A Double-blind, Randomized Controlled Trial of the Effect of Vaccine Inoculum on Oral Rotavirus Vaccine (Rotarix, GlaxoSmithKline) Take and Immunogenicity in Dhaka, Bangladesh
Phase: PHASE4
Conditions: Rotavirus Infection, Vaccine Response Impaired, Vaccine Virus Shedding
Sponsor: University of Vermont

 
A double-blind, randomized controlled trial of the effect of vaccine 
inoculum on oral rotavirus vaccine (Rotarix, GlaxoSmithKline) take 
and immunogenicity in Dhaka, Bangladesh  
 
 
Study protocol 
Version 9 
24 July 2019 
 
 
NCT02992197 
 
 
  
 

 
 
Table of Contents 
 
 
Project Summary ........................................................................................................................... 15 
Hypothesis to be tested: .................................................................................................................16 
Specific Objectives: .......................................................................................................................16 
Background of the Project including Preliminary Observations: ..................................................17 
Research Design and Methods .......................................................................................................19 
Retention of Records..................................................................................................................... 37 
Sample Size Calculation and Outcome (Primary and Secondary) Variable(s) ..............................37 
Data Analysis .................................................................................................................................38 
Data Safety Monitoring Plan (DSMP) ...........................................................................................39 
Ethical Assurance for Protection of Human rights ........................................................................40 
Use of Animals ..............................................................................................................................44 
Collaborative Arrangements ..........................................................................................................44 
Facilities Available ........................................................................................................................44 
Literature Cited ..............................................................................................................................45 
  Check here if appendix is included 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
List of Abbreviations 
Ab 
Antibodies 
AE 
Adverse event 
AR 
Adverse reaction 
BW 
Birth weight 
CCID50 
Cell culture infective dose, 50% 
COP 
Correlate of protection 
CPE 
Cytopathic effect 
CRF(s) 
Case report form(s) 
Ct 
Cycle threshold 
DGDA 
Directorate General of Drug Administration 
DNA 
Deoxyribonucleic acid 
DTP 
Diphtheria-tetanus-pertussis-vaccine 
DSMB 
Data and Safety Monitoring Board 
DSMP 
Data Safety Monitoring Plan 
EAE 
Expected Adverse Events 
EIA 
Enzyme immunoassay 
ELISA 
Enzyme-linked immunosorbent assay 
EPI 
Expanded Programme on Immunization 
ERC 
Ethical Review Committee (ICDDR,B) 
FDA 
Food and Drug Administration 
FFU 
Focus-forming unit 
FRA 
Field Research Assistant 
FWA 
Federalwide assurance 
GCP 
Good Clinical Practice 
HBGA 
Histo-blood group antigens  
HC 
Head circumference 
ICDDR,B 
International Centre for Diarrhoeal Disease Research, Bangladesh 
ICH 
International Conference on Harmonization 
ICF(s) 
Informed consent form(s) 
Ig 
Immunoglobulin 
IRB(s) 
Institutional Review Board(s) 
JHU 
Johns Hopkins University 
LAV 
Live-attenuated vaccines 
NIH 
National Institutes of Health 
NIPORT 
National Institute of Population Research and Training 
NSP 
Non-structural protein 

 
 
OHRP 
Office for Human Research Protections 
OPV 
Oral polio vaccine 
PBMC 
Peripheral blood mononuclear cell 
PCR 
Polymerase chain reaction 
PI(s) 
Principal Investigator 
PROVIDE 
Performance of Rotavirus and Oral Polio Vaccines in Developing Countries 
qRT-PCR 
Quantitative reverse-transcription polymerase chain reaction 
RV-IgA 
Rotavirus-specific serum IgA  
RRC 
Research Review Committee 
RT-PCR 
Reverse transcription polymerase chain reaction 
RV 
Rotavirus 
RV1 
Rotarix, GlaxoSmithKline 
RV5 
RotaTeq, Merck 
SAE 
Serious Adverse Event 
SAR 
Suspected Adverse Reaction 
SID 
Study identification number 
SUSAR 
Serious and unexpected adverse reaction 
UP 
Unanticipated problem 
UPnonAE 
Unanticipated Problem that is not an Adverse Event  
UAE 
Unexpected adverse event 
UVM 
University of Vermont 
VE 
Vaccine efficacy 
VP 
Viral protein 
WHO  
World Health Organization 
 

15 
 
 
Project Summary 
[The summary, within a word limit of 300, should be stand alone and be fully understandable.] 
Principal Investigator: Dr. Rashidul Haque 
 
Research Protocol Title: A double-blind, randomized controlled trial of the effect of vaccine inoculum on oral 
rotavirus vaccine (Rotarix, GlaxoSmithKline) take and immunogenicity in Dhaka, Bangladesh. 
Proposed start date:  March 1, 2017 Estimated end date: Dec 31, 2019 
Background: 
 
a. Burden: Oral rotavirus vaccines (e.g. Rotarix, RV1, GlaxoSmithKline) are highly efficacious in high-
income countries, but significantly underperform and/or are unavailable in low-income countries (including 
Bangladesh) where rotavirus remains the leading cause of diarrheal disease in children. 
b. Knowledge gap: The reasons for rotavirus vaccine underperformance remain unclear. Inadequate vaccine 
inoculum leading to diminished immunological effect is an unexplored possibility; improved immunogenic 
markers are needed to accelerate vaccine studies. 
c. Relevance: Improved vaccine performance and immunogenic markers will substantially impact rotavirus 
disease burden, vaccine research, and delivery in low-income countries.  
 
Hypothesis: Increased RV1 inoculum will increase rates of vaccine take (i.e. post-vaccination faecal RV1 
shedding or antibody seroconversion); antibodies targeting RV proteins VP4, VP5*, VP7, or NSP4, and post-
vaccination antigenemia/viremia will be superior immunogenic markers vs the current standard, anti-RV serum 
IgA (RV-IgA). 
 
Objectives: 
Primary (General) Objective 
1. To evaluate the effect of increased RV1 inoculum on vaccine take among infants in Dhaka. 
Secondary (Specific) Objectives 
1. To separately evaluate the effect of increased RV1 inoculum on:  
a. RV1 shedding and viral burden. 
b. RV-IgA seroconversion and concentration. 
Exploratory Objectives 
1. To evaluate alternate rotavirus immunogenic markers: 
a. VP4, VP5*, VP7, and NSP4 antibodies. 
b. Antigenemia/viremia.  
2. To explore the relationship between histo-blood group antigens (Lewis, secretor status) and: RV1 
shedding, immunogenicity, antigenemia/viremia. 
3. To explore RV1B and T cell responses.  
 
Methods: We will perform a double-blind, randomized (1:1), controlled trial among 220 infants in Dhaka to 
evaluate vaccine take and immunogenicity following standard- (106 FFU) vs high-dose (106.3 FFU) RV1.Infants 
will be followed through 14 weeks of life and evaluated pre- and post-vaccination for RV1 fecal shedding, serum 
RV-IgA, RV-VP4, VP5*, VP7, and NSP4 Abs, and antigenemia/viremia. 
 
Outcome measures/variables: Vaccine take; RV1 shedding; RV-IgA seroconversion and concentration; VP4, 
VP5*, VP7, and NSP4 antibody seroresponse and titre; RV1 antigenemia/viremia. 
 
 
 

16 
 
 
Description of the Research Project 
 
Hypothesis to be tested: 
In a hypothesis testing research proposal, briefly mention the hypothesis to be tested and provide the scientific basis of the hypothesis, 
critically examining the observations leading to the formulation of the hypothesis. 
 
Does this research proposal involve testing of hypothesis:
 No 
Yes (describe below) 
 
The hypothesis to be tested is that increasing the dosage of the oral, live-attenuated rotavirus vaccine 
Rotarix (RV1, GlaxoSmithKline) will lead to increased vaccine take and immunogenicity compared to 
standard dosing.  
 
It is now well-established that oral vaccines, including oral rotavirus (RV) vaccines, show significantly 
diminished efficacy in low-income countries. One possibility is that inhibitory factors in the infant gut, 
such as inflammation from enteric co infections or high levels of maternally-derived antibodies, might 
rapidly neutralize the vaccine and hinder vaccine “take.” Vaccine take refers to the ability of the vaccine 
to exert a biological effect on the host, and for oral vaccines is generally defined as seroconversion or 
detection of faecal vaccine shedding(1). Faecal shedding demonstrates replicating infection by the 
vaccine strain virus, a requirement for host recognition and subsequent immune response and long-term 
protection. Factors proposed to inhibit vaccine responses in the gut are prevalent in the developing world 
but might be overcome with an increased inoculum of vaccine.  
 
Specific Objectives: 
Describe the specific objectives of the proposed study. State the specific parameters, gender aspects, biological functions, rates, and processes 
that will be assessed by specific methods. 
 
Primary (General) Objective 
1. To determine whether the proportion of infants in Dhaka, Bangladesh with successful vaccine take 
is increased following high-dose RV1 vaccine administration compared to standard-dose RV1. 
Vaccine take is defined as detection of either post-immunization faecalRV1 shedding or rotavirus-
specific serum IgA (RV-IgA) seroconversion. 
 
Secondary (Specific) Objectives 
2. To separately evaluate the effect of increased RV1 inoculum on: 
a. Frequency of post-vaccination vaccine shedding and vaccine-strain virus burden in stool 
b. Frequency of RV-IgA seroconversion and RV-IgA concentration. 
 
Exploratory Objectives 
3. To evaluate alternate measures of rotavirus immunogenicity as candidate correlates of protection: 
a. Antibodies against RV1 outer capsid proteins VP4, VP7, and VP5* and RV1 non-structural 
protein NSP4. 
b. RV1 antigenemia and viremia following vaccination.  
4. To explore the relationship between the Lewis histo-blood group antigens (HBGA) and secretor 
status and RV1 faecal shedding, immunogenicity, and antigenemia/viremia.  
5. To explore B and T cell responses to RV1 immunization.  
 

17 
 
 
 
Background of the Project including Preliminary Observations: 
Provide scientific validity of the hypothesis based on background information of the proposed study and discuss previous works on the research 
topic, including information on sex, gender and diversity (ethnicity, SES) by citing specific references. Critically analyze available knowledge 
and discuss the questions and gaps in the knowledge that need to be filled to achieve the proposed aims. If there is no sufficient information on 
the subject, indicate the need to develop new knowledge.  
 
Diminished efficacy of oral rotavirus vaccines in low-income countries 
Rotavirus remains the leading cause of diarrhoeal disease in children worldwide and is responsible forup to 
a quarter of a million deaths yearly in children<5 years of age, primarily in Asia and sub-Saharan Africa 
(3, 4).Global introduction of oral live-attenuated 
vaccines (LAV) for rotavirus, such as RV1 and 
RotaTeq (RV5, Merck) have had remarkable success 
in reducing RV morbidity and mortality, particularly 
in high- and middle-income countries (5). RV 
vaccination is now recommended by the WHO in all 
national immunization programs (6), specifically at 6 
and 10 weeks of life in the Expanded Programme on 
Immunizations (EPI) schedule (7). However, for 
unclear 
reasons, 
vaccine 
efficacy 
(VE) 
is 
significantly diminished in low-income countries, 
where disease burden is highest (Figure 1). For 
example, in Europe, Latin America, and the United 
States, one-year VE against severe RV diarrhoea 
exceeded 95%, compared to approximately 50% in 
sub-Saharan Africa and Asia (8-11).  
 
