Effect of Probiotic on Rhinovirus Induced Colds
Phase: NA
Conditions: Common Cold
Sponsor: University of Virginia

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Version 5.0 
13Oct2017 
 
 
GCP SOP EPRIC 
_________________________________________________________________________________ 
 
SOP # 200 Form B 
Version no. 02 
Date: 08 December 2014 
 
Page 1 of 40 
DuPont – CRIM JB0004 
 
 
Effect of Probiotic on Rhinovirus Induced Colds 
 
NCT 02679807 
 
13 October 2017 
 

 
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Effect of Probiotic on 
 Rhinovirus Induced Colds 
 
 
 
 
Protocol number:       MK EPRIC 
 
Sponsor:                     Danisco Sweeteners Oy 
 
Sponsor Address:    Active Nutrition 
                                          Sokeritehtaantie 20 
                                          FI-02460 Kantvik, Finland 
 
Protocol version number:  5.0 
 
Date of issue:                    <October 13, 2017> 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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DECLARATIONS OF SPONSOR AND INVESTIGATORS 
 
Declaration of sponsor 
 
This clinical study protocol was subject to critical review and has been approved by the 
sponsors. The information it contains is consistent with: 
• 
The current risk-benefit evaluation of the study product 
• 
The moral, ethical, and scientific principles governing clinical research as set out in the 
Declaration of Helsinki and the principles of GCP as described in the ICH Harmonized Tripartite 
Guideline Topic E6: ‘’Guidelines for Good Clinical Practice’’, as well as in the applicable local 
guidelines. 
 
The investigator will be supplied with details of any significant or new findings, including 
adverse events, relating to treatment with the study product. 
 
Declaration of the investigators 
 
I confirm that I have read the above protocol.  I understand it, and I will work in accordance 
with the moral, ethical, and scientific principles governing clinical research as set out in the 
Declaration of Helsinki and GCP guidelines. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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RESPONSIBLE  PERSONNEL 
Principal Investigator 
Ronald B. Turner, MD, 
Department of Pediatrics P.O. 
Box 800386 University of Virginia 
School of Medicine 
Charlottesville, Virginia 22908, 
RBT2N@hscmail.mcc.virginia.edu 
Telephone: +1-434-243-9864, 
Fax:  +1-434-982-4246 
Study Manager / 
Immunoanalyses, 
Sponsor 
Markus Lehtinen, PhD, R&D 
Manager, Sokeritehtaantie 20 
02460 Kantvik, Finland, 
markus.lehtinen@danisco.com, 
Telephone: +358 40 1397813, 
Fax: +358 10 431 5555 
Project Manager 
 
 
Liisa Lehtoranta, PhD, Scientist 
Sokeritehtaantie 20 
02460 Kantvik, Finland 
Telephone: +358 407 667 418 
Fax: +358 10 431 5555 
liisa.lehtoranta@dupont.com 
Study Statistician 
Teppo Huttunen 
4Pharma, Ltd 
Lemminkäisenkatu 1 
FI-20520 TurkuFinland 
Telephone: +358 40 582 3793 
teppo.huttunen@4pharma.com 
Database Manager 
Jari Turunen 
4Pharma, Ltd 
Lemminkäisenkatu 1 
FI-20520 Turku 
Finland 
Telephone: +358 44 029 7783 
jari.turunen@4pharma.com 
Study Monitor 
Jackie Edwards, RN, MHA, 
CCRA,  
Office: 434 985-9890 
Mobile: 434 249-2564 
Fax: 434 990-5772 
jedwardscra@yahoo.com 
 
 

 
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PROTOCOL SYNOPSIS 
 
 
 
Study title 
Effect of Probiotic on Rhinovirus Induced 
Colds 
Study phase 
II 
Study objectives 
To study the effect of probiotic 
supplementation on common cold illness 
episodes induced by rhinovirus. 
Study population 
Healthy adults, aged 18-60 
Inclusion and exclusion 
criteria 
INCLUSION CRITERIA AT ENROLLMENT: 
• 
Subject must be 18-60 years of age 
• 
Subject must read and sign a copy of the 
approved Consent Form  
INCLUSION CRITERIA AT DAY -28 
• 
Female subjects must be using an effective 
birth control method 
INCLUSION CRITERIA AT CHALLENGE: 
• 
Female subjects must be using an effective 
birth control method 
• 
Subject must have a serum neutralizing 
antibody titer of less than or equal to 1:4 to 
rhinovirus type 39 
 
EXCLUSION CRITERIA AT ENROLLMENT: 
 
EXCLUSION CRITERIA AT DAY –28 
• 
Antibiotic use within 3 months prior to study 
start 
• 
Female subjects with a positive urine 
pregnancy screen 
• 
History of use of probiotics in the preceding 
two weeks  
• Current cancer diagnosis or 
immunosuppressive therapy in the last 6 
months 

 
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• 
Any clinically significant abnormalities of the 
upper respiratory tract 
• 
Any clinically significant acute or chronic 
respiratory illness 
• 
Any clinically significant bleeding tendency 
by history 
• 
Hypertension that requires treatment with 
antihypertensive medications 
• 
History of angina or other clinically significant 
cardiac disease 
• 
Any medical condition that in the opinion of 
the Principal Investigator is cause for 
exclusion from the study 
• 
History of regular use (more than 3 days in 
7) of tobacco products within the preceding 
two weeks 
• 
History of drug or alcohol abuse in the 6 
months preceding the study 
EXCLUSION CRITERIA AT CHALLENGE: 
• 
Any upper respiratory infection or allergic 
rhinitis in the two weeks prior to the 
challenge 
• 
Any medical condition that in the opinion of 
the Principal Investigator is cause for 
exclusion from the study 
• 
Positive pregnancy screen prior to challenge 
• 
Use of any anti-inflammatory (steroids or 
NSAIDs) or cough/cold or allergy preparation 
in the two weeks prior to the challenge 
Study design 
Randomized, double-blinded, placebo-controlled 
Study product 
Bifidobacterium animalis subsp. lactis Bl-04 as 
powder mixed into drink. 
Response variables and 
endpoints 
• Primary efficacy 
Primary efficacy 
• Incidence of clinically defined rhinovirus- 
associated cold episodes 

 
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• Secondary efficacy 
• Exploratory endpoints 
 
Secondary efficacy 
• Duration of illness episodes 
• Infection rate 
• Viral load 
• Proportion shedding the virus 
Exploratory endpoints 
• Blood transcriptomics 
• Fecal, nasal, and throat microbiota 
• Nasal cytokine response 
• Cold questionnaire comparison 
Safety 
AEs and SAEs will be followed throughout the trial 
Statistics 
Sample size  
A sample size of 95 completed subjects/arm would 
be expected to detect a reduction in rhinovirus-
induced illness of 20% (from 60% to 40%) with 80% 
power with pα= 0.05. 
 
Methdology 
The primary endpoint, the incidence of rhinovirus-
associated illness episodes between the treatment 
groups will be tested with a chi-square test and 
logistic regression model. 
 
T-test, Mann-Whitney U test, chi-square test and 
ANOVA will be used for secondary parameters. 
 
Descriptive statistics for safety and other variables 
will be reported. 
 
 
 
 
 
 

 
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ABBREVIATIONS 
 
 
 
ADR 
Adverse Drug Reaction 
AE 
Adverse Event 
Bl-04 
Bifidobacterium animalis subsp. lactis Bl-04 
eCRF 
Electronic Case Report Form 
CSR 
Clinical Study Report 
DMP 
Data Management Plan 
DVP 
Data Validation Plan 
EIA 
Enzyme Immunoassay 
ELISA 
Enzyme Linked Immunosorbent Assay 
GCP 
Good Clinical Practice 
HLA 
Human Leukocyte Antigen 
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonisation 
IEC 
Independent Ethical Committee 
IP 
Investigational Product 
IRB 
Institutional Review Board 
ITT 
Intented To Treat 
MedDRA 
Medical Dictionary for Regulatory Activities 
NSAID 
Nonsteroidal anti-inflammatory drugs 
PBMC 
Peripheral Blood Mononuclear Cells 
qPCR 
Quantitative Polymerase Chain Reaction 
RV 
Rhinovirus 
SAE 
Serious Adverse Event 
TMF 
Trial Master File 
 
 
 
 
 
 
 
 
 
 
 

 
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SCHEDULE OF EVENTS 
 
 
Screening 
 
Day  
-28 
Day 
-21 
Day 
-14 
Day 
-7 
Day 
0 
Day 
1 
Day 
2 
Day 
3 
Day 
4 
Day 
5 
Day 
6-14 
Day 
21-
28 
Consent 
x 
 
 
 
 
 
 
 
 
 
 
 
 
Review of 
inclusion/exclusion 
x 
 
 
 
 
x 
 
 
 
 
 
 
 
Serum neutralizing 
antibody 
x* 
 
 
 
 
x 
 
 
 
 
 
 
x 
Urine Pregnancy 
Screen for females 
 
x 
 
 
 
x 
 
 
 
 
 
 
 
Start of dietary run-in 
period 
x 
 
 
 
 
 
 
 
 
 
 
 
 
Compliance check/ 
resupply 
 
 
x 
x 
x 
x 
 
 
 
 
 
 
 
Probiotic/placebo 
treatment 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
Assessment of blinding 
 
 
 
 
 
x 
 
 
 
 
 
 
 
Nasal swab for 
microbiomics 
 
x 
 
 
 
x 
 
 
x 
 
 
 
 
Throat swab for 
microbiomics 
 
x 
 
 
 
x 
 
 
x 
 
x 
x(14) 
 
Throat swab for culture 
 
 
 
 
 
x 
 
 
 
 
x 
 
 
Fecal sample for 
microbiomics 
 
 
 
 
 
x 
 
 
 
 
 
 
 
Blood for transriptomics 
 
x 
 
 
 
x 
 
x 
 
 
 
 
 
Nasal lavage for viral 
PCR 
 
 
 
 
 
x 
 
 
 
 
 
 
 
Nasal lavage for IL-
8/multiplex 
 
x 
 
x 
 
x 
x 
x 
x 
x 
x 
 
 
Symptom assessment 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
 
 
Rhinovirus challenge 
 
 
 
 
 
x 
 
 
 
 
 
 
 
Nasal lavage for 
quantitative RV culture 
 
 
 
 
 
 
x 
x 
x 
x 
x 
 
 
Symptom diary 
 
 
 
 
 
 
 
 
 
 
 
x 
 
Assessment of Adverse 
Events 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Assessment of 
Concomitant 
Medications 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
x 
* Volunteers will be screened for neutralizing antibody under an existing screening protocol at 
the University of Virginia (IRB-HSR #9948).  Volunteers who are found to have antibody titers 
≤1:4 to the study virus will be invited to consent for participation in the EPRIC study.  Only 
volunteers who were tested more than 12 weeks prior to the screening visit will have 
serology repeated at this visit. 
 
