 
X 
X 
 
 
 
 
 
 
 
 
 
 
Demographic characteristics 
X 
 
 
 
 
 
 
 
 
 
 
 
Obtain estimation of socioeconomic status after enrollment 
 
X 
 
 
 
 
 
 
 
 
 
 
Record medications for infant 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Record antibiotics/probiotics taken by mother  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Anthropometric measurements e, bilateral pedal edema 
X 
X 
 
 
 
X 
 
 
 
X 
X 
X 
Full medical history f, full PE f, VS, and L and W 
X 
 
 
 
 
 
 
 
 
 
 
 
Interim history g, focused PE g as necessary, VS, L and W 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
Randomization (at hospital discharge)  
 
X 
 
 
 
 
 
 
 
 
 
 
Collect engraftment stool sample  
 
X 
 
 
 
X 
 
 
 
X 
X 
 
Collect exploratory stool samples h 
 
X 
 
 
 
X 
 
 
 
X 
 
 
Collect serum samples h 
 
X 
 
 
 
X 
 
 
 
 
 
 
Provide a week’s worth of doses for daily administration i 
 
X 
X 
X 
X 
 
 
 
 
 
 
 
Bi-26/placebo for daily administration j 
 
X 
X 
X 
X 
X 
 
 
 
 
 
 
Record all AEs and SAEs  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Provide feeding and dosing diary/review contents with 
parent(s)/legal guardian  
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Record number of doses used from diary  
 
 
X 
X 
X 
X 
 
 
 
 
 
X 
Record feeding history (number of feedings that are from 
breastmilk, and from other source/s) from diary k  
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Record dose storage information  
 
 
X 
X 
X 
X 
 
 
 
 
 
X 

 
 
 
 
 
 
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Activities 
Screen Clinic 
Visit 1 
Home 
Visits 
Clinic 
Visit 2 
Home 
Visit  
Home 
Visit 
Home 
Visit 
Clinic 
Visit 3 
Clinic 
Visit 4 
Discon 
Visitb 
Visit window Days (D) 
≤7 days 
before 
Day 1 
Day 1a 
 
Wk 1
 D 7  
Wk 2 
D 14 
Wk 3 
D 21  
M 1 
D 28 
Wk 5 
D 35 
Wk 6 
D 42 
Wk 7 
D 49 
D 56 
M 2 
D 90 
M 3 
 
 
±0 
±1 
±1 
±1 
±1 
±2 
±2  
±2 
±2 
±2 
 
Sub-study activities l: 
• 
Place box with temperature data logger in the home 
refrigerator  
 
 
 
 
 
 
X 
 
 
 
 
 
• 
Collect box with temperature data logger from the home 
refrigerator 
 
 
 
 
 
 
 
 
X 
 
 
 
D= day; Wk= week, M=month; AE= adverse event; L= infant’s length; PE = physical examination; VS= vital signs; W= infant’s weight 
a All study assessments related to determination of the participant’s health (medical history, PE, VS. L and W, anthropometric measures, bilateral 
pedal edema) and collection of all study samples should occur prior to administration of the study intervention.  
b Discon visit =A discontinuation visit will be scheduled, whenever possible, for participants who discontinue or withdraw from the study. 
c Informed consent and the screening visit may occur the same day as enrollment and randomization (Day 1). Study assessments should not be 
duplicated for visits completed on the same day. Participants who are out of window due to a change in expected discharge date, may be 
rescreened, if applicable. 
d Enrollment will occur on Day 1 after all eligibility criteria have been verified. Record whether discharged to home or nutritional rehabilitation 
unit at Visit 1. 
e Anthropometric measurements: WAZ, weight-for-length z score (WLZ), mid-upper arm circumference (MUAC), head circumference, and 
skinfold thickness 
f Refer to Section 8.3.1for full medical history and full PE 
g Refer to Section 8.3.2 for interim medical history and focused PE 
h Samples to measure exploratory biomarkers will be collected. 
i Doses to be delivered within 2 days before planned use to ensure no break/no delay in dosing regimen.  
j Monitor infants for 30 minutes after dosing, and a repeat dose will be administered if infant vomits within this observation period. If the daily 
dose is administered at the clinic during a visit, steps should be taken to ensure that blinding is maintained by not having any study personnel 
present during reconstitution and dose administration.  
k The dosing information described in Section 6.4 (Feeding history) should be recorded in the diary from Day 1 through Day 28.  
l Participants’ parent/legal guardian will be asked if they agree to participate in this sub-study and consent obtained at the enrollment visit. 
Approximately 20% of the participants at each site will be enrolled into the sub-study. Boxes with a temperature data logger will be placed in the 
home refrigerators at the participants’ homes and collected after 2 weeks. The study staff will return the box to the clinic so the information from 
the temperature data logger can be uploaded.

 
 
 
 
 
 
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2. 
Introduction 
Infant malnutrition, including undernutrition, can lead to impaired growth, immune and 
metabolic dysfunction, increased risk of infection, altered development of the central nervous 
system, and other abnormalities [Black 2008; Black 2013; Kerac 2015; Munirul Islam 2019]. 
Globally in 2020, 85 million children under 5 years of age were considered underweight (WAZ 
<-2 SD from median for WHO Child Growth Standards), the majority of whom lived in Asia and 
Africa [WHOa 2021]. As of 2020, the prevalence of underweight children under 5 years of age 
approached 40% in some low- and middle-income countries (LMICs) [WHOa 2021]. 
The burden of disease experienced by underweight children is significant, particularly in 
resource-poor countries. Undernutrition accounts for a global mortality of more than 3 million 
children every year [World Hunger Organization 2022] and around 45% of deaths among 
children under 5 years of age, most commonly in LMICs [WHOb 2021]. Underweight infants 
hospitalized with an acute illness are particularly at high risk of death. A meta-analysis from 23 
studies of over 33,000 children under 5 years of age hospitalized with pneumonia found that 
moderately underweight and severely underweight children (defined in this study as WAZ 
between -2 and -3 and WAZ less than -3) were 2 and 4.6 times more likely to die, respectively, 
compared with children of normal weight [Kirolos 2021]. This analysis further estimated that 
over half of in-hospital child pneumonia deaths in LMICs were attributable to being 
underweight, with higher deaths in those severely underweight (median 40.9%, range 14.7 to 
69.9) compared to those moderately underweight (median 18.3%, range 10.8 to 34.6). 
Several studies have reported that in severely underweight children <5 years of age the inpatient 
and post-hospital discharge mortality rates are similarly high. A study in Bangladesh in children 
who also had pneumonia demonstrated an in-hospital mortality of 8.6% and mortality of 8.7% 
within 3 months of discharge [Chisti 2014]. Mortality in severely underweight children 
hospitalized in Zambia and Zimbabwe for treatment of complicated acute malnutrition was 
nearly 10% in the year following discharge [Bwakura-Dangarembizi 2021]. 
Mortality is also high in the subset of underweight children less than 6 months of age. In a study 
of underweight Kenyan infants less than 6 months of age, in line with what was observed in 
studies in older children, in-hospital and post-discharge mortality within 1 year were similar 
(4.9% and 5.3%, respectively) [Mwangome 2017], indicating that these infants remain 
vulnerable even after acute inpatient management has completed. In the Kenyan study, WAZ 
was identified as a better predictor of inpatient and post-discharge mortality than some other 
anthropometric indices traditionally used to assess nutritional status. These findings were 
reinforced by subsequent studies [Hoehn 2021;Chowdhury 2021] and support that WAZ should 
be used for early diagnosis of underweight infants and to inform patient management decisions. 
Alterations in the gut microbiome have recently been strongly implicated in early childhood 
malnutrition and risk for intestinal inflammation [Kane 2015]. A healthy infant gut microbiome, 
dominated by Lactobacillus and Bifidobacterium, may contribute to metabolic functions that are 
essential to infant growth and development, such as development of the immune system, host 
metabolism and colonization resistance to enteric pathogens. Gut dysbiosis, an imbalance in 
microbial composition characterized by the overrepresentation of potentially pathogenic taxa, is 
implicated in mediating persistent pathophysiological and immune abnormalities [Casaburi 

 
 
 
 
 
 
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2021] and is thought to play a role in nutrient malabsorption leading to underweight infants and 
failure to thrive [Duar 2020; Casaburi 2021]. 
Existing therapies for infants that promote growth do so by delivering the requisite nutrients but 
do not address imbalances in gut bacteria that are critical for digesting and absorbing food and 
preventing pathogens from overgrowing and translocating. Children hospitalized for malnutrition 
are also most often treated with a regimen of antibiotics which can further disrupt the healthy 
microbes that protect from infection and establish a self-reinforcing cycle. With the growing 
recognition of the association between the gut microbiome and health, there has been an 
increasing focus on exploring products that are capable of improving health beyond providing 
basic nutrition and address the imbalance in the gut microbiome. Probiotics are defined as live 
microorganisms that, when administered in adequate amounts, confer a health benefit on the host 
[Hill 2014]. Bifidobacteria are common probiotic agents. 
The presence and dominance of bifidobacteria in the microbiota of the infant gut, as well as the 
Bifidobacterium composition at a species level, changes over time with relative abundance 
decreasing throughout development due to weaning and /or introduction of solid foods [Arrieta 
2014]. Breast-feeding supports growth of a healthy gut flora including Bifidobacterium strains 
that metabolize HMOs while supplemental feeding with formula is associated with less 
Bifidobacterium dominance [Arrieta 2014]. 
Bifidobacterium longum subspecies infantis (B. infantis) is a commensal bacterial strain common 
in the microbiome of breast-fed infants younger than 6 months of age in LMICs that has the 
unique ability to efficiently process HMOs. Although breast-feeding support is a key part of 
treatment of malnourished infants, this alone may be insufficient to support recovery. A decrease 
in abundance or absence of B. infantis could lead to inadequate HMO processing, elevating 
intestinal pH and increasing the risk of pathogen overgrowth. Intervention with a B. infantis 
probiotic strain such as Bi-26, in vulnerable malnourished infants could have a significant short-
term impact on nutritional recovery and mortality, and long-term impact on health and 
development outcomes by potentially breaking the cycle of impaired immunity, recurrent 
infections, and worsening malnutrition. 
2.1. 
Background and Study Rationale 
Bifidobacteria are gram-positive, heterofermentative, anaerobic bacteria that colonize the 
newborn gut within the first days and weeks of life. They are often the most abundant bacterial 
genus in a breast-fed infant’s intestinal flora. B. infantis metabolizes HMOs and produces short 
chain fatty acids (SCFA) that are thought to play an important role in nutrition and immune 
development, stimulating anti-inflammatory and inhibiting pro-inflammatory responses 
[Halloran 2019; Henrick 2021]. 
B. infantis Bi-26 strain is a nutritional probiotic intervention that will be evaluated in this trial for 
its ability to improve weight gain and health outcomes of underweight infants with WAZ <-2 
who have been hospitalized with acute illness. 
Clinical study data in the United States (US) have shown that B. infantis strain EVC001 
colonizes the gut and is associated with significant changes to fecal microbiome composition in 
healthy breast-fed infants [Frese 2017; Henrick 2019]. Supplementation of B. infantis EVC001 

 
 
 
 
 
 