Poor vaccines take as a potential factor in diminished oral vaccine efficacy 
Two issues currently frustrate efforts to improve RV vaccine performance: incomplete understanding of 
the variables impacting VE, and lack of reliable markers of host immune responses to vaccination 
(immunogenicity) in developing countries. Among variables impacting VE, one possibility is that inhibitory 
factors, such as maternally-derived antibodies, gut inflammation due to enteric coinfections, or 
environmental enteric dysfunction (12) might be limiting vaccine take in the infant gut, which is an absolute 
requirement for host recognition, subsequent immune response, and long-term protection (i.e. effective 
vaccination).  
 
Paucity of data regarding faecal vaccine shedding and lack of dose-ranging in low-income countries 
Faecal shedding of vaccine-strain virus has previously been evaluated, almost exclusively in high- and 
middle-income countries with good VE (13). In these studies, shedding was detected by RV-specific stool 
ELISA in approximately 60% of vaccinated infants 7 days following first RV1 dose (Table 1). One previous 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. Point estimates of Rotarix* and RotaTeq 
vaccine efficacy, and cases of severe rotavirus 
gastroenteritis prevented per 100 vaccinated infants 
by gross domestic product (GDP) per head. Ref: (2) 
Table 1. Selected studies showing rates of RV1 shedding by stool ELISA 7 days after first dose of RV1. 
Reference 
Study location 
Dose of RV1 
Percent shedding 
Vesikari 2004 (14) 
Finland 
105.8 FFU 
55% 
Dennehy 2005 (15) 
USA and Canada 
106.4 FFU 
58% 
Phua 2005 (1) 
Singapore 
106.1 FFU 
80% 
Ruiz-Palacios 2005 (16) 
Mexico 
105.8 FFU 
61% 
Hsieh 2014 (17) 
China 
106 FFU 
24% 

18 
 
study in Bangladesh evaluated RV1 shedding in a small number of infants following vaccination (N=76); 
less than 15% of vaccinated infants shed RV1 7 days following the first dose (18). Similarly, initial dose-
ranging studies for RV1 were conducted in Europe, Latin America, and East Asia, where VE is excellent; 
in these settings a range of doses (104.7– 106.4 FFU) of attenuated RV1 virus was shown to be safe (15, 19). 
Phase II trials in these countries assessed faecal RV1 shedding at a range of doses; in general, higher doses 
yielded slightly increased rates of shedding but the effects were extremely modest (1, 15, 20). Large-scale 
VE trials utilized 106.5 CCID50 of RV1, which is similar to the current commercial preparation of Rotarix 
and is equivalent to 106 FFU (17, 19). 
 
Significantly, the effects of vaccine inoculum on vaccine take, post-vaccination faecal RV1 
shedding, or immunogenicity have not been evaluated in low-income countries, where current vaccines are 
less effective. Factors proposed to inhibit vaccine responses in the gut are prevalent in the developing world 
but might be overcome with an increased inoculum of vaccine. This strategy has been successfully used 
with live oral cholera vaccines (21) but remains unexplored for RV.  
 
Need for improved markers of rotavirus immunogenicity and correlates of protection 
Efforts to improve vaccine efficacy are further hindered by lack of a highly reliable marker of 
immunogenicity for RV in developing countries. A reliable marker of immunogenicity must reflect host 
immune response to vaccination or natural infection. A 
marker of immunogenicity that correlates well with VE can 
be further employed as a correlate of protection (COP), a 
surrogate endpoint that would render clinical endpoints (i.e. 
RV diarrhoea) dispensable and greatly accelerate clinical 
vaccine trials. The standard marker of oral RV vaccine 
immunogenicity is RV-IgA. Typical assays measure 
antibodies (Ab) directed against the RV middle capsid 
structural protein VP6 (Figure 2). Serum RV-IgA has been 
shown to broadly correlate with VE when assessing 
population-level data (23). This association disappears, 
however, when assessing IgA responses specifically in 
low-income countries (24), as demonstrated in a large 
clinical trial in Africa where RV-IgA only explained a 
small fraction (32.7%) of vaccine effect (25). Improved markers of immunogenicity and improved COPs 
are required to expeditiously evaluate vaccine performance in low-income countries.  
 
A number of alternate markers have been proposed, including serum Ab against the RV outer capsid 
structural proteins VP4 and VP7 (24), which mediate viral attachment and may be more relevant to clinical 
immunity (Figure 2). RV antigenemia and viremia have also been noted following natural infection (26), 
but it is unknown if this occurs following vaccination. It seems probable that extra-intestinal antigen 
presentation and systemic immune responses would contribute to and enhance host protection from gut 
infection with RV; antigenemia or viremia could potentially serve as alternate markers of immunogenicity 
or COPs by identifying subjects more likely to develop these responses. Further, the Lewis HBGA and 
secretor status have recently been shown to modulate susceptibility to RV infection in a genotype-
dependent fashion, with Lewis and secretor-negative individuals protected from infection due to genotype 
P[8] rotavirus (27, 28). As RV1 is an attenuated G1P[8] virus, it is conceivable that Lewis and/or secretor 
status could similarly mediate susceptibility to vaccine take and subsequent protection from RV diarrhoea, 
but this has not been rigorously investigated. Finally, while previous work has demonstrated detection of 
RV-specific memory B cells and T cells expressing gut homing receptors following natural infection and 
vaccination, these aspects of RV immunity also remain significantly underexplored (reviewed in (24)). 
 
Preliminary Data 
We have preliminary data from the PROVIDE study, an RV1 efficacy trial conducted among infants in 
Dhaka, Bangladesh (29). In per-protocol analysis, VE against any RV diarrhoea at one year of life was 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2. Rotavirus structure. Ref: (22) 

19 
 
51%(30). No associations were found between RV-IgA seroconversion (RV-IgA > 20 U/mL following 
second RV1 dose vs baseline <20 U/mL) and VE, meaning many infants were protected from RV despite 
no RV-IgA response and vice versa. Overall immunogenicity was poor with only 27% seroconversion 
among vaccinated infants(30). In this context, there was a complete absence of RV1 shedding as detected 
by RV-specific stool ELISA following first RV1 dose (N=0/67). These data are in slight contrast with a 
previous RV1 immunogenicity study conducted in the same region of Dhaka, in which a much higher 
frequency of vaccine take (73.7%) was detected in a sub-cohort of infants, although total numbers were 
small (N=38) (18). Only 15% of infants were found to shed RV1 following the first RV1 dose, but a higher 
seroconversion rate of 56.5% was detected among infants concomitantly administered RV1 and OPV (as 
in PROVIDE) in the parent cohort. In that study, first vaccine dose was delayed until 12 weeks of life, and 
RV-IgA was performed using a different ELISA, which may account for some of the observed differences. 
Despite this, geometric mean concentration among infants vaccinated with concomitant OPV was relatively 
low at 46.6 U/mL. Overall, even in the study by Zaman et al., rates of RV1 shedding, RV-IgA 
seroconversion, and RV-IgA concentration remain well below those reported from high- and middle-
income countries. 
This population, in which vaccine take and immunogenicity were poor, therefore provides an ideal 
setting in which to test the effects of increased inoculum on vaccine take, immunogenicity, and alternate 
markers of immune response. Of note, higher rates of RV1 shedding are generally detectable when 
assessing stool shedding by RT-PCR rather than by ELISA. For example, in a study conducted in China, 
10.3% of subjects had detectable RV1 shedding 2-3 days following first dose when assessed by ELISA, 
compared to 72.4% with shedding detected in the same specimen when assessed by RT-PCR (17). At this 
time, RT-PCR evaluation of vaccine shedding from PROVIDE is in progress but data are not yet available.  
Using the PROVIDE study specimen bank, we are currently developing assays to interrogate serum 
Ab against RV outer capsid structural proteins VP4, VP7, and VP5*, and RV1 non-structural protein NSP4, 
all derived from the attenuated RV1 vaccine-strain virus, to identify a better COP. We are utilizing a 
baculovirus expression system to express target RV1 proteins in Sf9 insect cell culture; this method ensures 
antigen presentation in a manner that preserves proper conformation and glycosylation. We will thus have 
a new platform for evaluating host antibody responses to RV. From PROVIDE, remaining specimen 
volumes are limited and results will require validation in additional cohorts. This study will allow us to 
validate the performance of antibodies against these targets as markers of immunogenicity, relative to the 
standard RV-IgA. If these targets represent improved markers of immunogenicity, they would have 
significant potential as improved COPs.  
 
 
Research Design and Methods 
Describe the research design and methods and procedures to be used in achieving the specific aims of the research project. If applicable, mention 
the type of personal protective equipment (PPE), use of aerosol confinement, and the need for the use BSL2 or BSL3 laboratory for different 
part of the intended research in the methods.. Define the study population with inclusion and exclusion criteria, the sampling design, list the 
important outcome and exposure variables, describe the data collection methods/tools, and include any follow-up plans if applicable. Justify the 
scientific validity of the methodological approach (biomedical, social, gender, or environmental).  
 
Also, discuss the limitations and difficulties of the proposed procedures and sufficiently justify the use of them. 
 
STUDY DESIGN 
We will perform a double-blind, randomized controlled trial comparing a standard dose (106 FFU) to a high 
dose (106.3 FFU) of RV1 among 220 healthy infants in Dhaka, Bangladesh. The goal of this study is to 
evaluate the effect of increased vaccine inoculum on vaccine take and immunogenicity, and to explore new 
markers of immunogenicity against RV. Infants will be randomized 1:1 to receive 106(N=110) or 
106.3(N=110) FFU of RV1 at 6 and 10 weeks of life. A standard dose of commercially available RV1 
vaccine contains 106 FFU(17, 19). An inoculum of 106.3 FFU can be achieved by simultaneous 
administration of two standard doses of RV1. Importantly, RV1 doses up to 106.4 FFU have been evaluated 
in the United States and Canada and shown to be safe (15).  
 

20 
 
STUDY SITE AND POPULATION 
Two hundred twenty infants (aged 0-7 days) will be recruited from the urban Mirpur community of Dhaka 
and enrolled into the study. This population represents the same population studied in PROVIDE. 
Numerous important studies evaluating oral vaccine performance and enteric diseases have also been 
previously performed in this population. icddr,b already has an established clinic in Mirpur that continues 
to serve as an ongoing field site for vaccine trials. It is currently the study site for the Dengue in Dhaka 
Initiative, conducted in collaboration with the University of Vermont (UVM), Johns Hopkins University 
(JHU), and the National Institutes of Health (NIH). Due to the strong working relationship between this 
community and the icddr,b, the presence of an established field site, and a proven history in successful 
completion of multi-center, collaborative trials, this is an ideal site in which to conduct this study.   
 