 
 
 

 
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TABLE OF CONTENTS 
 
Contents 
1. 
INTRODUCTION ......................................................................................................................... 13 
1.1 
BACKGROUND .................................................................................................................................. 13 
1.2 
STUDY PRODUCT .............................................................................................................................. 14 
1.3 
SUMMARY OF KNOWN POTENTIAL RISKS AND BENEFITS TO HUMAN SUBJECTS ................................. 14 
2 
STUDY OBJECTIVES ................................................................................................................. 16 
2.1 
PURPOSE OF THE TRIAL .................................................................................................................... 16 
2.2 
OBJECTIVES ..................................................................................................................................... 16 
2.2.1 
Primary objective ............................................................................................................................ 16 
2.2.2 
Secondary objectives ....................................................................................................................... 16 
2.2.3 
Exploratory objectives ..................................................................................................................... 16 
3 
STUDY POPULATION ................................................................................................................ 16 
3.1 
INCLUSION CRITERIA ....................................................................................................................... 16 
3.2 
EXCLUSION CRITERIA ...................................................................................................................... 17 
3.3 
RECRUITMENT, SCREENING, AND RANDOMIZATION.......................................................................... 17 
3.4 
SUBJECT WITHDRAWAL AND REPLACEMENT OF SUBJECTS ............................................................... 18 
3.4.1 
Withdrawal criteria ......................................................................................................................... 18 
3.4.2 
Withdrawn subject data collection .................................................................................................. 18 
3.4.3 
Replacement of subjects .................................................................................................................. 18 
4 
EXPERIMENTAL DESIGN ........................................................................................................ 19 
4.1 
OVERALL STUDY DESIGN ................................................................................................................ 19 
4.1.1 
Number of subjects .......................................................................................................................... 19 
4.1.2 
Study center ..................................................................................................................................... 19 
4.2 
ASSESSMENTS AND PROCEDURES .................................................................................................... 19 
4.2.1 
Study Conduct ................................................................................................................................. 19 
4.2.2 
Study periods ................................................................................................................................... 19 
4.2.3 
Pre-study safety assessment (Screening) ......................................................................................... 21 
4.2.4 
Nasal lavage (Study Day -28, and 0-5) ........................................................................................... 21 
4.2.5 
Blood for transcriptomics (Study Days -28, 0, and 2) ..................................................................... 21 
4.2.6 
Nasal and throat swab sample collection (Study Day -28, and 0-5) ............................................... 22 
4.2.7 
Assessment of Compliance (Study Days -21,-14,-7, and 0-14) ........................................................ 22 
4.2.8 
Stool sample collection (Study Day 0) ............................................................................................. 22 
4.2.9 
Assessment of blinding (Study Day 0) ............................................................................................. 22 
4.2.10 
Challenge with rhinovirus (Study Day 0) ..................................................................................... 22 
4.2.11 
Symptom scoring (Study Days 0-14) ............................................................................................ 23 
5 
STUDY PRODUCT ....................................................................................................................... 23 
5.1 
STUDY PRODUCT AND COMPARATOR............................................................................................... 23 
5.1.1 
Investigational product.................................................................................................................... 23 
5.1.2 
Placebo ............................................................................................................................................ 23 

 
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5.2 
SUPPLY, PACKAGING, LABELING, HANDLING AND STORAGE ........................................................... 23 
5.2.1 
Study product supply and storage ................................................................................................... 23 
5.3 
DOSAGE AND ADMINISTRATION ....................................................................................................... 24 
5.3.1 
Duration of Treament ...................................................................................................................... 24 
5.3.2 
Methods of Assigning Subjects to Treament .................................................................................... 24 
5.4 
CONCOMITANT MEDICATIONS .......................................................................................................... 24 
5.4.1 
Concomitant diet ............................................................................................................................. 24 
5.4.2 
Prior medications ............................................................................................................................ 24 
5.5 
STUDY PRODUCT ACCOUNTABILITY ................................................................................................ 24 
6 
RESPONSE VARIABLES AND STUDY ENDPOINTS ........................................................... 25 
6.1 
ASSESSMENT OF EFFICACY .............................................................................................................. 25 
6.2 
PRIMARY ENDPOINT ......................................................................................................................... 25 
6.2.1 
Incidence of rhinovirus-associated illness episodes ........................................................................ 25 
6.3 
SECONDARY ENDPOINTS .................................................................................................................. 25 
6.3.1 
Duration of illness ........................................................................................................................... 25 
6.3.2 
Incidence of infection ...................................................................................................................... 25 
6.3.3 
Viral load during infection .............................................................................................................. 25 
6.3.4 
Proportion shedding the virus ......................................................................................................... 25 
6.4 
EXPLORATORY ENDPOINTS .............................................................................................................. 26 
6.4.1 
Cytokine response of nasal mucosa ................................................................................................. 26 
6.4.2 
Blood transcriptomics ..................................................................................................................... 26 
6.4.3 
Rhinovirus type-specific antibody response .................................................................................... 27 
6.4.4 
Fecal, throat and nasal microbiota ................................................................................................. 27 
6.4.5 
Cold questionnaire comparison ...................................................................................................... 27 
6.5 
ASSESSMENT OF SAFETY .................................................................................................................. 27 
6.5.1 
Adverse events ................................................................................................................................. 27 
7 
ADVERSE EVENTS ..................................................................................................................... 27 
7.1 
ADVERSE EVENT .............................................................................................................................. 27 
7.1.1 
Definitions ....................................................................................................................................... 27 
7.1.2 
Assessment of Adverse Events ......................................................................................................... 28 
7.1.3 
Halting rules for adverse events ...................................................................................................... 28 
7.1.4 
Monitoring of adverse events .......................................................................................................... 29 
7.2 
SERIOUS ADVERSE EVENTS AND UNEXPECTED ................................................................................. 29 
7.2.1 
Definitions ....................................................................................................................................... 29 
7.2.2 
Severity ............................................................................................................................................ 30 
7.2.3 
Causal relationship with trial medication ....................................................................................... 30 
The causal relationship will be rated as follows: .......................................................................................... 30 
7.2.4 
Reporting ......................................................................................................................................... 31 
8 
STATISTICAL METHODS ......................................................................................................... 31 
8.1 
ESTIMATION OF SAMPLE SIZE ................................................................................................. 31 
8.2 
RANDOMISATION ....................................................................................................................... 31 
8.3 
BLINDING AND CODE BREAKING INSTRUCTIONS .............................................................. 31 
8.3.1 
Blinding ........................................................................................................................................... 31 
8.3.2 
Emergency unblinding of a individual subject ................................................................................ 31 
8.4 
STATISTICAL ANALYSIS PLAN ................................................................................................ 32 
8.4.1 
PRIMARY ANALYSIS ...................................................................................................................... 32 
8.4.2 
SECONDARY ANALYSIS ................................................................................................................ 32 
8.5 
STUDY POPULATIONS................................................................................................................ 33 
8.6 
DATA MANAGEMENT ................................................................................................................ 33 
9 
REGULATORY AND ADMINISTRATIVE PROCEDURES .................................................. 34 

 
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9.1 
INSTITUTIONAL REVIEW .......................................................................................................... 34 
9.2 
SUBJECT INFORMATION/ INFORMED CONSENT .................................................................. 34 
9.3 
SUBJECT CONFIDENTIALITY ................................................................................................... 35 
9.4 
GCP AND RECORD RETENTION ................................................................................................ 35 
9.4.1 
The study will be carried out in accordance with: .......................................................................... 35 
9.4.2 
Record Retention ............................................................................................................................. 35 
9.5 
MONITORING AND QUALITY CONTROL ................................................................................ 36 
9.5.1 
Monitoring ....................................................................................................................................... 36 
9.5.2 
Audit-Inspection .............................................................................................................................. 36 
9.5.3 
Quality Control ............................................................................................................................... 37 
9.6 
INSURANCE AND LIABILITY .................................................................................................... 37 
9.7 
STUDY REPORT ........................................................................................................................... 37 
9.7.1 
Clinical Study Report ...................................................................................................................... 37 
9.8 
PUBLICATION AND DATA RIGHTS .......................................................................................... 37 
10 
REFERENCES .............................................................................................................................. 38 
11 
SIGNATURES ............................................................................................................................... 40 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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1. INTRODUCTION 
1.1 
Background 
The common cold is a ubiquitous illness of man that is associated with significant medical 
and socioeconomic consequences.  Current treatments for the common cold that have 
proven efficacy are limited to pharmacologic agents that are directed at specific symptoms.  
These treatments- antihistamines, nasal decongestants and analgesics- have limited 
effectiveness, generally relieving the target symptom by 15-25% at the peak of activity, and 
are associated with bothersome side effects.  There are no currently effective treatments for 
prevention of rhinovirus infections.   
 
Most of the URTIs (i.e. common colds) (30-80 %) are caused by rhinoviruses, but also 
coronaviruses (10-15 %) and influenza (5-15 %) are found in significant number of URTI 
cases. A recent systematic review addressed the efficacy of probiotics on upper respiratory 
tract infections (URTI): “Probiotics were better than placebo in reducing the number of 
participants experiencing episodes of acute URTIs, the rate ratio of episodes of acute URTI 
and reducing antibiotic use.” [1]. Positive effect on enhancing influenza vaccination efficacy 
has been shown for probiotics in human clinical studies [2-4], but so far no studies have been 
published on efficacy of probiotics against rhinovirus infections.  
 
The effect of Bl-04 on the incidence of respiratory tract illness symptoms in healthy active 
adults was studied in a randomized, double-blind, placebo-controlled clinical trial [5]. The 
study was conducted in Australia by the Griffith University. A total of 229 health active adults 
were randomized to placebo (n = 127) and Bl-04 (n = 102) groups. The probiotic was 
administered with a total daily dose of 2.0 × 109 CFU for 5 months throughout the primary 
cold and flu season. The participants reported selfassessed symptoms of respiratory illness – 
scratchy or sore throat, sneezing, runny or stuffy nose – using an online questionnaire. An 
upper respiratory tract illness (URTI) episode was recorded when two or more symptoms 
were present for three or more consecutive days. During the study period, subjects in the Bl-
04 group had 102 (59 single and 43 recurrent) URTI symptomepisodes compared to 127 (67 
single 60 recurrent) episodes in the placebo group. Recurrent events analysis showed a 27% 
risk reduction (p=0.022) for any URTI episode in the Bl-04 group compared to the placebo 
group. Further analyses indicated that in the Bl-04 group there was a significant delay in the 
onset of URTI symptoms (median time 3.2 months), compared to the placebo group (median 
time 2.5 months) [6]. 
  