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has been associated with a reduction in pro-inflammatory cytokines [Henrick 2019]. Similar 
observations have been reported for preterm infants given B. infantis EVC001 [Nguyen 2021]. 
In a pilot trial evaluating B. infantis EVC001 supplementation in Bangladesh in hospitalized 2- 
to 6-month-old infants with SAM, i.e., weight-for-length z score [WLZ] <-3, researchers 
observed that daily supplementation with B. infantis for 4 weeks was associated with B. infantis 
colonization and an increase in WAZ [Barratt 2022]. 
A number of B. infantis strains, including Bi-26, have been designated as generally recognized as 
safe (GRAS) based on review by a panel of independent experts in the field. Furthermore, the US 
Food and Drug Administration (FDA) did not have questions about the GRAS determination of 
Bi-26 (GRN 985). In the US, the GRAS determination and the FDA no question letter allow the 
use of Bi-26 under its intended conditions of use (healthy term infants) in infant formula. The 
strain bearing the GRAS designation can also be used in dietary supplements, including those for 
infants.  
In low-income countries, B. infantis strain Bi-26, evaluated in this study, could potentially be 
used as a dietary supplement in underweight infants if the safety and efficacy findings of the 
previous pilot study are confirmed. 
2.2. 
Benefit/Risk Assessment 
B. infantis supplements, including Bi-26 strain, that are designated as GRAS are considered safe 
for consumption in healthy term infants that consume breastmilk. Evidence supports that Bi-26 
displays common probiotic characteristics, and the history of B. infantis used in food, as well as 
clinical trials at the species level, have been reviewed to support the safety assessment of the Bi-
26 strain (International Flavors & Fragrances Inc. Bi-26 Investigator’s Brochure). 
Similar products to Bi-26 have been used in very vulnerable populations with increased intestinal 
permeability such as very low birth weight premature infants, with no safety signals detected 
[Garland 2011]. 
A Cochrane review of 24 trials of probiotic supplement administration for the prevention of 
necrotizing enterocolitis (NEC), late-onset sepsis and death in preterm infants noted that none of 
the studies reported systemic infection associated with probiotic use [AlFaleh 2014]. In this 
review, the majority of reported short-term adverse consequences associated with probiotic 
administration are limited to individual case reports of bacteremia a decrease in all-cause 
mortality, and no increased risk of culture-proven sepsis, and no sepsis due to probiotic species 
[Fleming 2019]. 
A large randomized controlled trial of a different supplement containing B. infantis in preterm 
infants under 1500g birth weight did not detect any safety signals and found reduced rates of 
NEC in the active treatment group [Jacobs 2013]. 
No safety concerns have been reported after B. infantis supplementation evaluated in infants and 
adults [Chichlowski 2020; Smilowitz 2017]. Stool Bifidobacterium spp. count was significantly 
higher and stool frequency significantly lower in infants receiving B. infantis and no differences 
in health and safety outcomes were detected between infants receiving B. infantis and lactation 
support, compared to those only receiving lactation support. One month after discontinuing 
feeding B. infantis, stool count of B. infantis persisted and was significantly higher compared to 

 
 
 
 
 
 
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the control group [Frese 2017]. The dominance of B. infantis influenced beta diversity (diversity 
between samples). However, there were no differences in terms of microbial species richness 
(alpha diversity) as the Shannon diversity index was similar between control and B. infantis 
groups. Lack of differences in alpha diversity between control and B. infantis is consistent with 
previous reports on breast-fed infants. Brink, et al, reported lower alpha diversity in infants 
receiving human milk compared to infant formula [Brink 2020]. 
Of note, the quality control of the study intervention will be important since contamination is a 
potential known risk for this type of product. 
Bacterial transmigration and bacteremia have been described in case reports of B. infantis 
products but are considered coincidental, and not pathogenic. No bacterial transmigration or 
bacteremia cases have been reported with Bi-26 in ongoing infant clinical trials. 
Potential benefits of supplementing infant feeding in low-income countries with B. infantis 
include a possible reduction in gut inflammation, and improved nutrient adsorption, potentially 
resulting in improved health outcomes. The findings from the SYNERGIE study with B. infantis 
strain EVC001 support the potential for positive clinical outcomes with B. infantis 
supplementation [Barratt 2022]. In the SYNERGIE study, following hospital discharge and acute 
phase management, infants were transferred to nutritional rehabilitation units and randomized to 
receive either 8 billion CFUs (target) of B. infantis EVC001 (n=20), 8 billion CFUs (target) or 
EVC001 plus probiotic LNnT (n=21), or lactose placebo (n=21). Infants received 4 weeks of 
supplementation and were followed for an additional 4 weeks for outcomes. Researchers 
observed that daily supplementation with B. infantis for 4 weeks was associated with B. infantis 
colonization and an increase in anthropometric measures, including WAZ [Barratt 2022]. This 
study provides support for a larger study assessing weight gain in underweight infants following 
supplementation with other B. infantis strains. 
3. 
Objectives and Endpoints 
See Table 1. 
4. 
Study Design 
4.1. 
Overall Design 
Disclosure statement: This is a Phase 3, randomized, double-blind, placebo-controlled study to 
demonstrate increased weight gain with Bi-26 supplementation versus placebo in underweight 
infants (WAZ<-2) discharged from hospital following acute illness and to evaluate the safety of 
Bi-26 supplementation in this population. 
Underweight infants (WAZ<-2), who are between 1 and 4 months of age at enrollment and are 
currently hospitalized for acute non-surgical illness, will be enrolled to receive daily 
supplementation of Bi-26 or placebo for 28 days with a total study duration of approximately 90 
days. 
Intervention: Refer to Section 6. 

 
 
 
 
 
 
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Study Population: Refer to Section 5. 
Screening: Screening will take place during the infant’s hospitalization, within a week before 
anticipated hospital discharge. A participant discharged to a nutrition rehabilitation unit would 
still be considered a hospital discharge.  
Enrollment and Randomization: Enrollment and randomization will take place on the day of 
hospital discharge (Day 1) and after the infant participant has completed the acute stabilization 
phase of treatment, including fluid rehydration and antibiotic course as needed. 
Note: Screening may be conducted on the same day as enrollment/randomization (Day 1). Study 
assessments should not be duplicated if screening and Day 1 occur on the same day.  
Blinding: Refer to Section 6.2. 
Total duration of study participation: The study duration for each participant is approximately 
3 months. 
Study sites: The study will be conducted at 4 or more sites in 2 or more low-income countries. 
Infant Feeding: Breast-feeding counseling as recommended by the WHO will be conducted by 
study staff. 
IDMC: Refer to Section 8.4.5. 
Optional sub-study: After the 28-day intervention period has completed, participants may be 
enrolled into an optional, descriptive sub-study to gather information on the reliability of 
refrigeration in the participants’ homes. Refer to Section 4.5. 
4.2. 
Scientific Rationale for Study Design 
Gut dysbiosis, or disrupted development of the gut microbiota, is hypothesized to be a 
contributing cause of infant malnutrition. Absolute B. infantis abundance is reported to be 
markedly lower in 3-to-24-month-old Bangladeshi infants and children under 24 months of age 
with SAM, compared to their healthy controls. The primary hypothesis to be tested in this study 
is Bi-26 supplementation for 28 days will lead to increased weight gain in underweight infants 
(WAZ <-2) who were hospitalized with acute non-surgical illness. 
This study is designed as a double-blind, placebo-controlled study to enable equal attention and 
an unbiased evaluation of AEs and health outcomes. 
WAZ was selected as the primary endpoint because it appears to be a better surrogate for 
mortality than WLZ [Mwangome 2019; Chowdhury 2021]. However, weight-for-age Z-scores 
require that the birth date of the infants enrolled is known and that weight is determined 
accurately. 
We anticipate that an accurate gestational age at birth will often not be known for the infants 
enrolled. The study population will be heterogeneous and will include prematurely born infants 
and infants with normal weight-for-gestational age, infants with intrauterine growth retardation 
born small-for-gestational age, and infants with insufficient postnatal weight gain or weight loss 
due to illness and/or undernutrition. 

 
 
 
 
 
 
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4.3. 
Justification for Dose 
The primary goal of this study is to confirm the findings of the initial study in Bangladesh 
(SYNERGIE study) in which 4 weeks of single daily feeding with B. infantis EVC001 resulted 
in significant weight gain relative to placebo, and in B. infantis colonization for at least 4 weeks 
post end of treatment. The target dose listed in the SYNERGIE study was approximately 8 
billion CFUs with a 28-day duration of treatment; however, the amount of product initially 
packaged, and the potential range of doses administered over the course of the study was not 
provided [Barratt 2022]). 
. Thus, the anticipated range of 
CFU per dose included in this study is expected be generally comparable to that in the 
SYNERGIE study. Refer to the Gates MRI IB addendum for additional information. 
4.4. 
End of Study Definition 
A participant is considered to have completed the study if he/she completes the last study visit at 
Day 90. 
The EoS is defined as the date of the last visit of the last participant in the study. 
4.5. 
Optional Sub-study  
The aim of this optional, prospective, observational sub-study is to collect information on 
reliability of refrigeration in the geographic areas where the study is being conducted and where 
B. infantis supplementation may be recommended by WHO if supported by clinical data.  
Ensuring universal access to affordable, reliable, sustainable, and modern energy by 2030 is a 
sustainable development goal. Access to sufficient electricity is essential to reduce poverty as 
well as improve public services, such as healthcare. While strides have been taken to decrease 
the number underserved, as of 2020, 733 million people did not have access to electricity in 
2020, over 75% (568 million people) of whom lived in sub-Saharan Africa [World Bank 2022]. 
Per estimates from a year prior, another 1 billion people live with unreliable or insufficient 
electricity [Sanni 2019]. 
Electricity deficits are greater in rural areas compared to urban areas [World Bank 2022]. In 
order to get a geographical cross-representation in this study, each clinical site will be asked to 
enroll up to 20% of their overall study population into the sub-study.  
The selected participants will receive a box containing a temperature data logger to store in the 
home refrigerator. Participants may refuse enrollment into the sub-study without effecting their 
participation in the main study.  

 
 
 
 
 
 
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The box with the temperature data logger will be placed in the refrigerator used by the 
participant’s parent(s)/legal guardian after the participant has completed the study intervention 
period and during the home visit on Day 35. The box will be collected from the refrigerator at 
the home visit on Day 49. Participants in the sub-study should keep the box unopened in the 
refrigerator until the box is collected by the study staff. The study staff will return the box to the 
clinic so the information from the temperature data logger can be uploaded. 
No formal statistical analysis is planned; data will be presented descriptively. 
5. 
Study Population and Eligibility Criteria 
A sufficient number of participants will be screened to randomize approximately 396 study 
participants. Screening will take place while the infant is hospitalized. 
Randomization will take place on the day of hospital discharge. Prospective approval of protocol 
deviations to recruitment and enrollment criteria, also known as protocol waivers or exemptions, 
is not permitted. 
The preferred method of reconstitution of the study intervention is in breastmilk and therefore 
study activities are assigned to the mother of the infant participant. However, other caretakers 
may perform certain study activities (e.g., picking up the study doses, assisting in completion of 
the feeding diary, etc.). 
5.1. 
Inclusion Criteria 
Participants are eligible to be included in the study only if all of the following criteria apply: 
Age 
1. Participant must be between 30 days and 120 days of age (inclusive), at the time of 
enrollment (study Day 1) 
Type of Participant and Disease Characteristics 
2. Hospitalized for acute non-surgical illness 
3. Completed acute stabilization phase of treatment, including fluid rehydration and antibiotic 
course, prior to enrollment (study Day 1) 
Weight 
4. WAZ at enrollment (study Day 1) is less than negative 2 (<-2) 
Sex 
5. Any 
Informed consent 
6. Participant’s parent(s)/legal guardian is capable of giving informed consent which includes 
agreement to comply with the requirements and restrictions listed in the informed consent form 
(ICF) and in this protocol 

 
 
 
 
 
 