ELIGIBILITY CRITERIA 
Inclusion criteria 
1. Generally healthy infant(as determined by research staff; any infant with potential health concerns 
will be assessed by a medical officer to determine health status) 
2. Age 0-7 days at enrolment 
3. Mother willing and able to provide signed informed consent 
4. Mother willing to allow infant to be vaccinated according to study schedule 
5. Mother willing to allow biological specimens, including blood, stool, and saliva, to be collected 
from infant according to study protocol 
6. Mother willing and able to adhere to study schedule  
 
Exclusion criteria 
1. Obvious congenital malformation 
2. Birth weight (if known) or enrolment weight (if birth weight unknown) < 2000 gm 
3. Known immunocompromising condition in infant  
4. Enrolment in other vaccine research trials  
5. Other household member enrolled in this study 
 
STUDY PROCEDURES 
Recruitment 
Recruitment will be conducted in the Mirpur community by trained field research assistants. Potential 
families will be identified and informed of the study during late pregnancy. In consideration of the literacy 
rate of this community (65.6% among married women in Dhaka, (31)), written recruitment materials will 
not be used, but recruitment will rely on word-of-mouth. 
 
Screening and enrolment 
Families recruited during the pre-natal periods who indicate an interest to participate will be screened 
following birth of the infant, within the first 7 days of life. Screening will be conducted by field research 
assistants in the home of the potential study participant. All potential study participants screened will 
receive a screening number as an identifier. The screening process begins with a review of the inclusion 
and exclusion criteria listed above to confirm the newborn’s eligibility to participate in the study. If the 
infant is eligible and the family wishes to participate, informed consent will then be obtained from the 
prospective study participant’s mother. The mother will be encouraged to take as much time as necessary 
to consult with any and all family members (e.g. husband) or other important individuals before making a 
final decision. Consenting will consist of a thorough review of the written consent form in a manner 
appropriate for the mother’s literacy level, as well as study-related education, including: 
1. Information on the risks or discomforts that may occur and the anticipated benefits of participation 
in the study; 
2. Explanation of the study participant’s right to leave the study at any time; 

21 
 
3. Assurance of respect for confidentiality and how the study will protect participants’ personal 
information; 
4. Training on the procedure and importance of accurate tracking and reporting of diarrhoea 
surveillance and infant health events within the household; 
5. Demonstration of stool specimen collection and explanation of study requirements for samples; 
6. Review of the study calendar and time commitment expected of participants; 
7. Instructions on how and when to contact study staff. 
 
Prior to signing the consent form, the mother will have an opportunity to ask questions about the study. If 
the field research assistant determines the mother has demonstrated adequate comprehension of the study, 
the consent form will be signed by both the field research assistant and the mother. If the mother is not 
sufficiently literate to sign the consent form, a thumbprint will be obtained in the presence of a witness to 
indicate consent. The witness may not be associated with the study. The mother will be provided a copy of 
the signed consent form. 
 
If the mother demonstrates adequate understanding of the study and indicates consent for participation by 
signing the consent form, screening continues with the following procedures: 
• 
Collection of study participant’s name, date of birth, sex, demographic information, and household 
socioeconomic information; 
• 
Collection of infant’s anthropometric measurements: body weight at birth (BW, if known), and 
body weight at enrolment, length, and head circumference (HC); 
 
At the conclusion of this process, the study participant is considered to be enrolled in the study. Immediately 
following enrolment, each study participant will be assigned a unique study identification number (SID).  
 
Randomization and blinding 
Prior to study initiation, each SID will be randomly assigned to the control or intervention arm (1:1 
assignment) by a designated data manager. Randomization will be performed using random permuted 
blocks. Only the designated data manager at UVM and research pharmacist or designee at icddr,b will have 
access to the randomization assignments. Double-blinding is required to minimize the chance for potential 
bias due to differential intensity of follow-up or evaluation in the intervention vs control arms, and to reduce 
the risk of potential over-reporting by families of potential adverse events if families recognize that their 
infant is receiving the study intervention, rather than control. The UVM data manager will have access to 
the randomization codes as a necessary requirement of generating the randomization. The pharmacist or 
designee requires access to the randomization assignments to ensure proper dosing according to the study 
protocol and to manage any events that may require unblinding (e.g. serious adverse event requiring 
unblinding). Upon enrolment, each study participant will be assigned to the lowest available SID number 
and corresponding randomization assignment. The child will remain assigned to the treatment group therein 
for the duration of the child’s participation in the study, and study participants and investigators will remain 
blind to intervention status.  
 
Study Participant Withdrawal or Termination from the Study 
Participants may withdraw voluntarily from the study at any time without penalty or consequence.  
 
If any of the following events occur, the child will be withdrawn from the study: 
1. The research is terminated by the Principal Investigator (PI) or by any regulatory authority. 
2. The child’s mother withdraws consent for the child to participate in the study. 
3. The child’s family moves out of the surveillance area. 
4. The child dies. 
5. The child misses any RV1 dose, unless dosing beyond the visit window is approved by the PI or 
designee (e.g. medical officer). Any study participant withdrawn for missing the second dose will 

22 
 
be followed for safety until the end of the study period. Any study participant who is withdrawn 
for having missed the first dose will not be followed for safety as in this scenario no study 
procedures will have been performed on the participant.  
6. The mother is not available to answer questions for more than 14 days and her whereabouts are 
unknown. 
7. An investigator, in consultation with the medical officers, determines that continued participation 
in the study jeopardizes the health or well-being of the child. In this case, the study team will ask 
permission to contact the family at the end of the study to confirm the health and well-being of the 
withdrawn child. 
 
Replacement of Study Participants 
Children who withdraw or are terminated from the study will not be replaced, as expected attrition has been 
incorporated into sample size and target enrolment calculation. 
 
Intervention 
All children enrolled in the study will receive oral RV1 vaccine. Children will be randomized 1:1 to receive 
either a standard dose of vaccine (106 FFU = control) or double the standard dose (106.3 FFU = intervention) 
of RV1 vaccine at 6 and 10 weeks of life, according to the WHO-recommended EPI schedule.  
 
Vaccine: 
RV1 (Rotarix, GlaxoSmithKline) is an oral, live-attenuated rotavirus vaccine. It is provided as a clear, 
colourless liquid formulation, containing 1.5 mL of vaccine in a pre-filled applicator syringe or tube. 
Vaccine must be stored at 4oC. Each vial contains 106-106.5CCID50 (corresponding to 106 FFU) of RIX4414, 
an attenuated G1P[8] strain of group A human rotavirus(17, 19). A double dose of vaccine thus represents 
up to 106.3 FFU of vaccine (106.3/106 = 1.995). All vaccine and placebo doses will be transferred to sterile, 
generic-appearing syringes prior to administration by the research pharmacist or designee at the central 
pharmacy, labeled with the SID, and transported cold to the study clinic. For study participants randomized 
to receive 2 doses of vaccine, 2 syringes of vaccine, totaling 3 mL volume will be administered. Study 
participants randomized to receive a standard dose of vaccine will receive one syringe of vaccine and one 
syringe containing 1.5 mL of sterile, pharmacy-grade water, for 3 mL total volume. In this manner, all study 
participants and study staff will thus remain blinded to intervention arm.  
 
Study Event Schedule 
A detailed work plan for the study is presented below (Table 2), including: the study immunization plan, 
schedule of clinic visits, and timetable of laboratory tests for all study indicators. Full description of home 
visits and clinic visits are provided after the summary table. A summary of the study participant 
participation timeline is provided (Figure 3). 
 
Specimen Collection 
Mothers will be provided specimen cups and trained in how to save stool specimens for retrieval at 
designated time points by field research assistants for faecal RV1 shedding analysis. In addition, mothers 
will be trained to save at least one diarrhoeal stool specimen for retrieval by field research assistants for 
evaluation of RV diarrhoea during each diarrhoeal episode (if any) for her infant. Per the discretion of the 
mother and the assessment of the field research assistant, mothers may bring stool specimens to scheduled 
clinic visits, as each visit requires collection of stool, or may request that the specimens be retrieved by 
field research assistants.  For all clinic visits except week 14, the specimen must be obtained within the 24 
hours prior to the visit. All blood specimens will be collected at clinic visits by trained personnel.  
 
Scheduled clinic visits 
RV1 vaccine will be administered at the week 6 and week 10 clinic visits. It has been described that OPV 
has a mild inhibitory effect on RV1 immunogenicity, as measured by RV-IgA, when administered 

23 
 
simultaneously versus when given in staggered fashion (18, 32). No effect of RV1 on OPV immunogenicity 
has ever been observed and we have no reason to suspect an increased dose of RV1 will have an effect. To 
ensure that there is no interference between OPV and RV1, and at the request of the Mirpur EPI clinic, each 
child will receive EPI vaccines (including OPV) in our study clinic or at an EPI office at least 7 days  
following each RV1 dose. This will ensure that each child receives all vaccines on a similar schedule, 
without interference from OPV, but that EPI vaccines and RV1 are all received during approximately the 
7thand 11thweeks of life for all infants. 
 
 
 
 
 
 
Every reasonable attempt should be made to collect scheduled specimens within the designated visit 
window for each scheduled clinic visit; however, scheduled specimens may be collected outside the visit 
window at the investigator’s discretion in cases where doing so will not affect study participant safety or 
the scientific objectives of the study. 
Recruitment of families
x
Informed consent, enrollment, randomization
x
Collection of demographic, socioeconomic 
information
x
Anthropometric measurements
x
x
x
x
EPI vaccines
x
x
Weekly home visits1
x
x
x
x
x
x
x
x
x
x
x
x
x
x2
Daily home visits3 
x
x
x
x
Rotarix
x
x
Venipuncture
3 mL
3 mL
Heelstick
0.5 mL
0.5 mL
0.5 mL
RV-IgA
x
x
RV-IgG
x
x
Zinc
x
x
VP4, VP7, VP5*, NSP4 Ab
x
x
Rotavirus EIA (antigenemia)
x
x
x
x
Rotavirus PCR (antigenemia)
x
x
x
x
Viral culture (viremia)
x
x
x
x
PBMC collection
x
x
Diarrheal surveillance
Stool collection for vaccine shedding assessment
x
x
x
x
x
x
x
x
x
Stool PCR, Rotarix shedding
x
x
x
x
x
x
x
x
Gut inflammatory markers
x
x
Stool PCR, gut pathogens
x
Stool EIA for vaccine shedding assessment
x
x
x
x
x
x
x
x
Stool EIA for rotavirus diarrhea
Saliva collection (Lewis/secretor status)
x
Continuous testing of diarrheal specimens throughout study following enrollment
5
Intervention 
Study Activity
Blood measures
11
12
7
13
14
2.) 72 hours following the final clinic visit (week 14) to perform final safety assessment, conclude study participation 
3.) For 7 consecutive days following vaccination; telephone contact is acceptable alternative, unless a safety concern requires evaluation
1.) AE screening, health and safety assessment, reminder of scheduled stool collections, visits, and procedures
Table 2. Summary of work plan
Week of life
Pre-
natal
0
1
2
3
4
5
6
8
9
10
Other
Blood collection
1
2
3
4
Stool collection
Stool measures
Study Visit #
Continuous throughout study following enrollment

24 
 
 
 