A recent study using the experimental rhinovirus challenge model at the University of Virginia 
(manuscript submitted) assessed the effect of Bl-04 on host responses to rhinovirus infection.  
One-hundred fifty-two seronegative volunteers who had received Bl-04 (n=73) or placebo 
(n=79) for 28 days were challenged with RV39.  Administration of study intervention was then 
continued for five days during collection of specimens for assessment of host response, 
infection, and symptoms. Fifty-eight probiotic and 57 placebo volunteers met protocol defined 
criteria for analysis.  Analysis of nasal lavage IL-8 concentration as the primary outcome 
variable revealed significantly higher concentrations of IL-8 on Day 0 prior to virus challenge 
in the probiotic supplemented volunteers (97 versus 58 pg/mL, respectively, p=0.25).  In 
contrast, the IL-8 response (change in IL-8 compared to day 0 over days 1-5) to RV39 
challenge was significantly reduced in the probiotic supplemented group (geometric mean 
ratio for change, probiotic: placebo=0.65, p=0.03).  The administration of probiotic was 
associated with a reduction in nasal lavage virus titer and the proportion of subjects with virus 

 
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shedding was lower in the probiotic treated subjects (76% in the probiotic group, 91% in the 
placebo group, p=0.04).  There was no effect of probiotic treatment on symptom severity, 
lower respiratory inflammation or serum antibody responses to the study virus.  This study 
demonstrates that ingestion of Bl-04 has a significant effect on the baseline state of innate 
immunity in the nasal mucosa and on the subsequent response of the host to rhinovirus 
challenge.  
 
1.2 Study product 
Probiotics have been defined by FAO/WHO as “Live microorganisms which when 
administered in adequate amount confer a health benefit on a host.” The most common 
probiotics belong to Lactobacillus or Bifidobacterium genera. Bifidobacteria are natural 
human gut inhabitants that were discovered over a hundred years ago from the feces of 
breast-fed infants. The study product Bifidobacterium animalis subsp. lactis Bl-04 (Bl-04) has 
been genetically characterized as B. animalis subsp. lactis by 16S rRNA gene sequencing 
and full genome sequence comparison [7, 8]. Bl-04 has been deposited in the American 
Type Culture Collections safe deposit as SD5219.  
 
1.3 
Summary of known potential risks and benefits to human subjects 
Bifidobacterium spp. has long been considered safe and suitable for human consumption 
with several published studies addressing its safety [9-12]. Bl-04 has been used safely in 
human clinical trials that have included children, adults, and elderly (Table 1). Furthermore, 
Bifidobacterium lactis has been present in human food for decades and is listed in the 
Inventory of Microorganisms With Documented History of Use in Human Food [13].  The 
European Food Safety Authority has also added the species to the Qualified Presumption of 
Safety list (efsa.europa.eu) and Bl-04 has self-affirmed GRAS (Generally Recognized As 
Safe) status from FDA. Harmful or toxigenic activities have not been associated with B. lactis 
and acquired antibiotic resistance was not detected in B. lactis Bl-04 during screening by the 
EU-funded PROSAFE project. 
 
In human clinical studies, Bl-04 has been safely used as a single entity and in combination 
with other probiotics and/or prebiotics (Table 1). The age of the subjects in these trials 
ranged between children 4.2 yrs and elderly 90 years. Most of the studies were conducted in 
healthy subjects, but some also involved subjects with allergic history. Table 2 below 
provides further details on these trials – number of subjects, dose and length of 
supplementation (single entity or combination treatment) and rate of adverse events.  
 
In the clinical trial by West and colleagues a thorough safety and tolerability analysis was 
conducted for sub-group of 84 subjects (39 in Bl-04 and 45 in placebo groups)[5, 14]. None 
of the participants in the sub-group had reported any clinically significant adverse events.  
Blood samples were taken before and after the 150 days of supplementation and routine 
haematology, and clinical chemistry markers (incl. electrolytes, liver and kidney function and 
metabolic markers, and C-reactive protein) were analysed. No significant changes were 
observed in the Bl-04 group over the supplementation period and all the values were within 
the normal ranges.  
 
In the Turner study (Turner et al. unpublished), AEs in 5 subjects (4 GI, 1 Respiratory illness) 
were judged, prior to unblinding, to be possibly or probably related to the study interventions. 
No serious adverse effects have been associated with the administration of Bl-04. 

 
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Table 1. Bl-04 supplementation in human clinical trials.  
Reference 
Number and 
characteristics of 
subjects consuming Bl-
04 
Supplementation 
dose and length 
Adverse events 
Turner 
et 
al. 
2015; 
Unpublished 
95/190 
Adults (Mean 22 yrs) 
2 × 109 CFU/d for 32 
d 
Gastrointestinal AEs occurred only in the active 
group (P<0.01).  AEs in 5 subjects (4 GI, 1 
Respiratory illness) were judged, prior to 
unblinding, to be possibly or probably related to 
the study interventions.   
[5, 6, 14] 
161/465 
Adults (average 36 y) 
Physically active 
2 × 109 CFU/d for 
150 d 
2 subjects withdrew because of uncomfortable GI 
symptoms 
[15] 
336/503 
Adults (30-70 y) 
Receiving antibiotics 
4.3 × 109 CFU/d 
(n=168) or  1 × 109 
CFU/d (n=168) in a 
mixture of four 
strains for 10 to 21 d 
AE rates were 7.2%, 4.2% and 4.2% in the 
placebo, high-dose and  low-dose groups, 
respectively (AEs were allergy to sea food, 
arrhythmia, fever, headache, left upper arm 
fracture, runny nose and vomiting) 
One SAE was reported (death due to myocardial 
infarction; the subject had a history of coronary 
heart disease) 
None of the AEs were considered to be related to 
the study product 
High-dose probiotic group had fewer drop-outs 
than low-dose or placebo group 
[16] 
24 / 47 
Children (mean 9  yrs) 
with history of allergy 
1.25 × 109 CFU/d 
with one other strain 
for 122 d 
2 subjects dropped out in probiotic group, no 
reason published 
[17] 
20/40 
Adults (18-45y) 
Consuming antibiotics  
1 × 1010 CFU/d in a 
mixture of five 
strains for 20 d 
No significant difference in AE rate between 
treatment groups (both groups consumed 
antibiotics) 
Most common AEs were diarrhea (3 in active and 
1 in placebo group), vaginal yeast infection (4 in 
active and 1 in placebo group) and abdominal pain 
(3 in probiotic and 3 in active group) 
[18] 
9/86 
Adults (18-64 y) 
Oral cholera vaccine given 
2 × 1010 CFU/d for 3 
w  
None documented 
[19] 
20/40 
Adults (>18y) 
Consuming antibiotics 
5 × 109 CFU/d in a 
mixture of five 
strains for 20 d 
None documented 
[7] 
9/18 
Healthy elderly 
3.5 × 1010 CFU/d  in 
a mixture with inulin 
and Bb-02 for 28 
days 
None documented 
 
 

 
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2 STUDY OBJECTIVES 
2.1 
Purpose of the trial 
To test the hypothesis that prophylaxis of human volunteers with probiotic Bifidobacterium 
animalis subsp. lactis Bl-04 will reduce the incidence of rhinovirus induced cold episodes.  
 
2.2 
Objectives 
2.2.1 Primary objective 
• To study the effect of probiotic supplementation on incidence of rhinovirus-
associated common cold illness episodes. 
2.2.2 Secondary objectives 
• To study the effect of probiotic supplementation on the infection rate and 
duration of illness episodes 
• To examine the effect of the probiotic on the rhinovirus load in the nose 
 
2.2.3 Exploratory objectives 
• 
To study the effect of the probiotic on blood transcriptome during basal state and 
infection. 
• 
To validate the effect of the probiotic on nasal cytokine profile at baseline and during 
infection 
• 
To characterize the nasal, throat, and intestinal microbiomes before and during 
rhinovirus infection and supplementation period.  
• 
To investigate correlations between specific microbiome profiles, immune responses 
and subsequent infection and illness. 
• 
To compare with a cold questionnaire 
 
 
3 STUDY POPULATION 
Approximately 900 healthy, adult (18-60 years) volunteers will be recruited for participation in 
these studies via a screening protocol (IRB #9948). All volunteers will be tested for the 
presence of serum neutralizing antibody to rhinovirus type 39.  Volunteer subjects with an 
antibody titer ≤1:4 will be eligible for participation.   
 
3.1 
Inclusion Criteria 
INCLUSION CRITERIA AT ENROLLMENT: 
• 
Subject must be 18-60 years of age. 
• 
Subject must read and sign a copy of the approved Consent Form  
• 
Subject must have a serum neutralizing antibody titer of less than or equal to 1:4 
to rhinovirus type 39 
 
INCLUSION CRITERIA AT DAY -28 
• 
Female subjects must be using an effective birth control method. 

 
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INCLUSION CRITERIA AT CHALLENGE: 
• 
Female subjects must be using an effective birth control method. 
 
3.2 
Exclusion Criteria 
EXCLUSION CRITERIA AT ENROLLMENT: 
          
EXCLUSION CRITERIA AT DAY -28 
• 
      Antibiotic use within 3 months prior to  day -28 
• 
Female subjects with a positive urine pregnancy screen 
• 
      History of use of probiotics in the preceding two weeks 
• 
      Current cancer diagnosis or immunosuppressive therapy in the last 6 months 
• 
      Any clinically significant abnormalities of the upper respiratory tract 
• 
      Any clinically significant acute or chronic respiratory illness 
• 
      Any clinically significant bleeding tendency by history 
• 
      Hypertension that requires treatment with antihypertensive medications 
• 
      History of angina or other clinically significant cardiac disease 
• 
Any medical condition that in the opinion of the Principal Investigator is cause 
for exclusion from the study 
• 
History of regular use (more than 3 days in 7) of tobacco products within the 
preceding two weeks 
• 
      History of drug or alcohol abuse in the 6 months preceding the study 
 
 
EXCLUSION CRITERIA AT CHALLENGE: 
• 
Any upper respiratory infection or allergic rhinitis in the two weeks prior to  the 
challenge 
• 
Female subjects with positive pregnancy screen prior to challenge 
• 
Any medical condition that in the opinion of the Principal Investigator is cause 
for exclusion from the study 
• 
Use of any anti-inflammatory (steroids or NSAIDs) or cough/cold or allergy 
preparation in the two weeks prior to the challenge 
 
3.3 
Recruitment, screening, and randomization 
Approximately 900 healthy, young adult (18-60 years) volunteers will be recruited for 
participation in these studies by posted, newspaper, or radio advertisements.  Volunteers will 
be tested for the presence of serum neutralizing antibody to rhinovirus type 39 under an 
existing screening protocol at the University of Virginia (IRB-HSR #9948).  Volunteer subjects 
with an antibody titer ≤1:4 will be invited to consent for participation in this (EPRIC) study.  
Volunteers who were screened for antibody more than 8 weeks prior to consent for the 
EPRIC study will have a repeat serology done at the screening visit to assure eligibility. 
 