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Additional requirements 
7. Participant’s parent(s)/legal guardian agrees to stay in contact with the study site for the 
duration of the study, provide updated contact information as necessary, and have no current 
plans to relocate from the study area for the duration of the study 
8. Participant’s parent(s)/legal guardian has easy access to reliable refrigeration (for storage of 
investigational product) 
9. Participant receives some feedings from breastmilk and mother intends to continue breast-
feeding. 
5.2. 
Exclusion Criteria 
Participants are excluded from the study if any of the following criteria apply: 
1. Congenital condition (suspected or confirmed) that the investigator considers likely to 
interfere with feeding or with normal growth and development 
2. Infant has not been discharged from hospital since birth or has not been at home for at least 
one week since birth 
3. Infant hospitalized with septic shock during current hospitalization 
4. Infant required mechanical ventilation during current hospitalization 
5. Infant with acute kidney injury on hospital admission 
6. Infant with severe jaundice and suspected kernicterus 
7. Infant receiving treatment for suspected or confirmed tuberculosis, or suspected or confirmed 
HIV infection 
8. Ongoing infant antibiotic (e.g. as prophylaxis in sickle cell disease) and/or probiotic usage 
9. Ongoing maternal antibiotic and/or probiotic usage for breast-feeding infants 
10. Inability of participant’s parent(s)/legal guardian to comply with protocol requirements, as 
per investigator assessment. 
5.3. 
Screen Failures 
A screen failure participant is defined as a participant whose parent(s)/legal guardian consented 
to participate in the clinical study, but the infant is not subsequently randomly assigned to group 
and study intervention on the Day 1 visit. 
A minimal set of screen failure information is required to ensure transparent reporting of screen 
failure participants to meet the Consolidated Standards of Reporting Trials (CONSORT) 
publishing requirements and to respond to queries from regulatory authorities. Minimal 
information includes demography, screen failure details, eligibility criteria, and any SAE. 
Infants who do not meet the eligibility criteria for participation in this study (screen failures) will 
not be rescreened. Infants whose screening visit is outside of the 7-day planned window, due to a 
delay in hospital discharge, may be rescreened and enrolled if all the eligibility criteria are met. 

 
 
 
 
 
 
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6. 
Study Intervention 
Study intervention is defined as any investigational intervention, marketed product or placebo, 
intended to be administered to a study participant according to the study protocol. The study 
intervention includes Bi-26 and placebo, given to approximately 198 participants, per group. 
All participants will be given either Bi-26 supplementation, or product matched placebo (lacking 
the active ingredient), based on randomization (refer to Section 6.2). A single dose (Bi-26 or 
placebo control) will be resuspended, and administered to each participant, daily, for 28 days. 
Participants will be monitored for 30 minutes, and a repeat dose will be administered if a 
participant vomits within this observation period. 
While a total of 7 doses are to be administered to the participant per week for a total of 4 weeks, 
2 additional doses will be supplied each week to account for repeat dose administration in the 
event of vomiting, or unexpected events which may render a dose unusable (e.g., spillage or 
otherwise compromised). Therefore, mothers will be dispensed 9 doses containing Bi-26 or 
placebo once a week, within 2 days before the doses are needed to ensure that doses are available 
when needed. Doses will either 1) be delivered to the mother by study staff, or 2) stored by staff, 
at the local health center to be picked up from the health center.  
The product is presented as a lyophilized powder that the mother mixes with approximately 3 
mL to 5 mL of breastmilk once daily and administers to the infant orally using a feeding syringe. 
If the mother is unable to express breastmilk, the powder may be mixed in approximately 3 mL 
to 5 mL of water. The participant’s mother should provide the dose with a feeding at 
approximately the same time each day. 
The study staff will counsel the mother on recommended breast-feeding practices as per WHO 
guidance and encourage the participant’s mother to continue breast-feeding during the study. 
The participant’s parent(s)/legal guardian will also be counseled on the importance of having 
access to reliable refrigeration and to contact the study staff if there is ever a problem with 
product storage. 
Further information on the study intervention dose and handling can be found in the Study 
Manual and Investigator’s Brochure. 
As noted in Section 5, because the preferred method of reconstitution of the study intervention is 
in breastmilk, study activities are assigned to the mother of the infant participant. However, other 
caretakers may perform certain study activities (e.g., picking up the study doses, assisting in 
completion of the feeding diary, etc.). 

 
 
 
 
 
 
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Table 3: Study Intervention 
Group Name 
Bi-26 
Control 
Intervention Name 
Bifidobacterium longum ssp. 
infantis* 
Bi-26TM strain 
Product code MNK01 
Placebo 
Type  
Dietary supplement, presented as 
lyophilized powder 
Product matched lyophilized 
powder lacking B. infantis 
Dose Formulation 
Doses consist of a powder that 
the mother mixes with 
approximately 3 mL to 5 mL of 
breastmilk or water  
Powder that the mother mixes 
with approximately 3 mL to 5 mL 
of breastmilk or water  
Unit Dose Strength 
Contains approximately 5 billion 
CFU of B. infantis at end of 
shelf-life, with potato 
maltodextrin as the excipient, 
 
Contains potato maltodextrin as 
the excipient 
Dosage  
A single dose per day  
A single dose per day 
Route of 
Administration 
Oral, using a feeding syringe, 
given directly into the infant’s 
mouth 
Oral, using a feeding syringe, 
given directly into the infant’s 
mouth 
Packaging and 
Labeling 
Provided as single doses, labeled 
per country requirement. 
Provided single doses, labeled per 
country requirement. 
Current/Former 
Name or Alias 
Not applicable 
Not applicable 
*Family: Bifidobacteriaceae, Genus: Bifidobacterium, Species: longum, Subspecies: infantis (B. infantis), Strain Bi-
26 
6.1. 
Handling, Storage, and Accountability 
Each participant’s mother will be dispensed 9 single doses of the study intervention (either Bi-26 
or placebo) once a week for a total duration of 4 weeks. 
Only authorized site staff may supply study intervention. Doses will either be delivered to the 
participant’s mother by study staff, or be stored by study staff at the local health center, to be 
picked up from the health center.  
The preparation of study interventions will be demonstrated to the participant’s mother at the 
first study visit and written instructions with images will be provided to each participant’s 
mother. These instructions will be provided to the sites in the Study Manual. 
Doses must be shipped and stored at 2°C to 8°C after delivery to the local health center or home. 
The investigator or designee must confirm appropriate temperature conditions have been 

 
 
 
 
 
 
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maintained during transit for all study intervention received and any discrepancies are to be 
reported and resolved before use of the study intervention. 
All study intervention taken to the local health center must be stored in a secure, controlled 
environment, and should be monitored (manual or automated) in accordance with the labeled 
storage conditions, with access limited to the authorized site staff. Monitoring will be addressed 
in the Study Manual. 
All study intervention delivered to the participant’s home, must be stored in a refrigerator at the 
participant’s or neighbor’s home with confirmation from the parent(s)/legal guardian that the 
electricity in the home is reliable for the duration of the study. 
The participants’ parent(s)/legal guardian will be asked to inform the site if refrigeration was off 
or unreliable at any time during dose storage, and for how long, and for how many of the doses. 
The investigator or designee is responsible for study intervention accountability, reconciliation, 
and record maintenance (i.e., receipt, reconciliation, and final disposition records). 
Further guidance and information for the final disposition of unused study interventions are 
provided in the Study Manual. 
6.2. 
Measures to Minimize Bias: Randomization and Blinding 
Randomization: On the day of hospital discharge (Visit 1, Day 1) participants will be 
randomized 1:1 to receive either Bi-26 or placebo. To minimize differences between Bi-26 and 
placebo groups within a given treatment arm, randomization will be stratified by: 
1) country 
2) age at enrollment (Study Day 1):30-59 days of age, 60-120 days of age 
3) discharge destination (home, nutritional rehabilitation unit) 
4) WAZ (≤ -3 versus > -3) 
Target randomization is approximately 2/3 of participants with WAZ between -2 and -3 and 
approximately 1/3 with WAZ ≤ -3. As such, a stratification blocking factor will be used in the 
ratio of 1:2 for (WAZ≤-3):(-3< WAZ< -2) within each treatment arm. Dynamic randomization 
will be utilized to balance the treatment groups for the other stratification factors. 
Blinding: The study is double-blind. The placebo doses will be prepared to be as similarly as 
possible in appearance and odor to Bi-26. The final packaging material and the final target fill 
weight for Bi-26 and placebo will be identical. Study participants, principal investigators and 
study site personnel will remain blinded to treatment assignments throughout the study. The 
Gates MRI Medical Monitor, study monitors, and any other Gates MRI and contract research 
organization (CRO) personnel who are regularly in contact with the study site will remain 
blinded. Selected individuals listed below will have access to treatment allocations while the 
study is blinded: 
• 
Biostatistician preparing the randomization list  
• 
Biostatistician preparing the IDMC data  
• 
IDMC members.  

 
 
 
 
 
 
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Additionally, in the event of a suspected unexpected serious adverse reaction (SUSAR), 
designated members of the sponsor or the sponsor representative’s pharmacovigilance 
department may be required to unblind a study participant to meet country-specific reporting 
requirements.  
Emergency unblinding: All participants will be centrally assigned to randomized study 
intervention using an Interactive Web Response System (IWRS). Before the study is initiated, 
the login information and directions for the IWRS will be provided to each site. 
The IWRS will be programmed with blind-breaking instructions. In case of an emergency, the 
investigator has the sole responsibility for determining if unblinding of a participants’ treatment 
assignment is warranted. Participant safety must always be the first consideration in making such 
a determination. If the investigator decides that unblinding is warranted, the investigator should 
make every effort to contact the sponsor prior to unblinding a participant’s treatment assignment 
unless this could delay emergency treatment of the participant. If a participant’s treatment 
assignment is unblinded, the sponsor must be notified within 24 hours after breaking the blind. 
The date and reason that the blind was broken must be recorded in the source documentation and 
the electronic case report form (eCRF), as applicable. 
6.3. 
Study Intervention Compliance 
Participant compliance with study intervention will be assessed at each visit. Compliance will be 
assessed by counting returned doses, and by asking the mother if her infant received a dose each 
day, and if any of the additional doses were used and why. Deviations from the planned dosing 
regimen will be recorded in the eCRF. 
The following data will also be collected at each visit specified in the SoA for study intervention 
storage information: 
• where the study intervention is being stored (at the local health center or at the home, or a 
neighbor’s home) 
• any issues with study intervention storage (and how many doses were compromised). 
6.4. 
Feeding History 
The mother of the participating infant will be asked to complete a diary to record the number and 
types of feedings (e.g., breast-feeding, formula feeding, other) daily through Day 90.  
The type of liquid (breastmilk or water) used to mix with the Bi-26 or placebo, whether the full 
dose was taken, and whether a repeat dose was administered will also be recorded in the diary for 
Days 1 through 28.  
The completed diary should be collected by the site staff.  
6.5. 
Concomitant Therapy 
Any medication or vaccine (including over-the-counter or prescription medicines, vitamins, 
and/or herbal supplements) that the participant is receiving at the time of screening or receives 
during the study must be recorded at the scheduled timepoints along with: 

 
 
 
 
 
 