 
Figure 3.Summary of study participant participation 
 
Visit 1: Age 6 weeks (+7 days) 
1. Confirm that correct, signed informed consent form is on file 
2. Measure infant’s weight, length, and HC 
3. Verify eligibility and that all inclusion and exclusion criteria have been met, and confirm that no 
contraindications to vaccination are present (per vaccine manufacturer instructions) 
4. Prior to vaccination, confirm stool sample for baseline faecal RV1 shedding analysis has been 
collected 
5. Prior to vaccination, collect approximately 3 mL of blood by venipuncture for the following: 
a. RV-IgA and RV-IgG 
b. RV VP4, VP7, VP5*, and NSP4 Ab 
c. Rotavirus plasma antigenemia/viremia 
d. PBMCs 
e. Zinc 
6. Administer oral RV1 vaccine, unless contraindications identified on screening 
 
Visit 2: Age 7 weeks (week 6 visit date +3-7 days): 
1. Perform safety assessment (for AEs) 
2. Confirm stool sample for faecal RV1 shedding analysis has been collected 
3. Collect approximately 0.5 mL of blood by heelstick for rotavirus antigenemia/viremia analysis 
4. If visit occurs on day +7, EPI vaccines can be given at that time if available; if visit occurs on day 
+3-6 or EPI vaccines unavailable in the study clinic, family may be escorted on or after day +7  to 
an  EPI clinic for EPI vaccines 
 
Visit 3: Age 10 weeks (week 6 visit date +28-35 days) 
1. Perform safety assessment (for AEs) 
2. Measure infant’s weight, length, and HC 
3. Confirm that no contraindications to vaccination are present (per vaccine manufacturer 
instructions).Prior to vaccination, confirm stool sample for faecal RV1 shedding analysis has been 
collected 
4. Prior to vaccination, collect approximately 0.5 mL of blood by heelstick for rotavirus 
antigenemia/viremia analysis 
5. Administer oral RV1 vaccine, unless contraindications identified on screening 

25 
 
 
 
Visit 4: Age 11 weeks(week 10 visit date +3-7 days): 
1. Perform safety assessment (for AEs) 
2. Confirm stool sample for faecal RV1 shedding analysis has been collected 
3. Collect approximately 0.5 mL of blood by heelstick for rotavirus antigenemia/viremia analysis 
4. If visit occurs on day +7, EPI vaccines can be given at that time if available; if visit occurs on day 
+3-6 or EPI vaccines unavailable in clinic, family may  be escorted on or after day +7  to an  EPI 
clinic for EPI vaccines 
 
Visit 5: Age 14 weeks (week 10 visit date +28-35 days) 
1. Perform safety assessment (for AEs) 
2. Measure infant’s weight, length, and HC 
3. Collect approximately 3 mL of blood by venipuncture for the following: 
a. RV-IgA 
b. RV VP4, VP7, VP5*, and NSP4 Ab 
c. Zinc 
d. PBMCs 
4. Collect saliva for Lewis/secretor status testing.  
 
Home visits 
 
Weekly home visits 
Home visits will be performed throughout each study participant’s participation in the study to perform 
safety assessments, perform study-related procedures, and help ensure protocol adherence. Any scheduled 
clinic visit can substitute for a home visit if the schedule for clinic visit coincides with need for home visit. 
Following enrolment and randomization, each study participant will be visited at home by a field research 
assistant at least once weekly for the duration of the study. Weekly home visits will consist of the following 
activities: 
• 
Record diarrhoea and health surveillance data to screen for adverse events following RV1 
vaccination (safety assessment). 
• 
Refer to the clinic, if indicated, for acute medical conditions. 
• 
Remind mother about stool specimen collection time points, and ongoing sample collection for 
diarrhoea surveillance, as indicated. 
• 
Remind mother of all scheduled clinic visits and procedures, as described elsewhere in the protocol. 
 
Daily home visits 
In addition to the weekly home visits listed above, for 7consecutive days following each dose of vaccine 
(with the exception of day +3-7 following each dose, which is a scheduled clinic visit), each study 
participant will be visited at home by a field research assistant to perform vaccine safety assessment and 
record signs or symptoms of possible adverse events (as described below in “Adverse Events”). If a home 
visit cannot be arranged, telephone contact will be an acceptable alternative, unless a safety concern is 
raised that requires evaluation (e.g. temperature measurement to rule out fever and need for medical 
evaluation).  
 
Retrieval of stool specimens for RV1 faecal shedding evaluation 
Retrieval of stool specimens for faecal RV1 shedding analysis will occur at the time of weekly or daily 
home visit at the following time points (see Figure 3): 
• 
Pre-vaccination:  
1) Age <14 days 
 
 
 
2) Age 6 weeks, immediately (0-3 days) prior to Clinic Visit #1 

26 
 
• 
Post-RV1 dose #1: 
3) Age 6 weeks, 3-4 days after Clinic Visit #1 
 
 
 
 
 
 
4) Age 7 weeks, 6-7 days after Clinic Visit #1  
 
 
 
5) Age 8 weeks, 13-14 days after Clinic Visit #1 
 
 
6) Age 10 weeks, immediately (0-3days) prior to Clinic Visit #3 
• 
Post-RV1 dose #2: 
7) Age 10 weeks, 3-4 days after Clinic Visit #3   
 
  
8) Age 11 weeks, 6-7 days after Clinic Visit #3  
  
9) Age 12 weeks, 13-16 days after Clinic Visit #3 
  
 
For stool collections that coincide with clinic visits, mothers may choose to bring stool specimens to the 
clinic visits if desired, rather than having an FRA retrieves it from the home. Pre-vaccination and post-RV1 
dose #1 specimens will also be evaluated for gut inflammatory markers and for additional enteric pathogens, 
including but not limited to enteroviruses. 
Ongoing 
• 
Diarrhoeal surveillance, including:  
 
Diarrhoea sample collection when infant is reported to have 3 or more abnormally loose 
stools in a 24 hour period, for testing for presence of rotavirus antigen (i.e. rotavirus 
diarrhoea) and potentially other pathogens; and  
 
Appropriate treatment/medical management for diarrhoea, as indicated. 
 
Conclusion of study participation 
A home visit will be conducted 72 hours following the final clinic visit to perform a final safety assessment 
following the week 14 clinic blood draw. If no concerns are raised, the study participant will be discharged 
from the study at that time. If a home visit cannot be arranged, communication via telephone will be an 
acceptable alternative, unless a concern is raised that requires evaluation.  
 
Maternal participation 
Recent data suggest that maternal histo-blood group antigen phenotype may also be important in infant 
susceptibility to RV. In a prior study involving the MAL-ED cohort in Dhaka, researchers demonstrated 
that maternal secretor and Lewis status had a much greater effect on infant susceptibility to RV infection 
than infant status (33). In a study from India, infants of mothers whose milk had higher levels of secretor-
dependent antigens were more likely to have symptomatic neonatal RV infection (34). Similarly, in our 
previous PROVIDE cohort, we have determined that infants of mothers whose breast milk was secretor-
negative had significantly higher odds of Rotarix seroconversion (manuscript in preparation). Therefore, 
newer data indicate that maternal secretor and Lewis status is an important variable in RV vaccine response 
and susceptibility. Therefore, we will request saliva sample collection from all mothers of infants who 
completed the study for saliva Lewis and secretor phenotyping and genotyping to further extend these 
findings.  
 
Laboratory assays (see Table 3 for summary) 
 
Table 3. Summary of Tests/Assays 
Test/Assay 
Type of sample 
Location 
performed 
Nucleic acid extraction 
Vaccine shedding stools 
icddr,b 
Rotavirus PCR 
Stool nucleic acid extraction 
icddr,b 
EIA for rotavirus antigen 
Vaccine shedding, diarrhoeal stool 
icddr,b 
EIA for rotavirus antigen 
Plasma 
icddr,b 
Zinc 
Plasma 
icddr,b 
Rotavirus gene sequencing  
Stool nucleic acid extraction 
UVM 
RV-IgA, RV-IgG ELISA 
Plasma 
UVM 

27 
 
Nucleic acid extraction 
Plasma 
UVM 
Rotavirus PCR 
Plasma nucleic acid extraction 
UVM 
Viral culture (viremia) 
Plasma 
UVM 
RV antibody immunocytochemistry assays 
(VP4, VP5*, VP7, NSP4) 
Plasma 
UVM 
EIA for gut inflammatory markers 
Stool 
UVM 
PCR (or other molecular methods) for gut pathogens 
Stool, stool nucleic acid extraction 
UVM 
Multi-color flow cytometry (B & T cell phenotyping) 
PBMCs 
UVM 
Lewis antigen and secretor status by ELISA 
Saliva 
icddr,b 
Lewis and secretor genotyping 
PBMCs or stool nucleic acid 
extracts 
UVM 
 
Stool total nucleic acid extraction for vaccine shedding detection and gut pathogen detection. Total nucleic 
acid extraction will be performed on all stool specimens collected during the study period, with the 
exception of the week 1-2 collection, for the purpose of vaccine shedding detection using the QIAmp Fast 
DNA stool Minikit (QIAGEN), similar to previously described methods (35). Extraction controls using 
MS2 bacteriophage (ATCC) will be added to each extracted specimen, as well as a blank specimen on each 
day of extraction to serve as a batch control. The 1-2 week stool will only be assessed for rotavirus positivity 
if the study participant is positive for rotavirus at the week 6 visit, before vaccination, to estimate how long 
prior to vaccination the participant was first exposed to rotavirus. 
 
Real-time quantitative RT-PCR and gene sequencing for vaccine shedding detection. Due to our previous 
experience that demonstrated poor sensitivity of stool ELISA to detect faecal vaccine shedding in this 
population (PROVIDE, unpublished data,(18)) shedding will be assessed by RT-PCR. Total nucleic acids 
extracted from stool specimens will be tested for the presence of RV using real-time quantitative RT-PCR 
(qRT-PCR) targeting the highly conserved NSP3 gene sequence using previously described primers and 
probes(36). Detection of MS2 extraction controls will be conducted as previously described (36). Absolute 
quantitation of viral burden will be performed based on analysis of target Ct values relative to a standard 
reference curve. PCR reactions will be performed using the UltraPlex 1-Step Toughmix PCR kit (Quanta 
Biosciences) and detection will be performed using the CFX96 real-time PCR detection system (Bio-Rad). 
Specimens that test positive for RV will have genotyping performed by the Sanger method for the RV VP4 
gene to confirm presence of vaccine-strain virus (RIX4414). Alternatively, specimens may be directly 
assayed for vaccine-strain virus using a primer/probe set specific for the NSP2 gene segment for the Rotarix 
vaccine strain (37). 
 
PCR for gut pathogen burden. Stool total nucleic acid specimens will be evaluated for other gut pathogens, 
including but not limited to enteroviruses, by conventional or real-time PCR (38)or other molecular 
methods.    
 
Stool RV-specific EIAfor detection of RV antigen in diarrhoeal stool specimens. All diarrhoeal stool 
specimens collected during the study period will be tested for the presence of rotavirus antigen using a 
commercial EIA kit (ProSpecT Rotavirus Microplate Assay, Oxoid), per manufacturer’s instructions.  
 