Volunteers enrolled in the study will be randomized 1:1 to the study product or placebo.  The 
sponsor will supply the study product and a matching placebo.  The randomization schedule 
will be provided by the sponsor.  The blinding code will be held in confidence by the School 
of Medicine Clinical Trials Office at the University of Virginia. 

 
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3.4 
Subject Withdrawal and replacement of subjects 
3.4.1 Withdrawal criteria 
Subjects may be withdrawn from the study (i.e. from any further study product or study 
procedure but not from analyses) for the following reasons:  
• 
At their own request or at the request of their legally authorized representative. 
• 
If, in the investigator's opinion, continuation in the study would be detrimental to the 
subject’s well-being. 
• 
If they are lost to follow-up. 
 
In all cases, the reason for and date of withdrawal must be recorded in the eCRF  and the 
sponsor’s representative must be notified within 5 days. The subject must be followed up to 
establish whether the reason was an adverse event, and, if so, this must be reported. 
 
The investigator must make every effort to contact subject lost to follow-up. Attempts to 
contact such subject must be documented in the subject’s records (e.g., dates and times of 
attempted telephone contact). 
 
Any subject may be withdrawn from the study at the discretion of the investigator. The 
subject is also free to terminate his participation at any time. An end of study visit will then be 
organized. 
 
3.4.2 Withdrawn subject data collection 
The principal investigator and/or other investigator involved in the study will document on the 
termination page of the eCRF the reason for the subject withdrawal as follows:  
• 
Lost to follow-up: subjects who leave the study unnotified or do not attend the 
study visit or cannot be contacted by phone. Intensive efforts should be made to 
locate and recall them if possible and to determine their health status at a 
minimum. 
• 
Adverse event: an adverse event form must be completed. 
• 
Adverse laboratory event: this reason should be stated when a laboratory value is 
interpreted as a clinically significant abnormal value not explained by a laboratory 
error or being not a known or abnormal value commonly observed in this type of 
population.  
• 
Deviation from protocol. 
• 
Consent withdrawn. 
• 
Other: if no above mentioned reasons are applicable, then the reason will be 
specified. 
3.4.3 Replacement of subjects  
Subjects withdrawn from the study will not be replaced. 
 
 

 
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4 Experimental Design 
4.1 
Overall Study Design 
This double-blinded, randomized, controlled study will be conducted at the University of 
Virginia.  The study will use the experimental rhinovirus human challenge model. 
 
4.1.1 Number of subjects 
Approximately 900 volunteers will be recruited for participation in these studies. Volunteers 
seronegative to rhinovirus type 39 will be randomly treated for 42 days with either: 
• 
probiotic (n=130) 
• 
placebo (n=130) 
4.1.2 Study center 
The study will take place at the University of Virginia, Charlottesville. 
 
4.2 
Assessments and Procedures 
4.2.1 Study Conduct  
The study will be done in five cohorts of 60-70 subjects each for logistical reasons.  The first 
cohort will be conducted in the Spring of 2016.  Subsequent cohorts will be done in Fall 2016, 
Spring 2017, Fall 2017 and Spring 2018.  The assays and data collection for analysis of the 
primary and secondary efficacy endpoints will be substantially complete by December, 2018. 
 
4.2.2 Study periods 
Screening: 
▪ 
Consent and enrollment (approximately 375 subjects) 
▪ 
Medical history questionnaire and demographics 
▪ 
Vital signs and brief physical exam 
▪ 
Physical activity question 
▪ 
Review of inclusion/exclusion criteria  
▪ 
Blood draw for screening neutralization antibody titer (if no antibody screening test 
has been done in the last 12 weeks) 
 
Day -42 ± 4: 
▪ 
All subjects begin dietary “run-in” period  
 
Day -28 ± 4:  Seronegative subjects only 
▪ 
Review of inclusion/exclusion criteria 
▪ 
Pre-probiotic baseline nasal lavage for IL-8 and other cytokines 
▪ 
Pre-probiotic baseline nasal swab for microbiomics 
▪ 
Pre-probiotic baseline throat swab for microbiomics   
▪ 
Blood draw for pre-probiotic transcriptomics studies  
▪ 
Assessment of concomitant medications 
▪ 
Randomized to active or placebo probiotic 
▪ 
Urine pregnancy screen for females 
 
 
Day -21 ± 2 

 
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▪ 
All subjects return to study site for new supply of probiotic and compliance review 
▪ 
AE assessment 
▪ 
Assessment of concomitant medications 
 
Day -14 ± 2: 
▪ 
All subjects return to study site for new supply of probiotic and compliance review 
▪ 
Nasal lavage for multiplex cytokine assays 
▪ 
AE assessment  
▪ 
Assessment of concomitant medications 
 
Day -7 ± 2: 
▪ 
All subjects return to study site for new supply of probiotic and compliance review 
▪ 
AE assessment 
▪ 
Assessment of concomitant medications 
 
Day 0: 
▪ 
Review of inclusion/exclusion criteria 
▪ 
Assessment of blinding 
▪ 
AE assessment 
▪ 
Assessment of concomitant medications 
▪ 
Urine pregnancy screen for females 
▪ 
Stool specimen for probiotic compliance and microbiomics 
▪ 
Nasal swab for microbiomics 
▪ 
Throat swab for microbiomics 
▪ 
Throat swab for culture 
▪ 
Nasal lavage for unanticipated viruses 
▪ 
Nasal lavage for pre-RV challenge baseline IL-8 
▪ 
Blood draw for measurement of neutralizing antibody 
▪ 
Blood draw for pre-RV challenge transcriptomics 
▪ 
Nasal lavage for multiplex cytokine assays  
▪ 
Symptom assessment 
▪ 
Challenge with rhinovirus  
▪ 
Compliance check/resupply 
▪ 
Continue probiotic treatment 
 
Day 1-4: 
▪ 
AE assessment 
▪ 
Assessment of concomitant medications 
▪ 
Nasal lavage for quantitative rhinovirus culture 
▪ 
Nasal lavage for IL-8 measurement 
▪ 
Nasal lavage for multiplex cytokine assays 
▪ 
Symptom assessment 
▪ 
Assessment of the effect of symptoms on physical activity 
▪ 
Nasal swab for microbiomics (day 3) 
▪ 
Throat swab for microbiomics (day 3) 
▪ 
Blood draw for post-RV challenge transcriptomics (Day 2 only) 
▪ 
Continue probiotic treatment 
 
 

 
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Day 5: 
▪ 
AE assessment 
▪ 
Assessment of concomitant medications 
▪ 
Nasal lavage for quantitative rhinovirus culture 
▪ 
Nasal lavage for IL-8 measurement 
▪ 
Throat swab for culture 
▪ 
Throat swab for microbiomics 
▪ 
Symptom assessment 
▪ 
Assessment of the effect of symptoms on physical activity 
▪ 
Assessment of common cold illness 
▪ 
Compliance check/resupply 
▪ 
Continue probiotic treatment 
 
Day 14 
▪ 
Return symptom diaries and any remaining study product 
▪ 
Discontinue probiotic treatment 
▪ 
Throat swab for microbiomics  
 
Day 21-30: 
▪ 
AE assessment 
▪ 
Assessment of concomitant medications 
▪ 
Blood draw for serum neutralizing antibody 
 
4.2.3 Pre-study safety assessment (Screening) 
Volunteers who agree to participate will have a brief medical history and brief physical 
assessment prior to participation in the study.  The purpose of these evaluations will be to 
detect any underlying condition that would either place the subject at increased risk from the 
study procedures or that would compromise evaluation of the study endpoints.  A medical 
history questionnaire will be completed by all study subjects.  The completed questionnaire 
will be reviewed with study personnel and all positive responses clarified.  A brief physical 
examination consisting of pulse, respiratory rate, body temperature, blood pressure, and 
visual examination of the nose will be performed on all subjects and all female subjects will 
have a urine pregnancy screen.  The urine pregnancy screen will be repeated on Day 0 prior 
to virus challenge. Subjects will be asked whether they exercise at least 30 minutes 3 times 
each week over the last 3 months. All subjects must meet the inclusion criteria and must not 
meet any of the exclusion criteria for participation in the study. 
 
4.2.4 Nasal lavage (Study Day -28, -14, and 0-5) 
Nasal lavage will be collected for virology and for determination of IL-8 and other cytokine 
concentrations.  These specimens are collected by instillation of 5 mL of sterile 0.9% saline 
into each nostril.  This wash is then immediately expelled into a waxed paper cup and kept 
chilled until processed.   
 
4.2.5 Blood for transcriptomics (Study Days -28, 0, and 2) 
Blood specimens will be collected into PaxgeneRNA extractor tubes for subsequent analysis 
of gene expression by microarray.  These specimens will be handled according to the 
manufacturer’s instructions and stored at -70C until they are analyzed. 
 

 
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4.2.6 Nasal and throat swab sample collection (Study Day -28, and 0, 2, 3,5, 14) 
Nasal swabs will be collected and stored at -70C.  Nasal swabs will be collected by inserting 
a moistened nylon flocked swab (FloQswabs, Copan Diagnostics Inc, Murrieta, CA) into each 
naris until resistance is met.  The swab will be gently rotated and then withdrawn.  
Pharyngeal swabs will be done using the same swab material to swab the posterior 
pharyngeal wall.  The nasal swabs and throat swabs will be collected before the daily dose of 
the study product.  All throat swabs will be collected by a study nurse experienced in the 
procedure. 
 