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• Reason for use 
• Dates of administration including start and end dates 
• Dosage information including dose and frequency. 
Any maternal use of antibiotics or probiotics should be captured through Day 90. 
The Medical Monitor should be contacted if there are any questions regarding concomitant or 
prior therapy. 
6.6. 
Dose Modification 
Dose modification is not planned. 
7. 
Discontinuation of Study Intervention and Participant 
Discontinuation/Withdrawal 
7.1. 
Discontinuation of Study Intervention 
In rare instances, it may be necessary for a participant to permanently discontinue study 
intervention earlier than the protocol planned duration. Stopping the study intervention may be 
initiated by either the participant’s parent(s)/legal guardian or the Investigator. The Investigator 
should discontinue the study intervention if the participant experiences intolerable AEs, or if the 
Investigator believes that continuing dosing would result in an unfavorable risk/benefit for the 
trial participant. If study intervention is permanently discontinued, the participant will remain in 
the study to be evaluated for safety and weight change. 
Refer to the SoA (Table 2) for data to be collected at the time of discontinuation of study 
intervention for any reason. 
7.2. 
Participant Discontinuation/Withdrawal from the Study 
A participant may withdraw from the study at any time at the request of the participant’s 
consenting parent/legal guardian, or may be withdrawn at any time at the discretion of the 
investigator for safety, or other reasons. This is expected to be uncommon. 
At the time of discontinuing from the study, if possible, an early discontinuation visit should be 
conducted, and data should be collected, if possible, as shown in the SoA (Table 2). The 
participant will be permanently discontinued both from the study intervention and from the study 
at that time. 
If the participant’s consenting parent/legal guardian withdraws consent for disclosure of future 
information, the sponsor may retain and continue to use any data collected before such a 
withdrawal of consent. 
If a participant’s parent/legal guardian withdraws participant from the study, they may request 
destruction of any samples taken and not tested, and the investigator must document this in the 
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7.3. 
Replacement of Participants 
Participants prematurely discontinued from the study will not be replaced.  
7.4. 
Lost to Follow-up 
A participant will be considered lost to follow-up if the participant’s parent(s)/legal guardian 
repeatedly fails to return for scheduled visits, whether at the clinic or at home, and is unable to be 
contacted by the study site. 
The following actions must be taken if a participant’s parent(s)/legal guardian fails to return to 
the clinic for a required study visit or is not at home when a home visit is scheduled, and the 
parent(s)/legal guardian cannot be found for the home visit: 
The site must attempt to contact the participant’s parent(s)/legal guardian and reschedule the 
missed visit as soon as possible and counsel the participant’s mother on the importance of 
maintaining the assigned visit schedule and ascertain whether the participant’s mother wishes to 
and/or should continue in the study. 
Before a participant is deemed lost to follow-up, the investigator or designee must make every 
effort to regain contact with the participant’s parent(s)/legal guardian (where possible, 3 
telephone calls and, if necessary, a certified letter to the participant’s parent(s)/legal guardian last 
known mailing address or local equivalent methods). These contact attempts should be 
documented in the participant’s medical record. 
Should the participant’s parent(s)/legal guardian continue to be unreachable, the participant will 
be considered to have withdrawn from the study. 
If the participant’s parent(s)/legal guardian is unreachable when the weekly doses are being 
delivered at the home visit, and the doses are not received, the investigator or designee must 
make every effort to regain contact with the participant’s parent(s)/legal guardian (as mentioned 
above) to deliver the doses. 
8. 
Study Assessments and Procedures 
In this study, visits will be comprised of both clinic visits and home visits, as shown in the SoA 
(Table 2). Home visits and all assessments at these visits will be performed by trained study team 
staff. 
Screening procedures will be conducted after informed consent is obtained (refer to Section 
10.1.3) and within 7 days before anticipated hospital discharge and enrollment/randomization 
(Day 1). If informed consent and screening activities are conducted on the same day as 
enrollment/randomization (Day 1), assessments should not be duplicated. All screening 
evaluations must be completed and reviewed to confirm that potential participants meet all 
eligibility criteria. The investigator will maintain a screening log to record details of all 
participants screened and to confirm eligibility or record reasons for screening failure, as 
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On Day 1, after all eligibility criteria are verified, eligible participants will be enrolled and 
randomized. Randomization should take place at the time of hospital discharge. Refer to Section 
6.2 for randomization. 
Procedures conducted as part of the participant’s routine clinical management (e.g., procedures 
while hospitalized) and obtained before the ICF is signed, may be utilized for screening or 
baseline purposes provided the procedures met the protocol-specified criteria and were 
performed within the time frame defined in the SoA (Table 2). 
Adherence to the study design requirements, including those specified in the SoA is essential and 
required for study conduct. 
Immediate safety concerns should be discussed with the sponsor immediately upon occurrence or 
awareness to determine if the participant should continue or discontinue study intervention. 
Study procedures and their timing are summarized in the SoA (Table 2). Protocol waivers or 
exemptions are not allowed. 
8.1. 
Demographic and Baseline Characteristics 
The following information pertaining to the participant will be obtained from the participant’s 
mother at screening: 
• Participant’s sex, race, ethnicity, date of birth (including day, month, and year), and 
whether the delivery was a C-section. 
• Participant’s gestational age in weeks, if known, and the participant’s birth weight (in 
kilograms). This information may be obtained from the hospital medical record if 
necessary. 
• The mother’s age will be obtained whenever possible. 
• The participant’s mother will be asked about breast-feeding practices from the time of the 
participant’s birth, and planned feeding practices during the study (and this information 
will be captured in the case report form). 
A full medical history and full physical examination will be performed at the screening visit (refer to 
Section 8.3.1). The participant’s anthropometric measurements will be taken at the screening visit and 
at other specified timepoints in the SoA (refer to Table 2 and Section 8.3.1for more details). 
If the eligibility criteria are met, the participant will be enrolled into the study on Day 1. After 
enrollment, an estimation of socioeconomic status will be obtained if possible.  
Hospital discharge to home, or to nutritional rehabilitation unit, will be collected on Day 1 and 
obtained from either the participant’s parent(s)/legal guardian, or the hospital medical record. 
The anthropometric measurements obtained at Day 1 will be considered baseline for the 
purposes of statistical analyses (refer to Section 9.2). 
8.2. 
Efficacy Assessments 
Efficacy assessments include calculation of the WAZ and assessments for re-hospitalizations for 
acute non-surgical illness at various time points per SoA (Table 2). 

 
 
 
 
 
 
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Refer to Section 9.7 for analyses. 
8.3. 
Safety Assessments 
Safety assessments include SAEs and AEs from the time the ICF is signed through Day 90 (or at 
the last visit [Discontinuation Visit]), if possible, if participant is discontinued/withdrawn early). 
Planned time points are provided in the SoA (Table 2).  
Any AEs and any SAEs that occur from the time of screening until Visit 1 may be obtained from 
the participant’s hospital records.  
Refer to Section 8.4 for details regarding AEs and SAEs. 
Refer to Section 9.7.1for analyses. 
8.3.1. 
Medical History and Full Physical Examination 
A full medical history, including medications and vaccinations received, will be recorded at 
screening to assess enrollment eligibility and to provide a baseline. 
All conditions that exist before and during screening will be recorded in the medical history. The 
infant’s HIV status, including history of HIV exposure, will also be recorded. Physical 
examination at screening will include, at a minimum, assessment of all planned anthropometric 
measurements, including weight to assess WAZ, length to measure WLZ score, MUAC, head 
circumference, and skinfold thickness. Details regarding standardized anthropometric 
measurements will be provided in the Study Manual. Presence of bilateral pedal edema will also 
be assessed. 
In addition, resting vital signs, including body temperature (refer to Section 8.3.3) and other 
assessments of eligibility criteria requiring a physical examination will be performed. 
A complete physical examination will include, at a minimum, assessments of the head, eyes, 
ears, nose and throat, cardiovascular, respiratory, gastrointestinal, musculoskeletal, and 
neurological systems. 
8.3.2. 
Interim History and Focused Physical Examination 
The interim medical history covers the period since the previous clinic visit. If the interim history 
raises any concerns, a focused physical examination will be conducted as appropriate if indicated 
by participant’s medical issues and will include assessments of body systems involved. AEs 
identified as a result of the review of the interim medical history should be recorded as outlined 
in the SoA (Table 2). 
8.3.3. 
Vital Signs 
Vital signs, length and weight will be assessed and recorded at all clinic visits. 
Vital sign measurements will include pulse rate, pulse oximetry, and body temperature. 
Measurements are to be repeated if clinically significant changes are observed or a machine error 
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Body temperature should be taken per standard of care at the site. If the participant passes a 
bowel movement during temperature collection at a clinic visit only, this stool sample should be 
collected for biomarkers (see Section 8.6). 
8.4. 
Adverse Events, Serious Adverse Events 
The definitions of an AE or SAE can be found in Section 10.2. 
AEs and SAEs will be assessed as further described in this section and recorded at timepoints 
indicated in Table 2. The infant’s parent(s)/legal guardian will be asked about any event that 
occurred since the previous clinic or home visit. 
The investigator and any qualified designees are responsible for detecting, documenting, and 
recording events that meet the definition of an AE or SAE and remain responsible for following 
up AEs that are serious, considered related to the study intervention or study procedures, or that 
caused the participant to discontinue the study intervention or the study (see Section 7). 
The investigator is solely responsible for assessment, including assignment of causality and 
intensity, reporting and management. 
8.4.1. 
Time Period and Frequency for Collecting AE and SAE Information 
All AEs and SAEs will be collected from the time the informed consent is signed through the 
EoS (Day 90 visit or at the Discontinuation Visit, as applicable). All AEs and SAEs will be 
classified according to the Medical Dictionary for Regulatory Activities (MedDRA). 
All SAEs will be recorded and reported to the sponsor or designee immediately within 24 hours, 
as indicated in Section 10.2.4. The investigator will submit any updated SAE data to the sponsor 
within 24 hours of it being available. 
Investigators are not obligated to actively seek AE or SAE information after conclusion of the 
study participation. However, if the investigator learns of any SAE, including a death, at any 
time after a participant has been discharged from the study, and if the investigator considers the 
event to be reasonably related to the study intervention or study participation, the investigator 
must promptly notify the sponsor. 
8.4.2. 
Method of Detecting AEs and SAEs 
The method of recording, evaluating, and assessing causality of AEs and SAEs and the 
procedures for completing and transmitting SAE reports are provided in Section  10.2. 
Care will be taken not to introduce bias when detecting AEs and/or SAEs. Open-ended and 
nonleading verbal questioning of the participant is the preferred method to inquire about a 
SAE occurrence. 
Day-to-day fluctuations in chronic conditions that were present at baseline, and do not represent 
a clinically significant change in the participant’s health status will not necessarily be reported as 
AEs. If the investigator determines that this change is related to the study intervention it will be 
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8.4.3. 
Follow-up of AEs and SAEs 
After the initial AE/SAE report, the investigator is required to proactively follow each participant 
at subsequent visits. All SAEs will be followed until resolution, stabilization, the event is 
otherwise explained, or the participant is lost to follow-up (as defined in Section 7.4). Further 
information on follow-up procedures is provided in Section 10.2.3. 
8.4.4. 
Regulatory Reporting Requirements for SAEs 
Prompt notification by the investigator to the sponsor of a SAE is essential so that legal 
obligations and ethical responsibilities toward the safety of participants and the safety of a study 
intervention under clinical investigation are met. Refer to Section 10.2.4 regarding reporting 
timelines. 
The sponsor has a legal responsibility to notify both the local regulatory authority and other 
regulatory agencies about the safety of a study intervention under clinical investigation. The 
sponsor will comply with country-specific regulatory requirements relating to safety reporting to 
the regulatory authority, Ethics Committees (ECs)/Institutional Review Boards (IRB), and 
investigators. 
Investigator safety reports must be prepared for any SUSARs according to local regulatory 
requirements and sponsor policy and forwarded to investigators as necessary. 
An investigator who receives an investigator safety report describing a SAE or other specific 
safety information (e.g., summary or listing of SAEs) from the sponsor will review it and will 
notify the EC/IRB, if appropriate according to local requirements. 