Stool RV-specific EIA for vaccine shedding detection in high-dose RV1 recipients. After initial analysis, 
EIA for detection of RV antigen will be performed on all stool specimens collected during the study period 
for the purpose of vaccine shedding detection that test positive by PCR, and on a subset of PCR-negative 
specimens. While ELISA was previously demonstrated to have poor sensitivity to detect faecal vaccine 
shedding in this population following standard-dose RV1 (PROVIDE, unpublished data,(18)), it is 
conceivable that vaccine shedding may be detected following high-dose RV1. If so, detection of shedding 
in future studies would be greatly simplified by use of EIA rather than real-time qRT-PCR. 

28 
 
 
Plasma rotavirus antigen and virus detection. Plasma specimens collected 7 days following each vaccine 
dose will be assessed for presence of rotavirus antigenemia using a commercial kit (ProSpecT Rotavirus 
Microplate Assay, Oxoid) (39) and by qRT-PCR targeting RV NSP3 following RNA extraction using the 
QIAmpViral RNA Mini Kit (QIAGEN). Plasma specimens collected 7 days following each vaccine dose 
will be assessed for the presence of infectious virus via serial passage on MA104 cells followed by antigen-
specific immunostaining, similar to previously described methods (26).  
 
Plasma rotavirus-specific IgA and IgG.RV-IgG will be assessed in pre-vaccination (week 6) plasma 
specimens, and RV-IgA will be assessed in pre-vaccination (week 6) and post-vaccination (week 14) 
plasma specimens by ELISA, as previously described (40). RV-IgA concentration (U/mL) will be 
calculated via interpolation from a standard reference specimen.  
 
VP4, VP7, VP5*, NSP4 antibody immunocytochemistry assays. IgA and IgG antibodies targeting RV1-
specific outer capsid proteins VP4, VP7, VP5*, and RV1-specific non-structural protein NSP4 will be 
assessed in pre-vaccination (week 6) and post-vaccination (week 14) plasma specimens using an 
immunocytochemical staining procedure, similar to previously described methods (41, 42). Briefly, 
recombinant baculovirus expressing epitopes cloned from the RV1 vaccine strain will be used to 
synchronously infect monolayers of Sf9 cells to yield recombinant protein expression. At the time of peak 
protein expression, cells will be fixed and permeabilized. Study participants’ plasma will be incubated on 
the fixed cell layers, followed by visual detection using conjugated secondary antibodies against human 
IgG and IgA. 
 
Determination of Lewis and secretor status. Saliva specimens collected from all study participants 
(including mothers who agree to provide a sample) will be tested for determination of Lewis antigen and 
secretor status by ELISA, similar to previously described methods (27). However, phenotyping assays, 
particularly in young infants, may be imprecise (43); an alternate method to more definitively identify 
Lewis and secretor status is to confirm the host’s genotype for the alleles of the genes that confirm the 
fucosyltransferase enzymes that determine Lewis (FUT3) and secretor (FUT2) status. Therefore, we will 
also performe sequence analysis of the FUT3 and FUT2 genes using the PBMC or stool nucleic acid extracts 
being collected for this study or from DNA extracts of infant and maternal saliva specimens to corroborate 
the phenotyping results. It is likely that additional, unknown genes in addition to FUT2 and FUT3 may be 
associated with rotavirus susceptibility or vaccine response. The only way to do this is to perform 
exploratory genetic analyses, which can be performed on the same specimens.  
 
B and T cell phenotyping. B and T cell phenotyping will be performed on baseline (week 6) and post-
vaccination (week 14) PBMCs by multi-color flow cytometry.  
 
Duration of participation 
Study participants will be active participants from the time of enrolment (0-7 days of life) until 3 days after 
the final visit at week 14 of life (approximately 3 months of life). Safety assessment will be performed by 
home visit (or telephone call, if home visit cannot be coordinated) after the final study visit. If no concerns 
are identified, the study participant will be discharged from the study and his or her participation in the 
study is concluded. 
 
Specimen preparation, handling, shipping and storage 
Preparation, handling, shipping, and storage of blood, stool, and saliva specimens will be described in the 
manual of procedures. All samples will be labelled by unique identifiers and not by name. 
 
During the study period, as needed for completion of the proposed assays, specimens may be housed at 
either at icddr,b or at UVM. After completion of the study, any specimens remaining at UVM will be 

29 
 
returned to icddr,b. Any specimens remaining that are of too low of volume to be usable any more will be 
destroyed. All specimens will be stored at icddr,b up to 5 years following the completion of the study.  
 
SAFETY MONITORING, ASSESSMENT AND REPORTING 
 
ADVERSE EVENTS: DEFINITIONS AND REPORTING 
 
Table 4 summarizes the definitions and required responses of any safety issues that might arise during this 
study. All AEs occurring from the time of the first study visit at 6 weeks of life through the end of the study 
period will be documented, recorded, and reported. All reports will include assessment of severity, 
relationship to study interventions, actions taken, and outcomes, as described below in “Assessment of 
adverse events.” The study team will not have primary responsibility for reporting safety submissions 
directly to the Data Safety and Monitoring Board (DSMB), except as specifically described below under 
Data Safety and Management Plan. Safety reports requiring DSMB attention will be submitted to DSMB 
by the ERC. All events outlined in Table 4. Adverse Events Definitions and Reporting will be reported to 
DSMB by the study team at annual review or otherwise directed. For reports to the Directorate General of 
Drug Administration (DGDA), only those events that are considered to have been possibly, probably, or 
definitely related to vaccination will be reported. 
 
 
Table 4. Adverse Events Definitions and Reporting 
Event 
Definition 
Reporting Requirements 
Adverse Event 
(AE) 
Any untoward or unfavourable medical occurrence in a 
study participant, including any abnormal sign, symptom, 
or disease, temporally associated with the study 
participant’s participation in the research, whether or not 
the event is determined to be related to the study 
participant’s participation in the research. 
 
Solicited AE’s. We will specifically query for symptoms 
listed below. Within 7 days of vaccination: 
1. Fussiness 
2. Cough or runny nose 
3. Fever 
4. Loss of appetite (decreased feeding compared to 
baseline) 
5. Vomiting  
6. Diarrhoea (defined as 3 or more stools looser than 
normal per mother’s judgement within 24 hours) 
 
Within 3 days of blood draw: 
7. Bleeding at puncture site 
8. Bruising at puncture site 
9. Signs of infection (redness, swelling, warmth, 
tenderness, purulent drainage, or fever) at puncture site 
 
Unsolicited AE’s. Any of the above symptoms if they 
should occur outside the specified time period for solicited 
AE’s, or any other signs or symptoms reported by the 
family.  
1. ERC:  Annual review  
2. DSMB: Annual review, or as 
otherwise directed. 
3. UVM: Annual review  
4. DGDA: Annual review  
 
Expected 
Adverse Event 
(EAE)* 
An AE that is a known reaction to the vaccine or 
phlebotomy, or that is generally common in this 
population. This includes the symptoms that appear above 
1. ERC:  Annual review  
2. DSMB: Annual review, or as 
otherwise directed. 

30 
 
in the list of solicited AE’s, even if they occur outside the 
specified time period for solicited AE’s. For example, 
diarrhea is a very common event in this population, and 
may be completely unrelated to study participation, and 
therefore is an EAE.  
 
As it is now accepted that RV1 is associated with a very 
small (1-5 excess cases per 100,000 children immunized) 
risk of intussusception, this will be categorized as an 
expected SAE. However, all cases of intussusception will 
be considered a pausing criteria (see below). 
3. UVM: Annual review 
4. DGDA: Annual review 
Serious 
Adverse Event 
(SAE) 
An AE that is determined to be “serious,” whether or not 
the event is related to study participation, including:  
1. Death  
2. Life threatening event that places a subject at 
immediate risk of death at the time of the event and 
does not refer to an event that hypothetically might 
have caused death were it more severe 
3. Inpatient hospitalization or prolongation of existing 
hospitalization, defined as at least an overnight stay in 
the hospital or emergency ward for treatment that 
would have been inappropriate if administered in the 
outpatient setting 
4. Persistent or significant disability or incapacity  
5. Other medically important event that may put the 
study participant at risk of harm or require 
intervention to prevent harm 
1. ERC*: As soon as possible (goal: 
within 24 hours of knowledge of 
their occurrence), including 
measures taken to 
mitigate/manage the event. In the 
event any participant experiences 
multiple SAEs, each event will be 
reported separately. If detailed 
information is not available at the 
time of submission of the initial 
report, follow-up report(s) will be 
submitted as soon as information 
is available. The first follow-up 
report will be made as soon as 
possible (preferably within 72-96 
hours whenever feasible) of the 
initial report.  Final outcome of 
the event will be reported within 
72 hours of knowledge of 
outcome. A follow up SAE report 
will be submitted to the ERC if 
SAE persists ≥ 2 weeks. 
2. DSMB: Annual review, or as 
otherwise directed. 
3. UVM: To be reported as soon as 
possible to UVM study staff. If it 
is determined that the event was 
unexpected AND related to study 
participation, the event will be 
reported to the UVM IRB as soon 
as possible by the UVM PI. 
Otherwise, it will be reported at 
annual review. 
4. DGDA: Reports of SAEs will be 
submitted by the PI at the same 
time they are reported to the ERC 
for any SAE related to the 
intervention. 
 
Unexpected 
Adverse Events 
(UAEs) 
An AE that does not qualify as an EAE, or an EAE that 
significantly exceeds the severity that has typically been 
observed. The PI will make this determination on a case-
by-case basis, should it arise.  
1. ERC: As for SAE. 
2. DSMB: Annual review, or as 
otherwise directed. 
3. UVM: As for SAE. 
4. DGDA: as for SAE 

31 
 
Serious and 
Unexpected 
Suspected 
Adverse 
Reactions 
(SUSAR) 
A Suspected Adverse Reaction (defined as an AE that for 
which there is a reasonable possibility that the AE was 
caused by the vaccine) that is both Serious and 
Unexpected. SUSARs are reported only if there is 
evidence to suggest a causal relationship between the 
vaccine and the adverse event, such as: 
1. A single occurrence of an event that is uncommon and 
known to be strongly associated with the vaccine. 
2. One or more occurrences of an event that is not 
commonly associated with the vaccine, but is 
otherwise uncommon in the exposed population. 
3. An aggregate analysis of specific events observed in a 
clinical trial that indicates those events occur more 
frequently in the intervention than control group. 
1. ERC: As for SAE.  
2. DSMB: Annual review, or as 
otherwise directed.  
3. UVM: As for SAE.  
4. DGDA: as for SAE 
Unanticipated 
Problem that is 
not an AE 
(UPnonAE) 
Does not fit the definition of an adverse event, but which 
may, in the opinion of the investigator, involve risk to the 
study participant, affect others in the research study, or 
significantly impact the integrity of research data. 
Examples might include occurrences of breaches of 
confidentiality or accidental destruction of study records. 
1. ERC: As soon as possible but no 
later than one week following the 
event 
2. DSMB: Annual review, or as 
otherwise directed.  
3. UVM: To be reported as soon as 
possible to UVM study staff. If it 
is determined that the event was 
unexpected AND related to study 
participation AND involve harm 
or have the potential to cause 
harm to others, the event will be 
reported to the UVM IRB as soon 
as possible by the UVM PI. 
Otherwise, it will be reported at 
annual review. 
4. DGDA: Annual review 
Protocol 
deviation or 
Non-
compliance 
A protocol deviation is any change, divergence, or departure 
from the IRB approved study procedures in a research 
protocol. A protocol deviation is considered severe if it 
meets the definition of a Serious Adverse Event or 
compromises the safety, welfare or rights of study 
participants or others. 
 