4.2.7 Assessment of Compliance (Study Days -21,-14,-7, and 0-14) 
At each study visit during the pre-challenge dosing period, volunteers will be asked to return 
unused study product to the study site.  Compliance will be assessed by counting missed 
doses of study product. For purposes of the per protocol analyses, the subject will be 
assessed as compliant with the protocol if at least 80% of the planned doses were taken. 
In addition, DuPont Nutrition and Health will measure the amount of Bifidobacterium animalis 
subsp. lactis from the fecal samples using qPCR.   
 
4.2.8 Stool sample collection (Study Day 0) 
Fecal samples will be collected into an air tight container and frozen. The subjects will be 
given stool sample containers, outer packaging for hygienic storage and delivery and written 
instructions including sampling and clinic visit dates at a visit prior to sample collection.  A 
prior notice as a reminder will be sent to all subjects before the sampling and clinic visit day. 
All samples will be marked with study subject code and date. 
 
The stool samples should be: 
• 
defecated on the same day 
• 
kept in a refrigerator or on ice before transport to the clinic 
• 
immediately frozen to -70°C at the clinic 
• 
kept frozen at all times until analysis 
 
4.2.9 Assessment of blinding (Study Day 0) 
Volunteers will be asked  whether they believe they are receiving active or placebo product.  
On study day 0, before challenge with the study virus, each volunteer will indicate their 
response to the following question: 
 
Which treatment do you think you are taking:  active    control    don’t know 
 
4.2.10 Challenge with rhinovirus (Study Day 0) 
The challenge virus to be used in this study is rhinovirus, type 39, a serotype that attaches 
via the ICAM-1 receptor.  Inoculation will be with a challenge pool of virus that has been 
thoroughly safety tested and approved for use for virus challenge experiments by the FDA 
(IND #12934).  Following a nasal wash, each subject will be given the challenge virus by 
intranasal drops.  The virus is administered in a volume of 0.25 mL/nostril with a calibrated 
pipette and two inoculations of virus are given several minutes apart.  The total virus 
inoculum will be 20-100 TCID50/volunteer contained in 1 mL of fluid.  This procedure is used 
routinely in the rhinovirus challenge model and results in an infection rate of 85-90% in 
susceptible volunteers.  
 

 
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4.2.11 Symptom scoring (Study Days 0-14) 
Symptom scores on study days 0-5 will be collected in an interactive interview with the study 
staff.  Symptoms will be assessed using the modified Jackson criteria.  On study days 6-14, 
volunteers will be asked to complete a symptom diary that will consist of the same symptoms 
assessed by the Jackson criteria.  Volunteers will also be asked whether their symptoms 
affected their physical activity. 
 
 
5 Study Product 
5.1 
Study Product and Comparator 
5.1.1 Investigational product 
The study product will be a 2*109 cfus of probiotic Bifidobacterium lactis Bl-04 (DuPont 
Nutrition and Health) mixed with 1g of sucrose as a carrier. The study product will be 
manufactured according to GMP and QC guidelines approved by FDA at DuPont production 
site located at Madison Wisconsin, USA. 
 
5.1.2 Placebo 
Placebo will be 1g of sucrose that has identical appearance, smell, and taste with the study 
product.   
 
5.2 
Supply, Packaging, Labeling, Handling and Storage 
Investigational product and placebo product will be released by DuPont Nutrition and Health 
Qualified Person (QP) and then sent to Ronald Turner, MD, Barringer 4441, University of 
Virginia School of Medicine, Charlottesville, VA, 22908. Study products will be packaged and 
labeled by number for each individual kit according to the study plan. The individual dose 
boxes (i.e. kits) will be labeled to include the following information: 
• 
Sponsor study code  
• 
Sponsor’s name and address 
• 
Investigational product (name) powder and/or corresponding placebo  
• 
Amount of powder etc. of  X g/mg and/or corresponding placebo 
• 
For oral administration only 
• 
Instruction of intake 
• 
Investigator’s name and study site 
• 
Kit and Batch number 
• 
Expiry date 
• 
Storage conditions 
• 
For clinical trial use only. Keep out of reach of children. 
 
5.2.1 Study product supply and storage 
Study product and placebo will be produced under GMP conditions and will be packaged 
ready for distribution to the volunteers.  The investigational product must be stored in the 
refrigerator until use (2-8 °C). Sponsor may control the stability of the investigational product 
by collecting samples from the returned containers after the study end to check the viability of 
the probiotic bacteria. Study product will be stored in a locked, limited access room at +4 °C 
under the supervision of the study coordinator. 

 
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5.3 
Dosage and Administration 
5.3.1 Duration of Treament 
All volunteers will sign consent and be screened for study eligibility before randomization to 
study product.  On day –28 of the study, all volunteers will come to the study site, be 
randomized to study treatment and instructed when to take the first dose of study product. 
Volunteers will be instructed to take daily one sachet of the study product mixed into a drink 
that is not hot and does not contain alcohol. The study product will have a minimal of 2*109 
cfus of Bl-04.  On days -21, -14 and -7 subjects will come to the study site for re-supply of the 
probiotic and for evaluation of compliance.  On day 0 the volunteers will be challenged with 
the study virus.  After the virus challenge all volunteers will continue the study treatment on 
through study day 14. 
 
5.3.2 Methods of Assigning Subjects to Treament 
The study product and placebo will be randomized and assigned sequential numbers by the 
sponsor.  Volunteers will be assigned a study subject ID number as they present for the day -
28 visit.  The randomization number and the number of the study material they are given will 
correspond to their study number. All study personnel will remain blinded to the product or 
placebo assignment until all data have been collected and entered into the study database, 
and the database has been locked. 
5.4 
Concomitant medications 
Volunteers who participate in this study will be asked to refrain from using common cold or 
allergy treatments for the duration of the study.  If in the opinion of the volunteer their 
symptoms are severe and require treatment, treatment should be limited to acetaminophen 
and the use of the medication should be reported to the study staff. The use of medications 
other than common cold treatments will be permitted at the discretion of the investigator.  All 
volunteers will be asked to refrain from the use of NSAIDs or steroids beginning two weeks 
prior to the virus challenge until the end of the symptom reporting period (study day 14). 
 
Use of antibiotics may have an impact on the investigational product functionality and 
therefore antibiotic use should be avoided unless explicitly needed as judged by a physician.  
Volunteers who take antibiotics during the study will be removed from the study. 
 
5.4.1 Concomitant diet 
During the dietary intervention period (days -28 to 14) subjects should not use any other 
probiotic or prebiotic containing products. A list of avoidable and acceptable yoghurt products 
will be provided to volunteers. Study subjects are advised not to mix the product or placebo 
with hot or alcohol containing drinks.  
 
5.4.2 Prior medications 
Continued use of topical agents, hormonal contraception or hormone replacement therapy, 
SSRIs, and other anti-depressants will generally be permitted. 
 
5.5 
Study Product Accountability 
The investigator is responsible for the accounting for all unused and all used investigational 
product containers. The study eCRFs must have an accurate dispensing/returning log. The 
third-party dispenser (can be a hospital pharmacy or a study nurse) uses this information to 

 
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maintain an accurate and complete dispensing and inventory record. The designated copies 
of the completed dispensing and inventory record will be returned to the sponsor. 
Supplies are shipped to the investigative site as needed. Study product accounting will be 
reviewed by the site monitor during routine monitoring visits. At the completion or termination 
of the study, a final study product accountability review and reconciliation must be completed, 
and any discrepancies must be investigated and their resolution documented. All full, partially 
full, and empty containers of study products must be returned to the sponsor with the 
appropriate form. The sponsor then destroys all and documents the destruction. 
 
6 Response Variables and Study Endpoints 
6.1 
Assessment of Efficacy 
6.2 
Primary endpoint 
6.2.1 Incidence of rhinovirus-associated illness episodes  
6.2.1.1 Rhinovirus infection:  Rhinovirus infection will be defined as the isolation of the 
challenge virus on any of the 5 days (study days 1-5) after virus challenge or a four-
fold rise in neutralizing antibody titer to the study virus. 
6.2.1.2 Symptomatic illness:  Symptomatic illness will be defined according to the modified 
Jackson criteria.  Volunteers who have a total symptom score of at least 6 over the 5 
days (study days 1-5) after virus challenge and either three days of rhinorrhea or the 
subjective impression that they have had a common cold illness will be considered 
to have a symptomatic illness. 
6.2.1.3 Rhinovirus-associated illness episodes:  Volunteers who have both a rhinovirus 
infection and a symptomatic illness will be defined as having a rhinovirus-associated 
common cold illness. 
6.3 
Secondary endpoints 
6.3.1 Duration of illness 
6.3.1.1 Duration of illness will be defined as the time between the virus challenge and the 
end of the illness episode in volunteers who meet the Jackson definition of 
symptomatic illness.  The end of the illness will be defined as the first of two 
consecutive days with a total symptom score ≤ 1 that occurs after the subject has 
met symptom criteria for a Jackson cold. 
6.3.2 Incidence of infection 
6.3.2.1    Rhinovirus infection will be defined as the isolation of the challenge virus on any of 
the 5 days (study days 1-5) after virus challenge or a four-fold rise in neutralizing 
antibody titer to the study virus. 
6.3.3 Viral load during infection 
6.3.3.1 Viral load will be assessed by comparing the quantitative virus titer in nasal lavage 
between the probiotic and placebo groups.  For purposes of this calculation subjects 
with no virus isolate on a study day will be defined as having a quantitative titer of -
0.5 (log10). 
6.3.4 Proportion shedding the virus 
6.3.4.1 The proportion of volunteers shedding virus in the placebo group will be compared 
with the  proportion in the probiotic group.  Virus shedding will be defined as the 