 
 
 
 
 
 
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8.4.5. 
Independent Data Monitoring Committee 
The IDMC will operate according to a charter. The IDMC structure, participants and other 
details will be provided in the charter. The charter will be available prior to study start. 
The role of the IDMC will be to review unblinded safety data and make recommendations to the 
sponsor. These recommendations may include continuing the study without modification, 
continuing the study with modifications, or terminating the study. Additionally, the sponsor will 
review data on an ongoing basis and may, on discussion with the IDMC, terminate the study if 
any clinically significant safety signal related to the study intervention is identified. The 
recommendations of the IDMC, along with the sponsor’s decision, will be communicated to the 
investigators, the ECs/IRBs and the national regulatory authorities as required. The sponsor or its 
designee agrees to abide by any directives issued by the national regulatory authority or the 
EC/IRB. 
8.5. 
Genetics 
Genetics are not evaluated in this study. 
8.6. 
Biomarkers 
To assess the biomarker study endpoints, serum and stool samples will be collected from all 
infants for biomarker research. All of these samples, or a subset of these samples from a 
representative cohort of all participants, may be tested to identify analytes of interest.  
At Visit 1 Day 1 and Visit 2 Day 28, as indicated in the SoA (Table 2), a blood sample will be 
collected and processed to serum from all participants. Restricted blood volumes may be 
dependent on the physician discretion and/or the participant’s health/condition. Testing will be 
determined by the amount of available blood collected.  
These serum samples will be tested for biomarkers including, but not limited to, host 
inflammatory biomarkers and metabolic analysis. No host genetic analyses will be performed. 
The maximum amount of blood collected from each participant over the duration of the study 
will not exceed approximately 4 mL. 
Repeat or unscheduled samples may be taken for safety reasons or for technical issues with the 
samples. 
Stool samples for measurement of exploratory biomarkers will also be collected at clinic visits as 
specified in the SoA (Table 2), in addition to stool samples collected for the secondary endpoint, 
(refer to Section 9.7.1). Collection will be performed by trained study team staff at specified 
clinical visits. Either taking temperature or a rectal swab will be performed to stimulate 
defecation. Defecation at any time during the clinical visit prior to stool collection can substitute 
for this rectal swab. 
Fecal pH and Bristol or similar stool scale will be measured and recorded by the trained study 
team staff, at the time of stool collection. Samples will be tested for microbiome composition 
including levels of total Bi-26 measured by nucleotide sequencing and/or polymerase chain 
reaction. Stool samples may be tested for biomarkers including, but not limited to, inflammatory 
biomarkers and host and/or bacterial metabolic analysis. 

 
 
 
 
 
 
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9. 
Statistical Considerations 
9.1. 
General 
All statistical analyses will be performed using SAS® (Version 9.3 or higher). 
Analyses of continuous data will be summarized using descriptive statistics where the following 
parameters will be reported: 
• 
number of observations 
• 
number of missing observations 
• 
mean 
• 
median 
• 
standard deviation (SD) 
• 
minimum (min) 
• 
maximum (max). 
Categorical data will be presented with absolute and relative frequency (n and %). 
For the summary statistics, the number of decimal places will be the same as for the source data 
for extreme values (min, max) and will exceed that of the source data by 1 for other parameters. 
Percentages will be presented with one decimal place and a percent sign (%). 
All tests will be two-sided and performed at the two-sided 5% significance level if not otherwise 
specified. When reporting the results of significance tests p-values will be reported. All 
confidence intervals will be two-sided at the 95% level, unless otherwise stated. 
All data will be presented in subject data listings. 
Summary statistics will be presented separately for the treatment and follow-up periods. 
CCI

 
 
 
 
 
 
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9.2. 
Subject Disposition, Demographics and Baseline Data, Concomitant 
Medication, and Compliance 
9.2.1. 
Subject Disposition 
Number of subjects who were enrolled and randomized, took at least one dose of study 
supplement, completed the treatment phase, completed the follow-up phase, withdrew including 
reasons for withdrawal and the number of participants in each analysis set will be summarized. 
Protocol deviations will be listed. 
9.2.2. 
Demographic and Baseline Data 
The demographic, background, characteristics, and baseline data will be presented descriptively. 
Corresponding summaries will be presented for all participants enrolled. 
9.2.3. 
Concomitant Medication 
Medications will be coded by site of action and therapeutic and clinical characteristics using 
WHO’s Anatomical Therapeutic Chemical (ATC) Classification, and will be presented 
descriptively 
9.2.4. 
Compliance 
Compliance will be presented descriptively. Compliance will be used in defining the per-
protocol population. 
9.3. 
Randomization 
Participants will be randomized in a ratio of 1:1 to each of the 2 treatment arms, Bi-26 or 
placebo. 
A stratification blocking factor will be used in the ratio of 1:2 for (WAZ ≤-3):(-3<WAZ<-2) 
within each treatment arm. Dynamic randomization algorithms will be used to balance treatment 
arms with respect to the other stratification factor levels (country, age at enrollment (Day 1), 
discharge destination) and ensure an overall treatment balance. 
9.4. 
Primary Statistical Hypotheses and Sample Size Determination 
The primary study hypothesis is that the difference in the mean change from baseline in WAZ is 
larger for participants randomized to at least 28 days of Bi-26 relative to placebo. 
For the sample size calculation for the primary hypothesis, it is assumed that the between group 
difference in the WAZ change from baseline is 0.45 with a standard deviation of 1.3. Based on 
the above assumption(s) using a 2-sided alpha of 5% and a power of at least 90%, a total of 354 
participants, 177 in each group, is required. 
Assuming a 10% loss to follow-up and a 3% mortality rate, a sufficient number of participants 
will be screened to ensure approximately 396 randomly assigned participants are enrolled, 198 in 
each treatment group. 

 
 
 
 
 
 
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Figure 2: Sample size sensitivity 
 
N/group, 1-sided α=2.5%, Power=90% 
9.5. 
Populations for Analyses 
For purposes of analysis, the following populations are defined: 
• Intention-to-treat (ITT) population: All participants randomly assigned to study 
intervention. 
• Modified intention-to-treat (mITT) population: All participants randomly assigned to 
study intervention, who received at least one dose (including a partial dose) of the study 
intervention. Participants will be analyzed according to the intervention they were 
randomized. 
• Per-Protocol (PP) population: All participants randomly assigned to study 
intervention, who received the study intervention, have a Day 90 visit, and did not 
substantially deviate from the protocol procedures. Participants will be analyzed 
according to the intervention they actually received. Details of criteria for defining the 
PP population will be specified in the Statistical Analysis Plan (SAP). The inclusion of 
participants in the PP population will be determined prior to unblinding the study. A 
participant data listing and a table with protocol deviations will be presented. 
• Safety population: All participants randomly assigned to study intervention, who 
received any dose, including a partial dose, of the study intervention. Participants will 
be analyzed according to the intervention they actually received. 

 
 
 
 
 
 
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9.6. 
Statistical Analyses 
The principal features of the statistical analysis are described in this section. A more technical 
and detailed document of the features will be found in a separate SAP, which is to be finalized 
before unblinding of the study. When the study is completed and the data are collected, a blinded 
review of the planned analysis will be carried out. The review will look at the definition of 
outliers, violators, and exclusion of participants or data from the analysis sets. Decisions made 
based on the review will be documented. 
9.7. 
Primary and Secondary Estimands 
Participants randomized to the group receiving 28 days of Bi-26 will be compared to participants 
randomized to 28 days of placebo for the primary analysis. 
Following International Council for Harmonization of Technical Requirements for 
Pharmaceuticals for Human Use (ICH) E9 (R1) addendum, the estimands relating to the study 
objectives are constructed by using the 5 following attributes: 
(a) treatment 
(b) population 
(c) variable/endpoint 
(d) population level summary 
(e) intercurrent events. 
These attributes were specified to translate the study objective(s) into a treatment effect that is to 
be estimated (estimand). Refer to Table 4 and Table 5 for a summary of the primary and 
secondary estimands, respectively.  
Table 4: Primary Estimands 
Objective 
Primary Estimand 
To evaluate the change in 
weight (standardized for 
age) of infants receiving 
Bi-26 
a. Population: The mITT population will be used for the analysis. 
b. Endpoint: WAZ change from baseline (Day 1) to the Day 56 visit in Bi-
26 group. 
c. Population level summary: Mean change from baseline in WAZ in the 
Bi-26 group compared to placebo with stratification blocking factors and 
feeding regimen covariate(s). 
d. Intercurrent event(s): Including but not limited to treatment 
discontinuation. Further details will be specified in the SAP. 

 
 
 
 
 
 
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Table 5: Secondary Estimands 
Objective 
Secondary Estimand 
To evaluate the change in 
weight of infants 
receiving Bi-26 
a. Population: The mITT population will be used for this analysis. 
b. Endpoint: weight change (in grams) from baseline (Day 1) to the Day 
56 visit in Bi-26 group. 
c. Population level summary: Mean change from baseline in weight in the 
Bi-26 group compared to placebo with stratification blocking factors and 
feeding regimen covariate(s). 
d. Intercurrent event(s): Including but not limited to treatment 
discontinuation. Details will be provided in the SAP. 
To estimate the treatment 
response over time 
associated with Bi-26 
e. Population: The PP population will be used for the analysis. 
f. Endpoint: WAZ change from baseline (Day 1) over time through the 
Day 90 visit (longitudinal assessment) by duration of dosing. 
g. Population level summary: Mean change from baseline in WAZ over 
time in the Bi-26 group compared to placebo with stratification blocking 
factors and feeding regimen covariate(s). 
h. Intercurrent event(s): Including but not limited to treatment 
discontinuation. Details will be provided in the SAP. 
To assess the proportion 
of infants who achieve a 
specified change in WAZ 
from baseline to Day 56 
a. Population: The PP population will be used for the analysis. 
b. Endpoint: Proportion of infants with a ≥ 0.3, 0.4, and 0.5 change in 
WAZ from baseline (Day 1) to Day 56 visit. 
c. Population level summary: Frequency and percentages of infants with a 
≥ 0.3, ≥ 0.4, and ≥0.5 change in WAZ from baseline (Day 1) to Day 56 
in the Bi-26 and placebo groups. 
d. Intercurrent event(s): Including but not limited to treatment 
discontinuation. Details will be provided in the SAP. 
To assess the proportion 
of infants who achieve a 
specified WAZ at Day 56 
a. Population: The PP population will be used for the analysis. 
b. Endpoint: Proportion of infants with a WAZ >-2 at Day 56 visit. 
c. Population level summary: Frequency and percentages of infants with a 
WAZ >-2 at Day 56 in the Bi-26 and placebo groups. 
d. Intercurrent event(s): Including but not limited to treatment 
discontinuation. Details will be provided in the SAP. 
To assess the re-
hospitalization rate 
a. Population: The PP population will be used for the analysis. 
b. Endpoint: Number of re-hospitalizations for acute non-surgical illness 
through the Day 56 visit. 
c. Population level summary: Frequency (and percentages) of re-
hospitalizations in the Bi-26 group compared to placebo. 
d. Intercurrent event(s): Including but not limited to treatment 
discontinuation. Details will be provided in the SAP. 

 
 
 
 
 
 
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9.7.1. 
Other Secondary Analyses 
The safety of Bi-26 supplementation will be evaluated through EoS in the Safety population, 
which includes, but is not limited to, the frequency (and percentages) of AEs, and all SAEs 
through the Day 90 visit. Individual results and descriptive statistics will be provided by 
treatment group. 
Individual results and descriptive statistics for presence of B. infantis in stool will be provided at 
each time point by treatment group. 
 