Non-compliance is defined as failure to comply with 
applicable IRB or regulatory requirements for protection of 
human study participants.  
1. ERC: As soon as possible but no 
later than one week following the 
event 
2. DSMB: Annual review, or as 
otherwise directed. 
3. UVM: To be reported as soon as 
possible to UVM study staff. If it 
is determined that the event 
involves harm or has the potential 
to cause harm to others, the event 
will be reported to the UVM IRB 
as soon as possible by the UVM 
PI. Otherwise, it will be reported 
at annual continuing review.  
4. DGDA:  Annual review for 
protocol deviation, as for ERC 
for non-compliance 

32 
 
Pausing 
Criteria 
Pausing is the suspension of any study interventions or 
procedures to a single study participant until a decision is 
made whether or not to resume study participation.  The 
pausing criteria for a single study participant include any 
of the following: 
1. Identification of a previously undiagnosed 
immunocompromising medical condition which no 
longer permits vaccination. 
2. Development of a chronic disease. 
3. Any severe (e.g. anaphylactic) reaction to previous 
vaccination 
4. Intussusception 
5. Any safety issue that the PI determines should pause 
further study intervention or procedures to be 
performed on a single study participant. 
1. ERC: As for SAE.  
2. DSMB: As directed by ERC. 
3. UVM: To be reported as soon as 
possible to UVM study staff. The 
event will be reported to the 
UVM IRB as soon as possible by 
the UVM PI.  
4. DGDA: as for SAE 
 
The PI and DSMB will determine if it 
is safe for the individual to resume 
study participation, and if so, will 
notify icddr,b ERC of the decision to 
resume participation. The UVM PI 
will be notified at the same time and 
will report the decision to UVM IRB 
as soon as possible. If a child meets 
pausing criteria and is unable to 
resume participation, the child will 
continue to be followed for safety 
until the end of the study.  
Stopping 
Criteria 
Stopping the study requires immediate discontinuation of 
all study interventions and procedures and suspension of 
further recruitment and enrolment until a decision is made 
whether or not to continue the study. Either IRB or the 
DSMB may stop the study at any time following review of 
any safety concerns. Stopping rules are as follows: 
1. Two or more study participants experience the same 
or similar SAEs that are unexpected and are possibly, 
probably, or definitely related to the vaccine. 
2. Any safety issue that the PI and/or designee 
determines should halt the study 
1. ERC: As soon as possible (goal: 
within 24 hours of knowledge of 
their occurrence). 
2. DSMB: As directed by ERC. 
3. UVM: To be reported as soon as 
possible to UVM study staff. The 
event will be reported to the 
UVM IRB as soon as possible by 
the UVM PI.  
4. DGDA: A report will be 
submitted by the PI at the same 
time it is reported to the ERC. 
 
The PI and DSMB will determine if it 
is safe to resume the study. If so, the 
PI will notify icddr,b ERC of the 
decision to resume participation. The 
UVM PI will be notified at the same 
time and will report the decision to 
UVM IRB as soon as possible. 
 
*Given the demographic profile of the participants for this study (Bangladeshi infants from birth until 
approximately 4 months of age), we might see mortality and morbidity-related SAEs in the study 
population. Of note, data from the 2014 Bangladesh Demographic and Health Survey report under-five and 
infant (< 1 year) mortality rates of 46 deaths per 1,000 live births and 38 per 1,000 respectively for the 
country as a whole; furthermore, 61% of all under-five deaths occur during the neonatal period in the first 
month after birth (31). We also anticipate a high baseline of morbidity in this population, particularly from 
diarrhoeal and respiratory illness, and malnutrition. 
 
MONITORING FOR ADVERSE EVENTS 
All study participants will be closely monitored for adverse events during the study. For 7 consecutive days 
following each dose of RV1, field research assistants will perform a home visit or contact the family by 
telephone to ask specifically about solicited AEs as described in Table 4. Families will also be encouraged 

33 
 
to report any additional signs or symptoms that may be concerning. Participants will be observed for 30 
minutes following each immunization to ensure no allergic reactions have taken place. Safety assessment 
will also be performed at each scheduled clinic visit and at weekly home visits. Additional assessments 
may be performed at the request of the family or study medical officer’s discretion, as conditions dictate, 
including assessments to confirm resolution of an adverse event. These assessments may involve specimen 
collection for the primary purpose of safety assessment (e.g. collection of complete blood counts, serum 
electrolytes) if it is felt to be indicated by the medical officer. A study clinician will be available to study 
participants by telephone 24 hours a day during the entire study period, and all participants will be provided 
with a telephone contact card during the initial recruiting and again at the enrolment visit. 
 
If the family is concerned the child has a fever, the family will be instructed to record the infant’s 
temperature. Families that do not have access to a thermometer will be provided a thermometer by the study 
clinic. Fever will be defined as axillary or rectal temperature >38oC on any measurement. If fever is 
confirmed, the family will be instructed to bring the child to clinic for evaluation and/or referral will be 
made for medical evaluation and management, according to local standards of care. Families will be 
provided with a card containing information for how to contact study staff with any concerns; families will 
have 24/7 access to study staff.  
 
Stable chronic conditions which are present prior to the first clinic visit (pre-existing conditions) and do 
not worsen are not considered AEs and will be accounted for in the study participant’s medical history.  
Exacerbation or worsening of pre-existing conditions are defined as AEs and are evaluated using the same 
criteria as for AEs. Given the study population (healthy infants from birth-3.5 months), it is unlikely that 
any such AEs will be detected as we anticipate that any infants with obvious medical conditions will be 
excluded. 
 
ASSESSMENT OF ADVERSE EVENTS 
All identified adverse events will be assessed for severity, relationship to study interventions, actions taken, 
and outcomes. Each category for AE assessment will be coded, according to the following grading systems: 
 
Determination of Severity 
The PI or designee will assess and grade AE severity using the classifications outlined in Table 5. For 
solicited AEs, the adverse event intensity scoring system will be adapted from Dennehy et al. (15), as 
described in Table 6, and used for scoring severity. 
 
Table 5. Adverse event severity definitions 
Severity 
Definition 
Grade 0 (Normal) 
No symptoms 
Grade 1 (Mild) 
Event that is easily tolerated, may require 1 dose of medication/treatment 
Grade 2 (Moderate) 
Event that interferes with normal activity or requires more than 1 dose of 
medication/treatment 
Grade 3 (Severe) 
Event that prevents normal activity and requires medical intervention 
Refer to Table 6 for a RV-specific Adverse Event Intensity Scoring System that specifies Grade 0-3 events 
Grade 4 (Life Threatening)  
Event which places the study participant at immediate risk of death 
Grade 5 (Death) 
Event which results in death  
 
 
 

34 
 
 
Note: The severity grades listed in Table 6 are equivalent to the severity grades listed in Table 5. 
 
Relationship to Study Interventions 
The PI or designee will assess the relationship between any study intervention or procedure and the 
occurrence of each AE. The PI will use clinical judgment to determine the relationship, based on the 
information available at the time. Alternative causes, such as undercurrent illnesses/infections, non-study 
related therapies (e.g. EPI vaccines), and other risk factors as well as the temporal relationship of the event 
to the study intervention or procedure will be considered and investigated. The PI may change his/her 
opinion of causality based on availability of follow-up information, and may amend/update reports 
accordingly.  
 
Causality of AEs will be assessed by the PI or designee using the following question, “Is there a reasonable 
possibility that the AE may have been caused by the intervention or procedure?” Causality will then be 
assigned to one of the following categories: 
Table 6. Adverse Event Intensity Scoring System. Adapted from: (15) 
  
Definition 
Fever                     
(Rectal or Axillary 
Temperature) 
  
0 
<38.0oC 
1 
38.0oC-38.5oC  
2 
38.6oC-39.5oC  
3 
>39.5oC 
Diarrhoea 
  
0 
Normal (0-2 looser than normal stools/day) 
1 
3 looser than normal stools/day 
2 
4-5 looser than normal stools/day 
3 
>6 looser than normal stools/day 
Vomiting  
  
0 
Normal (no emesis) 
1 
1 episode of vomiting/day 
2 
2 episodes of vomiting/day 
3 
>3 episodes of vomiting/day 
Fussiness/Irritability 
  
0 
Behaviour as usual 
1 
Crying more than usual/no effect on activity 
2 
Crying more than usual/interferes with normal activity 
3 
Crying that cannot be comforted/prevents normal activity 
Cough/runny nose 
  
0 
Normal 
1 
Cough/runny nose that is easily tolerated 
2 
Cough/runny nose that interferes with normal activity 
3 
Cough/runny nose that prevents normal activity 
Loss of appetite 
  
0 
Normal 
1 
Eating less than usual/no effect on activity 
2 
Eating less than usual/interferes with normal activity 
3 
Not eating at all 

35 
 
 
 
 
 
Adverse event action taken 
The PI/designee will assess the action taken by the study participant or the study staff in relation to the AE 
using the following classifications: 
1. None 
2. Therapy required, not requiring hospitalization (e.g. antibiotics for superficial infection at 
venepuncture site) 
3. Permanently discontinued from study participation 
4. Hospitalization 
5. Other 
 
Adverse event outcome 
The PI/designee will assess the outcome of the AE, either at resolution or at the end of the study participant’s 
participation, using the following classifications: 
1. Resolved 
2. Recovered with sequelae 
3. Continuing 
4. Death 
5. Unknown 
 
SAEs that have not resolved by the end of the follow-up period are followed until final outcome is known. If 
it is not possible to obtain a final outcome for an SAE (e.g., the study participant is lost to follow-up), the 
reason a final outcome could not be obtained will be recorded by the investigator on the AE case report form. 
 
DATA MANAGEMENT 
Source Documentation and Data Collection 
Clinical data. Complete source documentation will be required for every study participant for the duration 
of the study. The participant’s study record will record his/her participation in the clinical trial, concomitant 
medications or other interventions administered, as well as any adverse reactions experienced during the 
trial.  
 
Information from source documentation will be double data entered by trained, designated study team 
members into electronic case report forms (CRFs), which will be created using the REDCap software 
1. Related 
There is a reasonable possibility that the AE was related to the study intervention or 
procedure. AEs related to study participation can further be categorized, if necessary, 
based on strength of relatedness: 
1a. Definitely     
      Related: 
There is a clear-cut temporal association with the study intervention or procedure and the 
AE, and there are no other reasonable causes for the outcome in question. 
1b. Probably  
            Related: 
It is more likely than not that the study intervention or procedure contributed to the AE; 
the temporal association is suggestive but not definitive, and other causes remain a 
possibility but are less likely. 
1c. Possibly  
            Related: 
It is possible that the study intervention or procedure contributed to the AE; the temporal 
association is not clear cut and other causes remain a possibility. 
2. Unrelated: 
The AE is not causally or temporally related to the study intervention or procedure. There 
are other, more likely causes and the study intervention or procedure is not suspected to 
have contributed to the AE. 