 
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isolation of the challenge virus on any of the 5 days (study days 1-5) after virus 
challenge 
6.4 
Exploratory endpoints 
6.4.1 Cytokine response of nasal mucosa 
6.4.1.1    Interleukin-8 assay: Concentrations of interleukin-8 in nasal lavage are determined 
with a commercially available ELISA assay (R&D Systems, Minneapolis, MN).  The 
lower limit of detection for the assay is 31.25 pg/mL.  The precision of the assay for 
measurement of IL-8 in nasal wash has been determined with pools of nasal wash 
spiked with known concentrations of IL-8.  Intra-assay variability was found to range 
from 3.8-5.7% and inter-assay variability was found to range from 2.7-11% for nasal 
wash containing high and low concentrations of IL-8, respectively.  The recovery of 
IL-8 in nasal wash ranged from 88-136% of predicted.  Specimens that have IL-8 
concentrations greater than the operating range of the assay will be diluted 1:10 and 
1:100 and re-assayed. 
6.4.2 Multiplex assays: A panel of cytokines and chemokines (TGF-β1, GCSF, GMCSF, 
IFN-γ, IL-1α, IL-12p70, IL-15, CCL20, IL-1β, IL-6, IL-10, CXCL10, CCL2, CCL3, 
CCL5, TNF-α) may be measured in nasal lavage fluid using a commercially available 
multiplex assay (Aushon BioSystems, Inc., Billerica, MA).  A determination of which 
assays will be included will be made as a post-hoc exploratory analysis.Blood 
transcriptomics 
6.4.2.1 Collection of specimens:  The effect of probiotics on gene expression before and 
after rhinovirus challenge will be determined by comparing expression in the 
probiotic and placebo groups on study days -28, 0 and 2.  Expression analysis will 
be done on peripheral blood specimens by microarrays targeted at inflammatory and 
antiviral genes.  Blood samples will be collected in PAXgene RNA tubes 
(PreAnalytix) and stored at -80°C until shipping.  Samples will be shipped on dry ice 
to DuPont Haskell Global Centers for Health and Environmental Science and 
immediately stored at -80°C until further processing.  RNA will be isolated from 
blood stored in PAXgene tubes using a PAXgene Blood RNA isolation kit (Qiagen) 
and potential DNA contamination will be removed with on-column DNase treatment.  
RNA concentration and quality will be verified on a Nanodrop© spectrophotometer 
(NanoDrop) or with a Quant-iT™ RiboGreen® RNA Assay Kit (Thermo Fisher).  
RNA integrity will be determined with an Agilent Bioanalyzer 2100 (Agilent).  All RNA 
samples processed for RNA-sequencing should have a 260/280 absorption ratio 
greater than 2 and a RNA integrity number (RIN) greater than 9.8.   
6.4.2.2 RNA-seq:  Targeted RNA-seq library preparation will be conducted with Tempo-Seq 
(BioSpyder) detector oligos designed against a ~1500 human gene panel focused 
on innate immunity and antiviral defense genes.  Library preparation will be 
conducted in 96 sample batches.  Up to 2 library preparations (n=192 samples) will 
be pooled, purified, quantitated and stored frozen prior to RNA-sequencing.  The 
pooled libraries will be sent to the Pioneer core genomics facility in Johnston, Iowa 
for RNA-seq using an Illumina NextSeq, or HiSeq platform.  The target read-depth 
per sample will be 1.5 million reads to ensure 500-1000 reads per gene.  The reads 
will be quality filtered, trimmed and demultiplexed by the technical staff in Johnston.  
The resulting FASTQ files will be transferred via external hard drive, or through the 
DuPont network for further processing.  Backup FASTQ files will be archived at the 
Johnston facility.  The FASTQ files will be aligned to an annotated reference to 
determine the number of reads per gene.  The reads will be quantitated by 

 
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normalizing with control genes in the samples set.  Other quantitation methods may 
be considered as well.  Data will be stored in this format until the samples can be 
decoded for further analysis.  Once decoded, metadata will be added to the data 
files to add additional annotation to the sample identifiers and group samples 
according to experimental conditions.  Differential expression analysis will be 
conducted to determine transcripts that are up or down regulated between the 
experimental conditions.   
 
6.4.3 Rhinovirus type-specific antibody response 
6.4.3.1 The effect of probiotic on host antibody responses to the challenge virus will be 
determined by comparing the convalescent neutralizing antibody titer in the probiotic 
and placebo groups. 
 
6.4.4 Fecal, throat and nasal microbiota 
6.4.4.1 Bacterial DNA will be isolated from the swabs and feces by using MagMAX™ Total 
Nucleic Acid Isolation Kit (Applied Biosystems, Bridgewater, NJ). Human DNA may 
be quantified for analysing the ratio of human and bacterial DNA by using 
housekeeping genes that do not allow subject identification. The composition of 
microbiota will be analyzed by next-generation sequencing (or equivalent) 
technology for identification of microbial species in the samples. The data will be 
analyzed to determine effects of probiotic on both abundance and diversity of 
bacterial species. . In addition, single strain targeting PCR-based molecular 
methods (i.e. qPCR, digital PCR or equivalent) will be applied to detect the 
supplemented probiotic strain, Bl-04. 
6.4.4.2 Throat swabs will be collected on study days 0, 5, and 14 for detection of 
pharyngeal pathogens by culture and qPCR.  A single swab will be sent to the 
microbiology laboratory at the University of Virginia for detection of S. aureus, 
pneumococcus, H. influenza, meningococcus and group A streptococcus. 
 
6.4.5 Cold questionnaire comparison 
Performance of the questionnaire used in the West study [5] will be compared to 
endpoints of this study.  
 
6.5 
Assessment of safety 
6.5.1 Adverse events 
6.5.1.1 The safety of the study interventions will be assessed by monitoring of adverse 
events.  Volunteers will be asked at each study visit to report any unexpected events 
whether or not they are apparently study related.  Each event will be recorded and 
an assessment of relateness to the study interventions will be made by the 
investigator.  Adverse events will be coded according to established standards 
(MedDRA).  The analysis will compare both total and related events in the probiotic 
and the control group. 
 
7 Adverse Events 
7.1 
Adverse event 
7.1.1 Definitions 
7.1.1.1 An adverse event (AE) is any untoward, undesired, or unplanned event in the form 
of signs, symptoms, disease, or laboratory or physiologic observations occurring in a 

 
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person given an IP in a clinical study. The event does not need to be causally 
related to the IP or the clinical study. An AE includes, but is not limited to the 
following: 
• 
Any clinically significant worsening of a pre-existing condition. 
• 
An AE occurring from overdose of an IP, whether accidental or intentional. Overdose 
is a dose greater than specified in the protocol. 
• 
An AE occurring from abuse (e.g., use for non-clinical reasons) of an IP. 
• 
An AE that has been associated with the discontinuation of the use of an IP. 
 
7.1.1.2 Treatment Emergent Adverse Event and Repetition of Adverse Event: An adverse 
event will be regarded as treatment-emergent if it occurred for the first time after the 
study product administration or if it was present before, but worsened after the study 
product administration.  
 
7.1.2 Assessment of Adverse Events 
For the purposes of this study using experimental rhinovirus infection, physical signs and 
symptoms that are expected to be associated with the common cold should not be reported 
as adverse events. The following physical signs and symptoms are expected to be 
associated with the common cold: nasal obstruction (nasal stuffiness), coryza (runny nose), 
sore throat, cough, sneezing, myalgia, headache, feverishness, chills, loss of appetite and 
malaise.  
 
Information regarding common cold-related complications must be documented 
appropriately. Common cold-related complications include bronchitis, bronchospasm or 
exacerbation of asthma, sinusitis and otitis media. For common cold-related complications, 
the following information should be documented: onset date, severity, seriousness, 
relationship to study therapy, action taken regarding study therapy, outcome, and therapy 
given for event.  
 
Some subjects may experience gut discomfort during the study product consumption. The 
symptoms that have been associated with use of bifidobacteria include bloating, flatulence 
and abdominal pain. The subjects should  contact the study staff in the case that symptoms 
persist. 
 
Assessment of all adverse events will include recording the time of onset and resolution of 
the event, severity, intervention, and an attribution by the investigator of whether the event 
was related to the study product. 
 
7.1.3 Halting rules for adverse events 
Should any of the following occur, the entire study will be stopped 
• 
B1-04 bacteremia or invasive infection 
• 
Any single serious and unexpected adverse event for which attribution is 
assessed as possibly, probably, or definitely related to the study products. 
• 
10% of participants develop any severe (grade 3) gastrointestinal AE 
• 
10% of participants develop any severe (grade 3) non-gastrointestinal AEs of 
the same type. 

 
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7.1.4 Monitoring of adverse events 
All volunteers will be specifically asked about any unexpected medical events at each study 
visit after the study treatment is begun.  All volunteers will be instructed to contact the study 
site if they experience any unexpected symptoms during the time they are consuming the 
study product.  At the final study visit, volunteers will be asked if they have had any unusual 
symptoms or problems during the study.  All positive responses will be recorded.  Medically 
significant responses will be followed until the symptom/problem is resolved or judged 
unrelated to the study and the subject has been referred for appropriate care. 
 
7.2 
Serious adverse events and unexpected 
7.2.1 Definitions 
7.2.1.1 General definitions: 
A Serious Adverse Event (SAE) or Serious Adverse Drug Reaction (ADR) is any untoward 
medical occurrence that at any dose: 
• 
Results in death. 
• 
Is life-threatening (see below). 
• 
Requires inpatient hospitalization or prolongation of an existing hospitalization 
(see below). 
• 
Results in a persistent or significant disability or incapacity (see below). 
• 
Results in cancer. 
• 
Is a congenital anomaly or birth defect. 
 
Additionally, important medical events that may not result in death, be life-threatening, or 
require hospitalization may be considered SAEs when, based on appropriate medical 
judgment, they may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed in this definition. Examples of such events include 
allergic broncho-spasm requiring intensive treatment in an emergency room or at home, 
blood dyscrasias or convulsions that do not result in hospitalization, or development of drug 
dependency or drug abuse. 
 
Life-threatening refers to immediate risk of death as the event occurred per the reporter. A 
life-threatening experience does not include an experience, had it occurred in a more severe 
form, might have caused death, but as it actually occurred, did not create an immediate risk 
of death. For example, hepatitis that resolved without evidence of hepatic failure would not 
be considered life-threatening, even though hepatitis of a more severe nature can be fatal. 
Similarly, an allergic reaction resulting in angioedema of the face would not be life-
threatening, even though angioedema of the larynx, allergic bronchospasm, or anaphylaxis 
can be fatal. 
 
Hospitalization is official admission to a hospital. Hospitalization or prolongation of a 
hospitalization constitutes criteria for an AE to be serious; however, it is not in itself 
considered a serious adverse event (SAE). In absence of an AE, a hospitalization or 

 
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prolongation of a hospitalization should not be reported as an SAE. This is the case in the 
following situations: 
 
• 
The hospitalization or prolongation of hospitalization is needed for a procedure 
required by the protocol.  
• 
The hospitalization or prolongation of hospitalization is part of a routine procedure 
followed by the centre (eg, stent removal after surgery). This should be recorded 
in the study file. 
 
In addition, hospitalization for a pre-existing condition that has not worsened does not 
constitute an SAE. 
 
7.2.1.2 Disability is defined as a substantial disruption in a person’s ability to conduct 
normal life functions. If there is any doubt about whether the information constitutes 
a SAE, the information is treated as a SAE. 
 