9.7.2. 
Exploratory Analyses 
The stool and blood biomarkers at specified timepoints will be analyzed using descriptive 
statistics. 
Refer to Section 8.6 for biomarker testing. Details of the exploratory biomarker analyses, 
including potential additional exploratory engraftment analyses, will evolve from the initial test 
set to potential verification set. These will be included in separate operational and/or analysis 
plans. Results of these additional exploratory objectives may be reported separately from the 
main clinical study report. 

 
 
 
 
 
 
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10. 
Supporting Documentation and Operational Considerations 
10.1. 
Regulatory, Ethical, and Study Oversight Considerations 
10.1.1. 
Regulatory and Ethical Considerations 
This study will be conducted in accordance with the protocol and with: 
• 
consensus ethical principles derived from international guidelines including the Declaration 
of Helsinki and CIOMS International Ethical Guidelines 
• 
applicable ICH GCP Guidelines 
• 
applicable laws and regulations. 
The protocol, protocol amendments, ICF, and other relevant documents (e.g., advertisements) 
must be submitted to an EC/IRB by the investigator and reviewed and approved by the IRB/EC 
before the study is initiated. 
Any amendments to the protocol will require EC/IRB approval and approval by health 
authorities where applicable, before implementation of changes made to the study design, except 
for changes necessary to eliminate an immediate hazard to study participants. 
The investigator will be responsible for the following: 
Providing written summaries of the status of the study to the EC/IRB annually or more 
frequently in accordance with the requirements, policies, and procedures established by the 
IRB/EC 
Notifying the IRB of SAEs or other significant safety findings as required by EC/IRB 
procedures. 
Providing oversight of the conduct of the study at the site and adherence to requirements of ICH 
guidelines, the EC/IRB, and all other applicable local regulations. 
10.1.2. 
Financial Disclosure 
Investigators and sub-investigators will provide the sponsor with sufficient, accurate financial 
information as requested to allow the sponsor to submit complete and accurate financial 
certification or disclosure statements to the appropriate regulatory authorities. Investigators are 
responsible for providing information on financial interests during the course of the study and for 
1 year after completion of the study. 

 
 
 
 
 
 
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10.1.3. 
Informed Consent Process 
The investigator or his/her representative will explain the study to the participant’s 
parent(s)/legal guardian and answer all questions regarding the study. The participant’s 
parent(s)/legal guardian must be informed that their participation is voluntary.  
The participant’s parent(s)/legal guardian will be required to sign a statement of informed 
consent that meets the requirements of local regulations, ICH guidelines, and the ECs/IRBs or 
study centers. 
Note that the participant’s mother is the preferred individual to sign the informed consent given 
the protocol requirements for some feedings to be from breast milk and for collection of 
information on maternal antibiotic and probiotic usage.  
However, the participant’s other parent or legal guardian can provide consent if necessary. If the 
mother is not the consenting parent, the study requirements should be reviewed with the 
participant’s mother to ensure understanding of study activities and ability/willingness to 
comply. The participant’s mother will also sign the ICF for collection of her medical information 
described in the protocol.  
The medical record must include a statement that written informed consent was obtained before 
the participant was screened and enrolled in the study and the date the written consent was 
obtained. The authorized person obtaining the informed consent must also sign the ICF. 
Participant’s consenting parent(s)/legal guardian must be re-consented to the most current 
version of the ICF during their infant’s participation in the study. 
A copy of the ICF must be provided to the participant’s parent(s)/legal guardian prior to any 
study-related procedure. 
Any withdrawal of consent for sample testing will be documented in the eCRF. 
Refer to Section 5.3 regarding rescreening. 
10.1.3.1. Informed Consent Form 
Participant’s consenting parent/legal guardian will be told that they are free to refuse to have 
their infant participate and may withdraw their consent at any time and for any reason during the 
study period. Any test results from the samples collected before withdrawing consent may still 
be used for the study. 
The informed consent form will be obtained using a written ICF approved by the IRB/EC and 
signed and dated by the participant’s consenting parent/legal guardian. 
The ICF will contain a separate section that addresses the use of remaining mandatory samples 
for optional exploratory biomarker research. The investigator or authorized designee will explain 
the objectives of the exploratory research to the participant’s consenting parent/legal guardian.  
Participant’s consenting parent/legal guardian must agree to the exploratory research; otherwise 
the participant cannot be in the study. 

 
 
 
 
 
 
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10.1.4. 
Data Protection 
Participants will be assigned a unique identifier by the sponsor. Any participant records or 
datasets that are transferred to the sponsor will contain the identifier only; participant names or 
any information which would make the participant identifiable will not be transferred. The 
participant’s consenting parent must be informed that the infant’s medical records may be 
examined by Clinical Quality Assurance auditors or other authorized personnel appointed by the 
sponsor, by appropriate EC/IRB members, and by inspectors from regulatory authorities. 
The participant’s mother, and the other consenting parent or legal guardian if not the mother, 
must be informed that the mother’s and infant’s personal study-related data will be used by the 
sponsor in accordance with local data protection law. The level of disclosure must also be 
explained. 
10.1.5. 
Dissemination of Clinical Study Data 
10.1.6. 
Data Quality Assurance 
All participant data relating to the study will be recorded by an eCRF using an electronic data 
capture (EDC) system transmitted to the sponsor or designee electronically (e.g., laboratory 
data). The investigator is responsible for verifying that data entries are accurate and correct by 
electronically signing the eCRF. 
The investigator must maintain accurate documentation (source data) that supports the 
information entered in the eCRF. 
The study will be monitored regularly by the sponsor or its designee throughout the study period. 
The investigator must permit study-related monitoring, audits, IRB/EC review, and regulatory 
agency inspections and provide direct access to source data documents. 
Monitoring details describing strategy (e.g., risk-based initiatives in operations and quality such 
as Risk Management and Mitigation Strategies and Analytical Risk-Based Monitoring), methods, 
responsibilities and requirements, including handling of noncompliance issues and monitoring 
techniques (central, remote, or on-site monitoring) are provided in the Monitoring Plan. 
The sponsor or designee is responsible for the data management of this study including quality 
checking of the data. 
The sponsor assumes accountability for actions delegated to other individuals (e.g., CRO). 
Study monitors will perform ongoing source data verification to confirm that data entered into 
the eCRF by authorized site personnel are accurate, complete, and verifiable from source 
documents; that the safety and rights of participants are being protected; and that the study is 
being conducted in accordance with the currently approved protocol and any other study 
agreements, ICH GCP, and all applicable regulatory requirements. 

 
 
 
 
 
 
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10.1.7. 
Record Retention 
Records and documents pertaining to the conduct of this study must be retained by the 
investigator for a minimum of 10 years after study completion unless local regulations or 
institutional policies require a longer retention period. 
No records may be destroyed during the retention period without the written approval of the 
sponsor. No records may be transferred to another location or party without written notification 
to the sponsor. 
10.1.8. 
Source Documents 
Source documentation consists of existing medical records and/or study records developed and 
maintained by the investigator. Source documents provide evidence for the existence of the 
participant and substantiate the integrity of the data collected and are to be filed at the 
investigator’s site. Data recorded on source documents will be transcribed onto eCRFs using an 
EDC system. 
Data entered in the eCRF that are transcribed from source documents must be consistent with the 
source documents or the discrepancies must be explained. The investigator may need to request 
previous medical records or transfer records, depending on the study. 
For the purpose of monitoring and auditing the study, source documentation will consist of 
existing medical records and/or study records developed and maintained by the investigator. 
10.1.9. 
Study and Site Closure 
The sponsor designee reserves the right to close the study site or terminate the study at any time 
for any reason at the sole discretion of the sponsor. Study sites will be closed upon study 
completion. A study site is considered closed when all required documents and study supplies 
have been collected and a study site closure visit has been performed. 
The investigator may initiate study site closure at any time, provided there is reasonable cause 
and sufficient notice is given in advance of the intended termination. Reasons for the early 
closure of a study site by the sponsor or investigator may include but are not limited to: 
• 
failure of the investigator to comply with the protocol, the requirements of the EC/IRB 
or local health authorities, the sponsor's procedures, or GCP guidelines 
• 
inadequate recruitment of participants by the investigator 
• 
discontinuation of further study intervention development. 
At the discretion of the sponsor, all materials and supplies provided to the investigator will be 
returned or disposed of in compliance with local regulatory requirements upon authorization 
from the sponsor, upon study completion. The investigator or designated clinical site staff will 
notify the EC/IRB when the study has been completed. 

 
 
 
 
 
 
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10.1.10. 
Publication Policy 
The results of this study may be published or presented at scientific meetings. If this is foreseen, 
the investigator agrees to submit all manuscripts or abstracts to the sponsor before submission. 
This allows the sponsor to protect proprietary information and to provide comments. 
The sponsor will comply with the requirements for publication of study results. In accordance 
with standard editorial and ethical practice, the sponsor will generally support publication of 
multicenter studies only in their entirety and not as individual site data. In this case, a 
coordinating investigator will be designated by mutual agreement. 
Authorship will be determined by mutual agreement and in line with International Committee of 
Medical Journal Editors authorship requirements. 
10.1.11. 
Financing and Insurance 
Financing and insurance information is provided as a separate agreement. 
10.2. 
Adverse Events and Serious Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting 
10.2.1. 
Definition of AE 
AE Definition 
An AE is any untoward medical occurrence in a patient or clinical study participant, 
temporally associated with the use of study intervention, whether or not considered related to 
the study intervention. 
NOTE: An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the 
use of study intervention. 
Events Meeting the AE Definition 
Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or other 
safety assessments (e.g., ECG, radiological scans, vital signs measurements), including those 
that worsen from baseline, considered clinically significant in the medical and scientific 
judgment of the investigator (i.e., not related to progression of underlying disease). 
Exacerbation of a chronic or intermittent pre-existing condition including either an increase in 
frequency and/or intensity of the condition. 
New conditions detected or diagnosed after study intervention administration even though it 
may have been present before the start of the study. 
Signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction. 
Signs, symptoms, or the clinical sequelae of a suspected overdose of either study intervention 
or a concomitant medication. Overdose per se will not be reported as an AE/SAE unless it is 

 
 
 
 
 
 
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an intentional overdose taken with possible suicidal/self-harming intent. Such overdoses 
should be reported regardless of sequelae. 
Events NOT Meeting the AE Definition 
Any clinically significant abnormal laboratory findings or other abnormal safety assessments 
which are associated with the underlying disease, unless judged by the investigator to be more 
severe than expected for the participant’s condition. 
The disease/disorder being studied or expected progression, signs, or symptoms of the 
disease/disorder being studied, unless more severe than expected for the participant’s 
condition. 
Medical or surgical procedure (e.g., endoscopy, appendectomy): the condition that leads to the 
procedure is the AE. 
Situations in which an untoward medical occurrence did not occur (social and/or convenience 
admission to a hospital). 
Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or 
detected at the start of the study that do not worsen. 
10.2.2. 
Definition of SAE 
If an event is not an AE per definition above, then it cannot be an SAE even if serious conditions 
are met.  
An SAE is defined as any untoward medical occurrence that, at any dose: 
i. 
Results in death 
j. 
Is life-threatening 
The term 'life-threatening' in the definition of 'serious' refers to an event in which the 
participant was at risk of death at the time of the event. It does not refer to an event, which 
hypothetically might have caused death, if it were more severe. 
k. Requires inpatient hospitalization or prolongation of existing hospitalization 
In general, hospitalization signifies that the participant has been detained (usually involving at 
least an overnight stay) at the hospital or emergency ward for observation and/or treatment that 
would not have been appropriate in the physician’s office or outpatient setting. Complications 
that occur during hospitalization are not necessarily SAEs. If a complication prolongs 
hospitalization or fulfills any other serious criteria, the event is serious. When in doubt as to 
whether “hospitalization” occurred or was necessary, it should be considered as an SAE. 
Hospitalization for elective treatment of a pre-existing condition that did not worsen from 
baseline is not considered an adverse event. 
l. 
Results in persistent disability/incapacity 