36 
 
program. In some instances, the source document may directly mirror the electronic case report form for 
ease of entry.  
 
Corrections to the paper source documents must be made by striking through the incorrect entry with a 
single line (taking care not to obliterate or render the original entry illegible) and entering the correct 
information adjacent to the incorrect entry.  Corrections must be initialled and dated by the person making 
the correction.  Source documentation should support the data collected on the CRFs, and must be signed 
and dated by the person recording and/or reviewing the data.   
 
Signed consent forms, demographic data, and other data with personal identifiers will be maintained by the 
investigators in a secure study binder for all study participants in a secure, locked location. Since this study 
binder will have documentation with both personal identifiers and SIDs, the chart is one possible link 
between study data and a personal identifier. Investigators will also maintain individual study charts for 
each study participant. These study charts will be identified only by the study participant’s unique SID and 
not by personal identifiers. The study chart will contain all source documentation and CRFs. 
 
Information captured on source documents will be double data entered into the appropriate CRFs. QA/QC 
will be performed according to the manual of procedures. Any discrepancies or errors will be reviewed and 
clarified by the data manager, the staff who performed data entry, and the staff member who obtained the 
source documentation.  
 
Laboratory data. Raw laboratory data generated at icddr,b will be entered into pre-designed Excel templates. 
Local laboratory staff will perform initial QC/QA to ensure proper data entry, and then transferred to the 
local data manager for final review. A similar process will take place with any raw laboratory data generated 
at UVM, with completed documents transferred to the primary data manager at UVM.  
 
Database management 
The overall data warehouse for the study will reside at UVM. Data can be added via two mechanisms, via 
REDCap and via direct file transfer. For clinical data, all data will be uploaded via REDCap.  Only 
authorized personnel from the data management teams at icddr,b and UVM will have access to the REDCap 
database. The primary data manager will be located at UVM and is identified as key personnel. The primary 
data manager will have primary responsibility for the REDCap database, including overall design, 
maintenance, troubleshooting, and granting of access and permissions to others. The primary data manager 
will be off-site and will not perform data entry or be involved in clinical or laboratory assessments, so thus 
will be unblinded, generate the randomization assignments, and communicate the randomization to the 
icddr,b research pharmacist (or designee). At icddr,b, a local data manager will be identified as key 
personnel. The local data manager and designated data entry staff will have access to REDCap to enter and 
edit data. None of the study PIs, clinic staff, pharmacy staff, or laboratory staff will be able to edit REDCap 
data.  
 
Laboratory data from icddr,b will be sent to the UVM data warehouse via secure file transfer using the 
UVM secure file transfer service. Any laboratory data will only include SID as the identifier, and thus no 
lab personnel will have access to coded data with other identifiers (e.g. DOB). The primary data manager 
at UVM will thus be able to generate and compile composite reports combining clinical data exported from 
REDCap with directly uploaded laboratory data. Primary data analysis will take place at UVM, but access 
to final data files may be granted to PIs and other authorized personnel at icddr,b for analysis as well.  
 
A complete description of how confidentiality will be maintained with respect to data storage, management, 
and analysis is further provided below under “Ethical Assurance of Human Rights, Adequacy of Protection 
Against Risks.” 
 

37 
 
Study Documentation 
Study-related documentation will be completed as required by each IRB. Continuing review documentation 
will be submitted by the Investigators to each IRB at least once yearly as specified by each IRB. The PI and 
designated icddr,b personnel will maintain adequate records to account for the disposition of the vaccines, 
including dates of receipt and quantity, current inventory and storage, and dispensation to study participants, 
and destruction of remaining inventory, if applicable.  
 
Retention of Specimens 
All specimens will be stored at either icddr,b, or at UVM, according to the assays which will be performed 
at each institution (Table 3). Once it has determined that all laboratory assays have been completed and that 
no further use of the biological specimens are required, any specimens being stored at UVM will be returned 
to icddr,b.  
 
If a study participant becomes lost to follow-up, all specimens already collected will be tested as outlined 
in the protocol. Any loss or unanticipated destruction of samples (for example, due to freezer malfunction) 
or data (for example, misplacing a printout of data with identifiers) that compromises the scientific integrity 
of the study will be reported to each IRB. 
 
Access to research samples will be limited using either a locked room or a locked freezer.  Samples and 
data will be stored using codes assigned by the Investigators or their designee.  Data will be kept in 
password-protected computers.  Only Investigators or their designees will have access to the samples and 
data. 
 
Retention of Records 
The PI is responsible for retaining all essential documents listed in the International Conference on 
Harmonization (ICH) Good Clinical Practice (GCP) Guideline.  Trial-related documents will be maintained 
by the PI in a secure storage facility for a minimum of 5 years after completion or termination of the study. 
Records will also be maintained in compliance with IRB and Bangladesh national, state, and federal medical 
records retention standards. All trial-related documents will be stored in a way that ensures that 
confidentiality will be strictly maintained to the extent provided by Bangladesh national, federal, state, and 
local law.  
 
All study files, source documents, and trial master files will be maintained and stored in a secure manner 
in the icddr,b in the office of the Clinical Research Coordinator (or equivalent). No study documents will 
be destroyed without prior written agreement between the PI and the clinical research staff.   
 
Sample Size Calculation and Outcome (Primary and Secondary) Variable(s) 
Clearly mention your assumptions. List the power and precision desired. Describe the optimal conditions to attain the sample size. Justify the 
sample size that is deemed sufficient to achieve the specific aims. 
 
Sample size calculation was performed to ensure adequate power to evaluate our primary outcome measure, 
vaccine take. As noted above in Preliminary Data, the seroconversion rate in PROVIDE was 27% among 
vaccinated infants, and no faecal shedding was detected when assessed by ELISA, yielding a frequency of 
vaccine take of only 27%.  RV-IgA antibody responses were very variable in this population and difficult 
to interpret, similar to RV-IgA responses in other low-income settings. A clearer impact on vaccine take is 
more likely to be observed by effecting an increase in faecal vaccine shedding. Because of this, we 
calculated our sample size primarily to detect differences in rates of faecal shedding. No RV1 vaccine 
shedding was detected in this population when assessed by stool ELISA and results for RT-PCR assessment 
of stool shedding are not yet available. However, a Chinese study found 72.4% of subjects had detectable 
shedding by RT-PCR 2-3 days following first RV1 dose, compared to 10.3% of subjects with detectable 
shedding when assessed by ELISA (17). Our assumption is that while no shedding was detected by ELISA, 

38 
 
some degree of shedding would have been identified by RT-PCR. Guided by the Chinese study data, we 
estimated that the baseline rate of RV1 shedding as detected by RT-PCR in the Mirpur population would 
be 50%. To detect a 20% increase in shedding to 70%, we calculate that 93 study participants in each arm 
will provide 80% power at 2-sided α=0.05. In PROVIDE, an attrition rate of 15% was observed, with the 
majority of drop-outs occurring very early in the study. Therefore, we anticipate a similar rate, which would 
require enrolment of 107 study participants per arm. Our target enrolment will thus be 110 study participants 
per arm for a total of 220 study participants. 
 
Assuming a similar rate of seroconversion among infants who receive a standard dose of RV1 in this study, 
at2-sided α=0.05, this sample size will also provide 80% power to detect a 20% difference in RV-IgA 
seroconversion rate (i.e. an increase in seroconversion rate from 27% to >47% among infants in the 
intervention arm). Our other outcome variables are contingent upon faecal shedding (e.g. faecal viral 
burden) or are purely exploratory in nature and therefore were not considered further in sample size 
calculation.  
 
Data Analysis 
Describe plans for data analysis, including stratification by sex, gender and diversity. Indicate whether data will be analysed by the investigators 
themselves or by other professionals. Specify what statistical software packages will be used and if the study is blinded, when the code will be 
opened. For clinical trials, indicate if interim data analysis will be required to determine further course of the study. 
 
Definitions 
Vaccine take: Detection of faecal shedding of RV1 in any non-diarrhoeal stool specimen at any time point 
following vaccination, OR RV-IgA seroconversion, defined as post-immunization (week 14) RV-IgA > 20 
U/mL with pre-immunization (week 6) RV-IgA <20 U/mL. 
 
Faecal shedding: Positive qRT-PCR for rotavirus NSP3 with confirmatory gene sequencing result positive 
for vaccine-strain virus at any time point following vaccination, with negative week 6 pre-vaccination stool 
testing. Due to the low level of viral shedding anticipated, no threshold Ct value will be set to determine 
positivity, and all specimens with detection (up to Ct=40) will be considered positive if controls are 
appropriate. In the event that qRT-PCR is negative for RV but ELISA is positive, the specimen will be 
considered negative for faecal shedding for primary outcome analysis, but additional analysis will be 
performed to include ELISA positive specimens as well. qRT-PCR specimens positive for RV but that 
yields a non-vaccine strain will not be considered positive for faecal shedding. If the strain is untypeable, 
the specimen will be considered negative for faecal shedding for primary outcome analysis, but additional 
analysis will be performed to include these outcomes as positive to compare for differences in outcome 
relative to the more stringent criteria.  
 
RV-IgA seroconversion: RV-IgA ELISA >20 U/ml post-vaccination (i.e. at 14 weeks) with RV-IgA ELISA 
<20 U/ml pre-vaccination (i.e. at 6 weeks). 
 
Antigenemia: Detection of RV antigen in any plasma specimen by rotavirus ELISA following vaccination, 
or detection of RV in any plasma specimen by qRT-PCR following vaccination. 
 
Viremia: Detection of RV from any plasma specimen following vaccination, as evidenced by positive RV-
specific immunostaining following serial passage on MA104 cells or demonstration of cytopathic effect.  
 
VP4, VP7, VP5*, or NSP4 sero-response and end-point titer: Sero-response is defined as greater than or 
equal to four-fold rise in end-point titer post-vaccination (i.e. at 14 weeks) compared to pre-vaccination 
(i.e. at 6 weeks). End-point titre is defined as the inverse of the highest dilution that gives positive 
immunostaining detectable above background. 
 

39 
 
Diarrhoea. Three or more bowels movements looser than normal per mother’s judgement within 24 hours. 
 
RV diarrhoea. Detection of RV antigen using commercial EIA kit in any diarrhoeal stool specimen.  
 
Analysis plan 
Data analysis will be conducted by the investigators, using Excel (Microsoft), GraphPad Prism (GraphPad 
Software, Inc.), and/or SAS (SAS Institute).As this study does not assess treatment efficacy, no scheduled 
interim analysis will be necessary to determine further course of the study. Study participants and 
investigators will remain blinded throughout the course of the study. Unblinding will not occur until all 
study-related laboratory procedures required for analysis of primary and secondary outcomes have been 
performed and results have been finalized after undergoing QA/QC. Study participant or group unblinding 
might potentially occur however if deemed necessary for safety review by the DSMB.  
 