7.2.1.3 Other Reportable Information. Certain information, although not considered an 
SAE, must be recorded, reported, and followed up as indicated for an SAE. This 
includes:  
• 
Overdose of an investigational product as specified in this protocol with or without 
an AE. 
• 
Inadvertent or accidental exposure with or without an AE. 
7.2.1.4 Definition of expected SAE: No type of expected SAE was defined for the purpose 
of this study; all SAE will therefore be considered unexpected. 
 
7.2.2 Severity 
The severity of adverse events will be graded on a three–point scale (mild, moderate, 
and severe) and reported in detail as indicated on the eCRF. A description of scales 
can be found below.  
• 
Mild: discomfort noticed but no disruption of normal daily activity. 
• 
Moderate: discomfort sufficient to reduce or affect daily activity. 
• 
Severe: inability to work or perform normal daily activity. 
 
7.2.3 Causal relationship with trial medication  
The causal relationship will be rated as follows: 
• 
Definitely not related 
• 
Probably not related 
• 
Possibly related. 
• 
Probably related. 
• 
Definitely related.  

 
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7.2.4 Reporting 
Any serious adverse event occurring in a subject after providing informed consent until end of 
trial  must be reported. Any adverse event should be recorded on the appropriate eCRF. 
Unexpected serious adverse events that are fatal or life-threatening must be filed as soon as 
possible and the sponsor will ensure all required information are edited in a initial report 
submitted to the Ethics Committee as soon as possible but not later than 7 calendar days 
after first knowledge, followed by as complete a report as possible within 8 additional 
calendar days.  
Unexpected serious adverse events that are not fatal or life-threatening must be filed as soon 
as possible but no later than 15 calendar days after first knowledge by the sponsor. 
Sponsor will be notified within 24h of awareness of any SAE, and Sponsor will be informed of 
the situation and its development on a running basis. 
 
8 STATISTICAL METHODS 
8.1 
ESTIMATION OF SAMPLE SIZE  
The sample size for this study was calculated on the primary endpoint of rhinovirus-induced 
illnesses.  The challenge model results in an infection rate of approximately 90% in the 
control group.  Approximately 67% of the infected volunteers (60% of all challenged 
volunteers are expected to develop a symptomatic illness and will meet the study definition of 
a rhinovirus-induced illness.  A sample size of 95 completed subjects/arm would be expected 
to detect a reduction in rhinovirus-induced illness of 20% (from 60% to 40%) with 80% power 
with pα= 0.05. 
8.2 
RANDOMISATION 
The study product and placebo will be randomized and assigned sequential numbers by the 
sponsor.  Volunteers will be assigned a study number as they present for the day -28 visit 
and the number of the study material they are given will correspond to their study number. All 
study personnel will remain blinded to the product or placebo assignment until all data have 
been collected and entered into the study database. 
 
8.3 
BLINDING AND CODE BREAKING INSTRUCTIONS 
8.3.1 Blinding 
The study product and a placebo that is identical in appearance, taste and smell will be 
provided by the sponsor.  The study material will be identified only by a number.  All study 
personnel will remain blinded to the study material administered until all data have been 
collected and entered into the study database. 
 
8.3.2 Emergency unblinding of a individual subject 
The randomization schedule will be provided by the sponsor and the randomization code will 
be available from the School of Medicine Clinical Trials Office in case of emergency. If it is 
medically imperative to know which study treatment the subject is receiving, emergency 
unblinding will be done. The Director of the Clinical Trials Office will unblind the code when 
medically needed, without identifying other subjects’ treatment. Every attempt must be made 

 
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to contact the sponsor before the code is unblinded. The person who unblinds the 
randomization code must record the date, time and the reason for emergency unblinding in 
the subject’s medical record, and on the Note-to-File, and in the data management system. 
In such cases, the use of the investigational product must be stopped and the sponsor must 
be contacted immediately to determine whether the subject should be withdrawn from the 
study. 
 
8.4 
STATISTICAL ANALYSIS PLAN 
In this chapter the most important analyses are described. Before locking the database and 
opening the treatment code a statistical analysis plan (SAP) document will be created, where 
all the analyses including the exploratory analyses will be described in detail. At least the 
descriptive statistics will be reported for all variables.  
 
8.4.1 PRIMARY ANALYSIS 
 
As the primary analysis, the incidence of rhinovirus-associated illness episodes (Yes/No) 
between the treatment groups will be investigated. The null hypothesis to be rejected is that 
there is no relationship between the treatment and the incidence of rhinovirus-associated 
illness episodes. The hypothesis will be tested by using a chi-square test. In case the cell 
frequencies, such as the expected number of subjects experiencing an illness episode or the 
expected number of subjects without any episodes in either of the treatment groups, turn out 
5 or lower, a Fisher’s exact test will be used for the comparison instead, which is seen as a 
more suitable method with low expected frequencies.    
 
As a sensitivity analysis a logistic regression will be computed for the same dichotomous 
response (incidence of rhinovirus-associated illness episodes) but in addition to the treatment 
effect also adjusted with potential confounding factors. Potential confounding factors can be 
such as antibody status, amount of virus given on day 0, gender or incidence of wild-type 
virus.  
 
In the primary analysis a p-value ≤ 0.05 is considered as significant.  
 
8.4.2 SECONDARY ANALYSIS 
 
The secondary analyses will be concentrating on duration of illness, incidence of infection, 
viral load during infection and proportion shedding the virus.  
 
The duration of illness between the treatment groups will be investigated by comparing the 
group mean durations with a two sample independent t-test. In case the durations are not 
normally distributed a non-parametric Wilcoxon-Mann-Whitney test will be used for the 
comparison. 
 
The dichotomous variables, proportion of infection incidence and virus shedding (days 1-5),  
will be both evaluated exactly the same way as the primary response (described in 8.4.1).  
 
The quantitative viral titer load during infection will be evaluated with repeated measures 
analysis of variance model (RM-ANOVA) including the daily and overall (study days 1-5) titer 
load as a response, treatment, day and the interaction of day and treatment as explanatory 

 
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variables. The random effect of subject will be taken into account in the model. Model 
assumptions will be checked with model diagnostics and normality plots. 
 
The secondary analyses are ordered in a hierarchical order by their clinical importance as 
described in 6.3. Thus no multiplicity correction is needed and a p-value ≤ 0.05 can be stated 
as statistically significant as long taking into account the hierarchy with the conclusions.  
 
8.5 
STUDY POPULATIONS 
The study population for this study will be healthy adult (18-60 y) volunteers.  The volunteers 
will be recruited from the University of Virginia community. 
8.6 
DATA MANAGEMENT 
The handling of data will comply with applicable regulatory guidelines. A more detailed data 
management plan (DMP) for the activities described below will be prepared. 
 
Data will be collected from the study visits using Viedoc™ Clinic (PCG Solutions AB, 
Uppsala, Sweden) electronic case report form (eCRF) system. Edit checks will be defined 
and programmed in the eCRF system to reveal possible discrepancies (missing, incomplete 
or illogical data) according to an agreed data validation plan (DVP). eCRF system will be 
used for recording data from each subject meeting the eligibility criteria and being included in 
the study, and for subjects who fail to meet eligibility criteria (i.e., screen failures) up to non-
eligibility decision. The study staff responsible for entering data into the eCRF system will be 
trained by the Study Monitor prior to the site initiates subject enrollment. A personal log-in will 
be provided for all responsible personnel to allow for an audit trail relating to the study data. 
The list of responsible persons in this study alongside their responsibilities will be 
documented and stored in the Trial Master File (TMF). No real-time study data will be 
transferred via the eCRF system until the site has been qualified through the training and 
completion of a test eCRF before “go-live”. 
 
Evaluations performed shall be entered in a timely manner into the eCRF by the site staff 
with delegated responsibility for this specific task. It is the responsibility of the site staff and 
Study Monitor to ensure that the eCRFs are properly completed. The data in the eCRFs 
should be consistent with the relevant source documents. The Investigator will sign the eCRF 
data entry screens to confirm that the captured data on each screen is accurate and 
complete. All data in the eCRF system must be stored in an unidentifiable form and treated 
with strict confidentiality in accordance with applicable data protection regulations. 
 
Captured data will be monitored and reviewed electronically. Any inconsistencies will be 
presented as queries; either automatically generated queries raised by the automated edit 
checks of the eCRF system, or by manually generated queries raised by the Study Monitor 
and Data Manager. Queries shall be resolved in a timely manner by the trained site staff. The 
eCRF may contain source data for certain assessments as agreed in writing between the 
Study Monitor and the staff at the study site. 
 
Recorded adverse event, medical history and concomitant medication data will be coded 
according to MedDRA and WHO ATC/DDD Index. 
 
The clean database will be locked after all data have been entered, queries have been 
resolved/closed and the database updated accordingly. 

 
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9 REGULATORY AND ADMINISTRATIVE PROCEDURES 
9.1 
INSTITUTIONAL REVIEW 
Prior to study initiation the protocol and ICF will be reviewed and approved by the Institutional 
Review Board for Health Sciences Research (IRB-HSR) at the University of Virginia. 
 
9.2 
SUBJECT INFORMATION/ INFORMED CONSENT 
Subjects will be informed of the nature of the study, its aim, its possible risks and restrictions, 
its duration. The protocol will be explained during a meeting prior to the study and each 
subject must be informed that participation in the study is voluntary and that he/she may 
withdraw from the study at any time. The investigator or his staff must explain to potential 
subjects or their legal representatives the aims, methods, reasonably anticipated benefits 
and potential hazards of the trial and any discomfort it may entail. They will be told that 
refusal to participate in the study will not prejudice future treatment. They will also be told that 
their study records may be examined by competent authorities and authorized persons but 
that personal information will be treated as strictly confidential and will not be publicly 
available. Subjects must be given the opportunity to ask questions. After this explanation and 
before entry into the trial, consent should be appropriately recorded by means of the subject's 
or his/her legal representative's dated signature. If a subject and his/her legal representative 
are unable to read, an impartial witness must be present during the entire informed consent 
discussion. The signature of the impartial witness will certify the subject's consent. The 
subject should receive a signed and dated copy of the informed consent form. No subject can 
enter the study before his informed consent has been obtained. 
 
The information sheets and original signed informed consent form will be retained by the 
investigator.  It will be kept for 15 years by the investigator and a copy will be given to the 
subject. The completed “subject screening log” will be signed by the investigator to attest that 
consent has been obtained from all subjects. Before being enrolled in the clinical study, 
subjects must consent to participate after the nature, scope, and possible consequences of 
the clinical study have been explained in a form understandable to them. 
 