 
 
 
 
 
 
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• The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions. 
• This definition is not intended to include experiences of relatively minor medical 
significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, 
and accidental trauma (e.g., sprained ankle) which may interfere with or prevent 
everyday life functions but do not constitute a substantial disruption. 
m. Is a congenital anomaly/birth defect 
n. Other situations: 
• Medical or scientific judgment should be exercised in deciding whether SAE reporting 
is appropriate in other situations such as important medical events that may not be 
immediately life-threatening or result in death or hospitalization but may jeopardize the 
participant or may require medical or surgical intervention to prevent one of the other 
outcomes listed in the above definition. These events should usually be considered 
serious. 
Examples of such events include invasive or malignant cancers, intensive treatment in 
an emergency room or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalization, or development of drug dependency or 
drug abuse. 
10.2.3. 
Recording and Following Up SAEs 
 SAE Recording 
• Care will be taken not to introduce bias when detecting SAEs. Open-ended and 
non-leading verbal questioning of the participant is the preferred method to inquire 
about occurrences. 
• When an SAE occurs, it is the responsibility of the investigator to review all 
documentation (eg, hospital progress notes, laboratory reports, and diagnostics reports) 
related to the event. 
• The investigator will then record all relevant SAE information in the eCRF. 
• After the initial SAE report, the investigator is required to proactively follow each 
participant at subsequent visits/contacts. All SAEs will be followed until resolution, 
stabilization, the event is otherwise explained, or the participant is lost to follow-up. 
• It is not acceptable for the investigator to send photocopies of the participant’s medical 
records to the sponsor in lieu of completion of the SAE eCRF page. 
• There may be instances when copies of medical records for certain cases are requested 
by the Medical Monitor, the IDMC or the sponsor. In this case, all participant 
identifiers, with the exception of the participant number, will be redacted on the copies 
of the medical records before submission. 

 
 
 
 
 
 
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• The investigator will attempt to establish a diagnosis of the event based on signs, 
symptoms, and/or other clinical information. Whenever possible, the diagnosis (not the 
individual signs/symptoms) will be documented as the SAE. 
• SAEs will be assessed for intensity and causal relationship to the study intervention.  
SAE Follow-up and Resolution 
The investigator is obligated to perform or arrange for the conduct of supplemental 
measurements and/or evaluations as medically indicated to elucidate the nature and/or 
causality of the SAE as fully as possible. This may include additional laboratory tests or 
investigations, histopathological examinations, or consultation with other health care 
professionals. 
If a participant dies during participation in the study or during a recognized follow-up 
period, the investigator will provide the sponsor with a copy of any post-mortem findings 
including histopathology, if available. 
New or updated information will be recorded in the originally completed eCRF. 
The investigator will submit any updated SAE data to the sponsor within 24 hours of 
receipt of the information. 
The onset and resolution dates of the event and medical care taken in response to the event 
will be documented. 
SAEs will be considered resolved when the condition returns to normal or returns to the 
participant’s baseline status as established on Study Day 1, or when the condition has 
stabilized with the expectation that it will remain chronic. 
If the event has not resolved by the final study visit, it will be documented as “ongoing” on 
the eCRF, however, follow-up of the SAE must continue until resolved or the condition has 
stabilized. Information recorded on the eCRF must be substantiated in the source 
documents. 
The resolution date to be recorded on the eCRF is the last date on which the participant 
experienced the SAE. 

 
 
 
 
 
 
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Assessment of Intensity 
The investigator will make an assessment of intensity for each SAE reported during the study 
and assign it to 1 of the following categories: 
• Grade 1, Mild: An event that is easily tolerated by the participant, causing minimal 
discomfort and not interfering with everyday activities. 
• Grade 2, Moderate: An event that causes sufficient discomfort and interferes with 
normal everyday activities. 
• Grade 3, Severe: An event that prevents normal everyday activities. An AE that is 
assessed as severe should not be confused with a SAE. Severe is a category utilized for 
rating the intensity of an event; and both AEs and SAEs can be assessed as severe. 
• Grade 4 Potentially life-threatening symptoms, causing inability to perform basic self-
care functions with intervention indicated to prevent permanent impairment, persistent 
disability, or death. 
An event is defined as ‘serious’ when it meets at least 1 of the predefined outcomes as 
described in the definition of an SAE, NOT when it is rated as severe. 
Assessment of Causality 
The investigator is obligated to assess the relationship between study intervention and each 
occurrence of each SAE. The investigator will assess whether the SAE is either: 
Related: An SAE is considered related to study intervention if there is a reasonable possibility 
that the study intervention contributed to the SAE. 
or 
Not related: There is no reasonable possibility that the SAE is causally related to 
administration of the study intervention. There are other more likely causes for the SAE. 
The investigator will use clinical judgment to determine the relationship. 
Alternative causes, such as underlying disease(s), concomitant therapy, and other risk factors, 
as well as the temporal relationship of the event to study intervention administration will be 
considered and investigated. 
For each SAE, the investigator must document in the medical notes that he/she has reviewed 
the SAE and has provided an assessment of causality. There may be situations in which an 
SAE has occurred and the investigator has minimal information to assess causality to include 
in the initial report. However, it is very important that the investigator always provide an 
assessment of causality with the initial submission of the SAE data. 
The investigator may change his/her assessment of causality when considering additional 
follow-up information and submit an SAE follow-up report with the updated causality 
assessment. The causality assessment is one of the criteria used when determining regulatory 
reporting requirements. 

 
 
 
 
 
 
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Assessment of Outcome 
The outcome of each SAE must be reported to the sponsor. For analysis purposes, the outcome 
for serious adverse events will be determined on the final study visit. 
Outcome of all SAEs will be classified as one of the following: 
•Resolved 
•Resolved with sequelae 
•Ongoing 
•Death. 
10.2.4. 
Reporting SAEs 
SAE Reporting to CRO via an Electronic Data Collection Tool 
• The primary mechanism for reporting an SAE to the CRO will be the electronic data 
collection tool. 
• If the electronic system is unavailable, then the site will use the paper SAE data 
collection tool (see next section) in order to report the event within 24 hours. 
• The site will enter the SAE data into the electronic system as soon as it becomes 
available. 
• After the study is completed at a given site, the electronic data collection tool will be 
taken off-line to prevent the entry of new data or changes to existing data. 
• If a site receives a report of a new SAE from a study participant or receives updated 
data on a previously reported SAE after the electronic data collection tool has been 
taken off-line, then the site can report this information on a paper SAE form (see next 
section) or to the Medical Monitor by telephone. 
• Contacts for SAE reporting can be found in the Study Manual. 
SAE Reporting to CRO via Paper Case Report Form 
• Email transmission of the SAE paper CRF is the preferred method to transmit this 
information. 
• In rare circumstances and in the absence of email, notification by telephone is 
acceptable with a copy of the SAE data collection tool sent by overnight mail or 
courier service. 
• Initial notification via telephone does not replace the need for the investigator to 
complete and sign the SAE CRF pages within the designated reporting time frames. 
• Contacts for SAE reporting can be found in the Study Manual. 

 
 
 
 
 
 
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11. 
References 
AlFaleh K, Anabrees J. Probiotics for prevention of necrotizing enterocolitis in preterm infants. 
Cochrane Database Syst Rev. 2014 Apr 10;(4):CD005496. doi: 
10.1002/14651858.CD005496.pub4. Update in: Cochrane Database Syst Rev. 2020 Oct 
15;10:CD005496. PMID: 24723255. 
Arrieta MC, Stiemsma LT, Amenyogbe N, et al. The intestinal microbiome in early life: health 
and disease. Front Immunol. 2014;5:427. doi: 10.3389/fimmu.2014.00427. 
Barratt MJ, Nuzhat S, Ahsan K, et al. Bifidobacterium infantis treatment promotes weight gain in 
Bangladeshi infants with severe acute malnutrition. Science Translational Medicine 
2022;14(640). doi 10.1126/scitranslmed.abk1107. 
Black RE, Allen LH, Bhutta ZA, et al. Maternal and Child Undernutrition Study Group. 
Maternal and child undernutrition: global and regional exposures and health consequences. 
Lancet. 2008 Jan 19;371(9608):243-60. doi: 10.1016/S0140-6736(07)61690-0. 
Black RE, Victora CG, Walker SP, et al. Maternal and Child Nutrition Study Group. Maternal 
and child undernutrition and overweight in low-income and middle-income countries. Lancet. 
2013;382(9890):427-451. doi:10.1002/14651858. 
Brink L, Mercer K, Piccolo B, et al. Neonatal diet alters fecal microbiota and metabolome 
profiles at different ages in infants fed breast milk or formula. Am J Clin Nutr. 
2020;111(6):1190-1202. doi: 10.1093/ajcn/nqaa076. 
Bwakura-Dangarembizi M, Dumbura C, Amadi B, et al.Risk factors for postdischarge mortality 
following hospitalization for severe acute malnutrition in Zimbabwe and Zambia. Am J Clin 
Nutr. 2021;113:665–674. 
Casaburi G, Duar R, Brown H, et al Metagenomic insights of the infant microbiome community 
structure and function across multiple sites in the United States. Scientific Reports. 
2021;11(1):1472. 
Chichlowski M, Shh N, Wampler J, et al. Bifidobacterium longum Subspecies infantis (B. 
infantis) in Pediatric Nutrition: Current State of Knowledge. Nutrients. 2020;12(6):1581. 
https://doi.org/10.3390/nu12061581. 
Chisti MJ, Graham SM, Duke T, et al. Post-discharge mortality in children with severe 
malnutrition and pneumonia in Bangladesh. PLoS One. 2014;9(9):e107663. Published 2014 Sep 
16. doi:10.1371/journal.pone.0107663. 
Chowdhury R, Nitika, Choudhary TS, et al. Diagnostic measures for severe acute malnutrition in 
Indian infants under 6 months of age: a secondary data analysis. BMC Pediatrics.2021;21:158. 
doi.org/10.1186/s12887-021-02629-9. 
Duar R, Henrick B, Casaburi, G. et al. Integrating the ecosystem services framework to define 
dysbiosis of the breastfed infant gut: the role of B. infantis and human milk oligosaccharides. 
Front Nutr. 2020;7:33. 

 
 
 
 
 
 
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Fleming P, Berrington J, Jacobs S. Addressing safety concerns of probiotic use in preterm 
babies. Early Hum Dev. 2019;135:72-74. doi: 10.1016/j.earlhumdev.2019.05.016. 
Frese S, Hutton A, Contreras L, et al. Persistence of Supplemented Bifidobacterium longum 
subsp. infantis EVC001 in Breastfed Infants. Amer Soc for Microbiol Msphere. 
2017;2(6):e00501-17.Doi: 10.1128/msphere.00501-17. 
Garland SM, Tobin JM, Pirotta M, Tabrizi SN, Opie G, Donath S, Tang ML, Morley CJ, Hickey 
L, Ung L, Jacobs SE; ProPrems Study Group. The ProPrems trial: investigating the effects of 
probiotics on late onset sepsis in very preterm infants. BMC Infect Dis. 2011;11:210. doi: 
10.1186/1471-2334-11-210. PMID: 21816056; PMCID: PMC3199779. 
Henrick B, Chew S, Casaburi G, et al. Colonization by B. infantis EVC001 modulates enteric 
inflammation in exclusively breastfed infants. Pediatr Res.2019;86:749–757. 
https://doi.org/10.1038/s41390-019-0533-2. 
Henrick B, Rodriguez L, Lakshmikanth T, et al. Bifidobacteria-mediated immune system 
imprinting early in life. Cell. 2021;184:1-15. 
Hill C, Guarner F, Reid G et al. The International Scientific Association for Probiotics and 
Prebiotics consensus statement on the scope and appropriate use of the term probiotic. 2014 
Nature vol 11 506-514. Nat Rev Gastroenterol Hepatol. 2014;11:506–514. 
doi.org/10.1038/nrgastro.2014.66. 
Hoehn C, Lelijveld N, Mwangome M, et al. Criteria for identifying infants under 6 months of age 
at risk of morbidity and mortality: a systematic review - 2021 Clinical Medicine Insights: 
Pediatrics. January 2021. doi:10.1177/11795565211049904. 
Jacobs SE, Tobin JM, Opie GF, et al, ProPrems Study Group. Probiotic effects on late-onset 
sepsis in very preterm infants: a randomized controlled trial. Pediatrics. 2013 Dec;132(6):1055-
62. doi: 10.1542/peds.2013-1339. 
Kane AV, Dinh DM, Ward HD. Childhood malnutrition and the intestinal microbiome. Pediatr 
Res. 2015;77(1-2):256-62. doi: 10.1038/pr.2014.179. 
Kerac M, Mwangome M, McGrath M, et al. Management of acute malnutrition in infants aged 
under 6 months (MAMI): current issues and future directions in policy and research. Food And 
Nutrition Bulletin 2015;36(1_suppl1):S30-S34. 
Kirolos A, Blacow RM, Parajuli A, et al. The impact of childhood malnutrition on mortality 
from pneumonia: a systematic review and network meta-analysis. BMJ Glob Health. 
2021;6(11):e007411. doi:10.1136/bmjgh-2021-007411. 
Halloran K, Underwood M. Probiotic mechanisms of action. Early Human Development. 
2019;135:58-65.doi.org/10.1016/j.earlhumdev.2019.05.010. 
Munirul Islam M, Arafat Y, Connell N, et al. Severe malnutrition in infants aged <6 months-
Outcomes and risk factors in Bangladesh: A prospective cohort study. Matern Child Nutr. 
2019;15(1):e12642. doi:10.1111/mcn.12642. 
Mwangome M, Ngari M, Fegan G, et al. Diagnostic criteria for severe acute malnutrition among 
infants aged under 6 mo. Am J Clin Nutr. 2017 Jun;105(6):1415-1423. doi: 
10.3945/ajcn.116.149815. 

 
 
 
 
 
 
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Mwangome M, Ngari M, Bwahere P, et al. Anthropometry at birth and at age of routine 
vaccination to predict mortality in the first year of life: A birth cohort study in Burkina Faso. 
PLoS One. 2019;14(3):e0213523. doi: 10.1371/journal.pone.0213523. PMID: 30921335. 
Nguyen M, Holdbrooks H, Mishra P, et al. Impact of Probiotic B. infantis EVC001 Feeding in 
Premature Infants on the Gut Microbiome, Nosocomially Acquired Antibiotic Resistance, and 
Enteric Inflammation. Front Pediatr. 2021 Feb 16;9:618009. doi: 10.3389/fped.2021.618009.  
Sanni M, Neureiter K (CDC Group). Insight: How innovation in off-grid refrigeration impacts 
lives in Kenya Impact Study: 007. 28 October 2019. https://assets.cdcgroup.com/wp-
content/uploads/2019/10/29165356/How-innovation-in-off-grid-refrigeration-impacts-lives-in-
Kenya.pdf. 
Smilowitz JT, Moya J, Breck M, et al. Safety and tolerability of Bifidobacterium longum 
subspecies infantis EVC001 supplementation in healthy term breastfed infants: a phase I clinical 
trial. BMC Pediatr. 2017;17(1):133. doi: 10.1186/s12887-017-0886-9. Erratum in: BMC Pediatr. 
2017 Aug 15;17 (1):180. PMID: 28558732; PMCID: PMC5450358. 
World Health Organization (WHOa). Global Health Observatory Data repository: Prevalence of 
underweight children under 5 years of age (% weight-for-age <-2 SD) (%) (country survey 
results) (who.int)]. https://apps.who.int/gho/data/view.main.CHILDUNDERWEIGHTv. 07Apr 
2021. Accessed on 31 March 2022. 
WHOb. WHO fact sheet. Malnutrition. 09 Jun 2021. https://www.who.int/news-room/fact-
sheets/detail/malnutrition. Accessed on 31 March 2022. 
WHO. Breastfeeding: recommendations. https://www.who.int/health-
topics/breastfeeding#tab=tab_1. Accessed on 31 March 2022. 
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Report. World Bank, Washington DC. © World Bank. License: Creative Commons 
Attribution—NonCommercial 3.0 IGO (CC BY-NC 3.0 IGO). 2022 International Bank for 
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World Hunger Organization. World Child Hunger Facts. https://www.worldhunger.org. 
Accessed on 31 March 2022.

 
 
 
 
 
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12. 
Protocol Amendments 
Overall Rationale for changes in Version 3.0: 
The primary purpose of this protocol update was to combine the enrollment activities with the 
Day 1 (randomization) visit and clarify other study procedures. The major changes incorporated 
into this protocol (Version 3.0) relative to the prior approved version (Version 2.0) are 
summarized below. Editorial and formatting changes are not included in this summary. 
Section # and 
Name 
Description of Change 
Brief Rationale 
Page 3  
Added Principal Investigator signatory page. 
To have signature recorded 
together with other study 
documents in a single 
location  
Throughout the 
document 
Removed the word “unsolicited” before ‘AEs’. 
This study does not include 
solicited AEs, so all AEs are 
unsolicited. However, to 
avoid any confusion, the 
word “unsolicited” was 
removed to clarify that all 
AEs will be recorded. 
Synopsis, 
Objectives and 
Endpoints (Table 1) 
and Section 9.7 
Table 5 Secondary 
Estimands 
Updated text in Table 1 and Table 5 to align 
wording across the tables. 
For clarification 
Synopsis, Schedule 
of Activities (SOA 
Table 2) and Main 
Study Schema 
(Figure 1), and 
Section 4.1  
Combined enrollment activities with the Day 1 
(randomization) visit. Added text to indicate 
that screening may occur on Day 1 with 
enrollment and randomization, or separately 
within the protocol-specified window.  
To clarify the timing of 
screening, enrollment, and 
randomization activities  
Synopsis, SoA 
(Table 2) 
Updated procedural details to align throughout 
the protocol. Updates include separating 
medication collection for the infant and the 
infant’s mother, specifying time points for 
provision and collection of diaries, and 
addition of explanatory footnotes. 
For clarification  
Section 4.4 End of 
Study Definition 
Clarified the definition of study completion for 
an individual participant. 
For clarification 

 
 
 
 
 
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Section # and 
Name 
Description of Change 
Brief Rationale 
Section 5.3 Screen 
Failures 
and  
Section 10.1.3 
Informed Consent 
Process 
Replaced text that stated ‘rescreening will not 
be allowed’, to allow for rescreening if 
screening visit is outside of the 7-day planned 
window, due to a delay in hospital discharge.  
For flexibility during the 
screening process  
Section 5.1 
Inclusion criteria 
Specified that enrollment is on Day 1. 
To clarify when the 
participant is enrolled in the 
study 
Section 5.1and 5.2, 
Inclusion/exclusion 
criteria, Section 6 
Study intervention 
and throughout 
document 
Replaced “mother” with “participant’s 
parent/legal guardian” unless only the mother 
can perform a particular task. 
To allow more flexibility at 
participant’s home so that 
another parent or legal 
guardian may perform some 
study activities 
Section 6.2Measures 
to Minimize Bias: 
Randomization and 
Blinding 
Clarified individuals who will be blinded and 
unblinded. 
For clarification 
Section 6.3 Study 
Intervention 
Compliance 
Removed confirmation of refrigeration 
temperature using a temperature logger. 
Temperature data loggers will 
not be used to monitor the 
study intervention.  
Section 6.4 Feeding 
history 
Added details to be recorded, including 
whether the full dose was taken, and whether a 
repeat dose was administered.  
For clarification 
Section 7.1 
Discontinuation  
Added details regarding investigator decision 
to stop the study intervention for an individual 
participant. 
For clarification 
Section 7.3 
Replacement of 
Participants 
Added new section “Replacement of 
Participants” and a statement that participants 
will not be replaced. 
To specify that participants 
will not be replaced if early 
discontinuation from study 
occurs 
Section 8.1 
Demographic and 
Baseline 
Characteristics 
Reworded text and added details regarding 
what will be collected at screening and at Day 
1. 
For clarification 
Section 8.3.3 Vital 
Signs 
Replaced respiratory rate with pulse oximetry 
For clarification 

 
 
 
 
 
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Section # and 
Name 
Description of Change 
Brief Rationale 
Section 8.4.5 
Independent Data 
Monitoring 
Committee (IDMC) 
Added further details about possible IDMC 
recommendations and about sponsor decision 
to terminate the study for safety reasons. 
For clarification 
Section 10.1.11 
Financial Disclosure  
Added this new section. 
For clarification 
Section 10.1.3 
Informed Consent 
Process 
Included a statement that the participant’s 
mother will also sign the Informed Consent 
Form to allow collection of her medical 
information. 
For clarification 
Protocol Version 
History and 
Summary of 
Changes page 4, and  
Section 12 Protocol 
Amendments 
Moved text from page 3 to newly created 
Section 12, Protocol Amendments. 
To move details from 
previous amendment changes 
to end of document and 
provide new amendment 
changes at the beginning of 
the document 
Minor editorial changes were made throughout the protocol for corrections or to clarify text. 

 
 
 
 
 
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CONFIDENTIAL 
 
 
 
Overall Rationale for changes in Version 2.0: 
The protocol was amended to include a sub-study to monitor the temperatures of the refrigerators 
used in the main study. The aim of this optional, prospective, observational sub-study is to 
collect information on reliability of refrigeration in the geographic areas where the study is being 
conducted and where B. infantis supplementation may be recommended by WHO, if supported 
by clinical data. As of 2020, 733 million people did not have access to electricity, and over 75% 
of people living in sub-Saharan Africa did not have access. In order to get a geographical cross-
representation in this sub-study, each clinical site will be asked to enroll up to 20% of their 
overall study population into the sub-study. 
Section # and 
Name 
Description of Change 
Brief Rationale 
1.1 Synopsis 
4.4 Optional 
Sub-Study 
and Table 2 
Schedule of 
Activities. 
This section was added to the 
protocol to specify use of a 
temperature data logger over a 2-
week period in the home 
refrigerators of a subset of the study 
participants. 
To collect information on reliability of 
refrigeration in the geographic areas where 
the study is being conducted. 
Minor changes were made throughout the protocol including the following: 
Throughout 
protocol 
 
The term ‘formula’ was replaced 
with ‘water’ for product 
reconstitution. 
 
To specify that water can be used to mix with 
the Bi-26 or placebo, instead of formula, if 
breastmilk is not available for a given dose. 
Section 2.1 
Background 
Text added to clarify GRAS status 
information. 
 
To provide additional information on the 
GRAS designation 
Section 2.2. 
Risk Benefit 
Assessment 
New data from the SYNERGIE 
study added. 
To provide evidence to support the potential 
for positive clinical outcomes with B. infantis 
supplementation. 
Section 8.6 
Biomarkers 
Blood volume changed from 2 mL to 
4mL. 
To allow for adequate blood volume for 
testing.  
Minor editorial changes were made throughout the protocol for corrections or to clarify text. 
 
 

Signature Page for:
Gates MRI-MNK01-301 Protocol Version 4.0 (12 Sep 2022)
Gateway RIM Document #:
MNK01-CLIN-000003 v4.0
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