An informed consent document that includes information about the study will be prepared 
and given to the subject. This document will contain all the elements required by the ICH E6 
Guideline for Good Clinical Practice and any additional elements required by local 
regulations. The document must be in a language understandable to the subject and must 
specify who informed the subject. Where required by local law, the person who informs the 
subject must be a physician. 
 
After reading the informed consent document, the subject must give consent in writing. The 
subject's consent must be confirmed at the time of consent by the personally dated signature 
of the subject and by the personally dated signature of the person conducting the informed 
consent discussions. The copy of the signed consent document must be given to the subject. 
The original signed consent document will be retained by the investigator. The investigator 
will not undertake any measures specifically required only for the clinical study until valid 
consent has been obtained. 
 

 
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The investigator should inform the subject’s primary physician about the subject’s 
participation in the study if the subject has a primary physician and if the subject agrees to 
the primary physician being informed. 
 
9.3 
SUBJECT CONFIDENTIALITY 
Subject names will not be supplied to the sponsor. Only the subject number will be recorded 
in the eCRF, and if the subject name appears on any other document (e.g., laboratory 
report), it must be obliterated on the copy of the document to be supplied to the sponsor. 
Study findings stored on a computer will be stored in accordance with local data protection 
laws. The subjects will be informed that representatives of the sponsor, independent ethics 
committee (IEC)/ institutional review board (IRB), may inspect their medical records to verify 
the information collected, and that all personal information made available for inspection will 
be handled in strictest confidence and in accordance with local data protection laws. The 
investigator will maintain a personal subject identification list (subject numbers with the 
corresponding subject names) to enable records to be identified. 
 
9.4 
GCP AND RECORD RETENTION 
9.4.1 The study will be carried out in accordance with: 
 
• 
The text of the Declaration of Helsinki adopted by the World Medical Assembly 
in June 1964, amended in Tokyo, October 1975, in Venice, October 1983, in 
Hong-Kong, September 1989, in Somerset West, October 1996 and in 
Edinburgh, October 2000, updated with the clarification note, Washington 
2002, Tokyo 2004 and Seoul 2008. 
 
• 
The ICH recommendations: Good Clinical Practice (E6), applied since January 
17th, 1997. 
 
9.4.2 Record Retention 
In compliance with the ICH/GCP guidelines the investigator/institution will maintain all eCRF 
data and all source documents that support the data collected from each subject, and all trial 
documents as specified in Essential Documents for the Conduct of a Clinical Trial and as 
specified by the applicable regulatory requirement(s). The investigator/institution will take 
measures to prevent accidental or premature destruction of these documents. Essential 
documents must be retained until at least two years after the last approval of a marketing 
application in an ICH region or at least two years have elapsed since the formal 
discontinuation of clinical development of the investigational product. These documents will 
be retained for a longer period if required by the applicable regulatory requirements or by an 
agreement with the sponsor. It is the responsibility of the sponsor to inform the 
investigator/institution as to when these documents no longer need to be retained. If the 
responsible investigator retires, relocates, or for other reasons withdraws from the 
responsibility of keeping the study records, custody must be transferred to a person who will 
accept the responsibility. The sponsor must be notified in writing of the name and address of 
the new custodian.  
 

 
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9.5 
MONITORING AND QUALITY CONTROL 
9.5.1 Monitoring 
The investigator will allow the representative of the sponsor and the study monitor: 
• 
To inspect the site, the facilities and the material used for the study, 
• 
To meet all members of the team involved in the study, 
• 
To consult all the documents relevant to the study, 
• 
To check that the eCRFs have been correctly completed, 
• 
To have direct access to source documents for comparison of data therein 
with the data in the eCRFs, 
• 
To check that AE have been documented, 
• 
To verify that the study is carried out in compliance with the protocol. 
 
This study will be monitored at regular intervals, by mutual agreement of the investigator and 
Monitor. All information dealt with during these visits will be treated as strictly confidential. 
 
The investigator will provide the sponsor with the following: 
• 
Progress reports at regular intervals, 
• 
Adequately completed eCRFs. 
Monitoring will be done according to the monitoring manual by a representative of the sponsor 
(study monitor) who will check the eCRFs for completeness and clarity, and cross-check them 
with source documents. In addition to the monitoring visits, frequent communications (letter, 
telephone, fax, e-mail), by the study monitor will ensure that the investigation is conducted 
according to protocol design and regulatory requirements. Study close-out will be performed 
by the study monitor upon closure of the study. 
 
9.5.2 Audit-Inspection 
The investigator will be informed that an audit may be carried out, at the request of the 
sponsor, before, during or after the study. The investigator will be informed that the 
Regulatory Authorities may also carry out an inspection. In this case, the investigator must 
inform the sponsor as soon as he receives the notification of inspection. 
The investigator must allow the representatives of the Regulatory Authorities and persons 
responsible for the audit to: 
• Inspect the site, facilities and material used for the study, 
• Meet all members of his team involved in the study, 
• Have direct access to study data and source documents, 
• Consult all the documents relevant to the study. 
Domestic and foreign regulatory authorities, the IEC/IRB, and an auditor authorized by the 
sponsor may request access to all source documents, eCRF data, and other study 
documentation for on-site audit or inspection. Direct access to these documents must be 

 
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guaranteed by the investigator, who must provide support at all times for these activities. 
Medical records and other study documents may be copied during audit or inspection provided 
that subject names are obliterated on the copies to ensure confidentiality. 
 
9.5.3 Quality Control 
The investigator or the appointed persons agree to complete the subject's eCRFs, at each 
investigation. Only the investigator or appointed persons in his team may fill out or correct the 
eCRFs. The eCRFs will display the subject number corresponding to the order of inclusion in 
the study. All corrections and alterations of data on the eCRFs must be made by the 
investigator or by the appointed persons. It is the responsibility of the monitor to make certain 
that all data are completed on the eCRFs. 
At the end of each study period, the investigator must sign the eCRF in order to attest 
respectively to: 
• 
Authenticity of the data collected in the eCRF , 
• 
Coherence between the data in the eCRF and those in the source documents. 
 
At the end of the study, the investigator will keep a copy of the correctly completed eCRF 
data for his own records. Archiving of the original eCRF data will be a responsibility of the 
sponsor. 
The investigator will keep a log of study volunteers screened for study participation and as 
appropriate, will indicate the reason individual study volunteers did not enter the study. 
No change made in the medical files by the investigator should obscure the original 
information. The record must clearly indicate that a change was made and clearly provide a 
means to locate and read the prior information. The investigator will save data at regular 
intervals. 
The investigator must guarantee the safety of the study data in the medical files by 
implementing security measures to prevent unauthorised access to the data and to the 
computer system. 
 
 
9.6 
INSURANCE AND LIABILITY 
Terms will be included in the research agreement. 
 
9.7 
STUDY REPORT 
9.7.1 Clinical Study Report 
The results of the study will be reported in a Clinical Study Report (CSR). This report will be 
prepared in co-operation with DuPont Nutrition and Health, Kantvik Active Nutrition team. In 
compliance with the regulations, the final report will be produced within one year of 
completing the study. The final report will be provided to all investigators having included 
subjects in the study. The CSR will be provided in printing and as Word and PDF files.  
 
9.8 
PUBLICATION AND DATA RIGHTS 
Terms for scientific publications are recorded in Research Agreement. 

 
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10 REFERENCES 
1. 
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infections. Cochrane Database Syst Rev 2015; 2: CD006895. 
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Davidson LE, Fiorino AM, Snydman DR, Hibberd PL. Lactobacillus GG as an immune 
adjuvant for live-attenuated influenza vaccine in healthy adults: a randomized double-
blind placebo-controlled trial. Eur J Clin Nutr 2011; 65(4): 501-7. 
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Olivares M, Diaz-Ropero MP, Sierra S, et al. Oral intake of Lactobacillus fermentum 
CECT5716 enhances the effects of influenza vaccination. Nutrition 2007; 23(3): 254-
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West NP, Horn PL, Pyne DB, et al. Probiotic supplementation for respiratory and 
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6. 
West NP, Horn PL, Barrett S, et al. Supplementation wiht a single and double strain 
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Bartosch S, Woodmansey EJ, Paterson JC, McMurdo ME, Macfarlane GT. 
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8. 
Ventura M, Zink R. Rapid identification, differentiation, and proposed new taxonomic 
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Aguirre M, Collins MD. Lactic acid bacteria and human clinical infection. J Appl 
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10. 
Borriello SP, Hammes WP, Holzapfel W, et al. Safety of probiotics that contain 
lactobacilli or bifidobacteria. Clinical Infectious Diseases 2003; 36(6): 775-80. 
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Boyle RJ, Robins-Browne RM, Tang ML. Probiotic use in clinical practice: what are 
the risks? Am J Clin Nutr 2006; 83(6): 1256-64; quiz 446-7. 
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Salminen S, von Wright A, Morelli L, et al. Demonstration of safety of probiotics -- a 
review. Int J Food Microbiol 1998; 44(1-2): 93-106. 
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Mogensen G, Salminen S. Inventory of microorganisms with a documented history of 
safe use in food. Bull Internat Dairy Fed 2002; 377: 10-9. 
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Cox AJ, West NP, Horn PL, et al. Effects of probiotic supplementation over 5 months 
on routine haematology and clinical chemistry measures in healthy active adults. 
European Journal of Clinical Nutrition 2014; 68(11): 1255-7. 
15. 
Ouwehand AC, DongLian C, Weijian X, et al. Probiotics reduce symptoms of antibiotic 
use in a hospital setting: a randomized dose response study. Vaccine 2014; 32(4): 
458-63. 
16. 
Ouwehand AC, Nermes M, Collado MC, Rautonen N, Salminen S, Isolauri E. Specific 
probiotics alleviate allergic rhinitis during the birch pollen season. World J 
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Engelbrektson A, Korzenik JR, Pittler A, et al. Probiotics to minimize the disruption of 
faecal microbiota in healthy subjects undergoing antibiotic therapy. J Med Microbiol 
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18. 
Paineau D, Carcano D, Leyer G, et al. Effects of seven potential probiotic strains on 
specific immune responses in healthy adults: a double-blind, randomized, controlled 
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19. 
Engelbrektson AL, Korzenik JR, Sanders ME, et al. Analysis of treatment effects on 
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11 Signatures 
 
Principal Investigator 
 
__________________________________ 
Ronald B Turner 
 
Date:  August 17, 2016 
 
 
Sponsor Representative 
 
 
___________________________________ 
Markus Lehtinen 
 
Date