Primary analysis will compare the proportion of infants with vaccine take (detection of faecal shedding of 
RV1 in any non-diarrhoeal stool specimen at any time point following vaccination, OR RV-IgA 
seroconversion, defined as post-immunization (week 14) RV-IgA > 20 U/mL with pre-immunization (week 
6) RV-IgA <20 U/mL) in the intervention versus control arm in all study participants who completed the 
study per-protocol. Analysis will be performed by Chi-square test and an estimate of proportion difference 
with corresponding 95% confidence interval will be calculated. Secondary analysis will compare frequency 
of faecal RV1 shedding alone and of RV-IgA seroconversion alone between intervention and control 
groups, using similar methods, and will compare both RV-IgA concentration and faecal viral burden among 
infants in the intervention arm versus control using independent samples t-test or Mann-Whitney test, as 
appropriate depending on the data distribution.  
 
Exploratory analyses, including by intention-to-treat, evaluating categorical outcomes according to 
intervention versus control assignment (e.g. VP4, VP7, VP5*, and NSP4 sero-response, presence of 
antigenemia, presence of viremia) will be performed by Chi-square or Fisher’s exact test, as appropriate. 
Additional analyses evaluating continuous variables according to intervention versus control assignment 
(e.g. VP4, VP7, VP5*, and NSP4 titres) will be performed by independent samples t-test or Mann-Whitney 
test, as appropriate. Associations between categorical and continuous variables (e.g. RV-IgA 
seroconversion and quantitative RV1 viral burden in stool specimens) will be evaluated by logistic 
regression analysis. Associations between continuous variables (e.g. RV-IgA concentration and 
quantitative RV1 viral burden in stool specimens) will be investigated by linear regression analysis. Any 
final models will be appropriately penalized for multiple testing.  
 
Data Safety Monitoring Plan (DSMP) 
All clinical investigations (research protocols testing biomedical and/or behavioural intervention(s)) should include the Data and Safety 
Monitoring Plan (DSMP). The purpose of DSMP is to provide a framework for appropriate oversight and monitoring of the conduct of 
clinical trials to ensure the safety of participants and the validity and integrity of the data. It involves involvement of all investigators in 
periodic assessments of data quality and timeliness, participant recruitment, accrual and retention, participant risk versus benefit, performance 
of trial sites, and other factors that can affect study outcome. 
 
A Data and Safety Monitoring Board (DSMB) will be convened for this study by the Ethical Review 
Committee (ERC) of the icddr,b. The ERC DSMB reflects the disciplines and medical specialties necessary 
to interpret the data from the clinical trial and may include expertise in clinical aspects of disease/patient 
population being studied, biostatistics, and clinical trials conduct and methodology. The ERC selects the 
DSMB chair and determines the terms of membership.  The ERC DSMB will review and evaluate the 
accumulated study data for participant safety, study conduct, and progress, and make recommendations to 
the ERC concerning the continuation, modification, or termination of the trial. The ERC DSMB will meet 
once before the study opens to make recommendations as to whether the study can proceed, and the DSMB 
schedule for meetings during and at completion of the study will be agreed between the PI and DSMB. 
Additionally, the DSMB may meet as needed to identify or review any potential safety concerns. The study 

40 
 
investigators will report to the DSMB per the Board’s requirements and expectations. All adverse events 
and unanticipated problems will be reviewed and reported to the respective IRBs of both icddr,b and UVM 
and/or to the DMSB according to each institution’s reporting guidelines and as further directed by the 
DSMB. AEs that are considered to be related to vaccination will also be reported to the DGDA according 
to the same reporting guidelines as for the icddr,b ERC. 
 
In addition, at the request of the DGDA, post-vaccination solicited AE safety data for approximately twenty 
children will be submitted to both DSMB and DGDA for initial review. Further enrolment will be paused 
once a sufficient number of children have been enrolled to ensure that approximately 20 will have 
completed vaccination for this review period. No additional children will be vaccinated until the initial 
safety data have been reviewed and approved by both DMSB and DGDA. Children already enrolled will 
continue additional study procedures according to the protocol schedule. 
 
 
Ethical Assurance for Protection of Human rights 
Describe the justifications for conducting this research in human participants. If the study needs observations on sick individuals, provide 
sufficient reasons for using them. Indicate how participants’ rights will be protected, and if there would be benefit or risk to each participants 
of the study. Discuss the ethical issues related to biomedical and social research for employing special procedures, such as invasive procedures 
in sick children, use of isotopes or any other hazardous materials, or social questionnaires relating to individual privacy. Discuss procedures 
safeguarding participants from injuries resulting from study procedures and/or interventions, whether physical, financial or social in nature. 
[Please see Guidelines] 
 
Assurance of Protection 
This study will be conducted in compliance with the Declaration of Helsinki and in full conformity with 
the principles of the Belmont Report: Ethical Principles and Guidelines for the Protection of Human 
Subjects of Research of the National Commission for the Protection of Human Subjects of Biomedical and 
Behavioral Research (April 18, 1979) and codified in 45 CFR 46, 21 CFR 312, and/or ICH E6; 62 Federal 
Regulations 25691 (1997).  Each participating institution will hold a current FWA issued by OHRP.  All 
key study staff will be trained in Good Clinical Practice. In addition, this study will be registered with 
ClinicalTrials.gov.  
 
Each participating institution’s RRC, ERC or IRB will review and approve this protocol and associated 
informed consent documents prior to study initiation.  The DGDA also will approve this protocol prior to 
study initiation. Any amendments to the protocol or consent materials will also be approved before they are 
implemented. The PI will be responsible for obtaining all necessary regulatory approvals. A copy of the 
study approvals (including approval of the informed consent form) is to be maintained in the Investigator 
study document binder. During the study, the PI is responsible for providing each regulatory body with all 
documents subject to review (e.g., Protocol Amendments, informed consent form updates, advertisements, 
and any written information that may be provided to the participant).  Annual Continuing Review Reports 
on the progress of the study will be submitted by the PI to each regulatory body in accordance with each 
respective institution’s guidelines. A final report will be submitted to each regulatory body at the 
completion of the study.  
 
Protocol Compliance 
The PI will conduct the trial in exact compliance with the written protocol. The PI will not implement any 
deviation from or change to the protocol without agreement, prior review, and documented approval by 
each IRB that granted original approval for the study.  The DSMB and DGDA will be notified of all protocol 
revisions (other than administrative) and will review any changes to the protocol that involve DSMB or 
DGDA oversight or involve changes to the data and safety monitoring plan (DSMP) of the study.   
 
The PI may implement a deviation or change in the protocol in order to eliminate an immediate hazard to 
study participants without prior IRB, DMSB, or DGDA approval or when the change involves only 
logistical or administrative aspects of the trial, such as a telephone number change. In the event of a medical 

41 
 
emergency, the PI and/or designated medical officer(s) shall perform any medical procedures deemed 
medically appropriate.   
 
As soon as possible, the implemented deviation or change, the reasons for the change, and, if appropriate, 
the proposed protocol amendment(s) should be submitted to each IRB, the DSMB, and DGDA. 
 
Risk to human subjects 
Human subjects involvement, characteristics, and design. This is a double-blind, randomized, controlled 
vaccine immunogenicity trial comparing standard vs high dose oral RV1 vaccine among healthy infants in 
Dhaka, Bangladesh. The clinical aspects of this study will be performed at the field clinic of the icddr,b in 
the Mirpur neighbourhood of Dhaka, using the target enrolment goal calculated to ensure adequate power 
to detect our primary outcome measure. Inclusion criteria include: 1) generally healthy infant; 2) age 0-7 
days at enrolment; 3) mother willing and able to provide signed informed consent; 4) mother willing to 
allow infant to be vaccinated; 5) mother willing to allow biological specimens, including blood, stool, and 
saliva, collected from infant according to study protocol; 6) mother willing and able to adhere to study 
schedule. Exclusion criteria include: 1) congenital malformations; 2) known immunocompromising 
condition in infant; 3) prior rotavirus vaccination; 4) other household member enrolled in the study.  
 
 
Inclusion of women, minorities, and children. RV gastroenteritis is almost exclusively a disease of infants 
and young children, and RV vaccine is indicated only for infants >6 weeks age. Therefore, by necessity this 
study will enrol infant children of both sexes, reflecting the racial and ethnic make-up of the local 
population.  
 
Sources of materials: Vaccines will be purchased in Bangladesh through the local GlaxoSmithKline 
distributor, and all laboratory materials and reagents will be obtained via commercial vendors. Blood, stool 
and saliva specimens will be collected from study participants at the indicated time points by trained 
personnel. Recombinant baculovirus constructs expressing RV1 VP4, VP7, VP5*, and NSP4that have 
already been constructed by the UVM investigators will be utilized in assessing VP4, VP7, VP5*, and 
NSP4 antibody responses. 
 
Potential Risks. 
Vaccination: The risks of RV1 vaccine to immunocompetent infants is low. Infants with known 
immunocompromising conditions may be at higher risk since RV1 contains live, attenuated virus; our 
exclusion criteria should be sufficient to exclude any potentially higher-risk infants. In clinical and post-
licensure evaluation, common adverse events following either dose of vaccine were fussiness, cough or 
runny nose, fever, loss of appetite, vomiting, or diarrhoea, but these rates did not differ from placebo 
(product insert, Rotarix, GlaxoSmithKline). Further, the rates of SAEs in vaccinated infants and in placebo 
recipients did not differ. The most severe adverse event following rotavirus vaccination is intussusception, 
which led to the withdrawal of the first licensed oral RV vaccine (RotaShield, Wyeth) from the market in 
1999. Extensive post-licensure surveillance of RV1 has demonstrated a very low increased risk of 
intussusception following RV1 vaccination of 1-5 excess cases per 100,000 children vaccinated; this 
incidence rate is substantially lower than the number of deaths prevented yearly due to rotavirus vaccine 
(5). An evaluation of background intussusception rates in Bangladesh has previously been conducted; in 
that analysis, the upper limit for estimated incidence of possible intussusception among children <2 years 
of age was 97 cases per 100,000 children per year (1 per 1030 children per year) (44). Given these numbers, 
it is extremely unlikely that any participant in this study would experience intussusception.   
 
The intervention group will receive double the standard dose of RV1. A similar dose has been previously 
evaluated in a phase II trial conducted in the United States and Canada (15). In that study, infants received 
either 105.2 FFU (N=212) or 106.4 FFU (N=209) of RV1, or placebo (N=108), whereas our study proposes 
to use 106 FFU (standard dose) or 106.3 FFU (high dose). In the previous study, no differences in either 
solicited or unsolicited AEs or SAEs were detected between treatment groups. No cases of intussusception 

42 
 
were detected, although the study was not powered for that outcome. One infant had gastroesophageal 
reflux first noted 7 days after receiving the first dose of 106.4 FFU and withdrew from the study. Two others 
had abdominal tenderness following the second dose of vaccine; imaging studies were negative for 
intussusception but positive for mesenteric adenitis, and stool specimens for both infants were negative for 
rotavirus but positive for other enteric viruses (enterovirus and adenovirus, respectively). In summary, both 
doses of vaccine were safe and well-tolerated. We have no reason to suspect that reactogenicity or safety 
risks will be increased in our population; on the contrary, given the difficulty in establishing vaccine take 
with standard doses in this population (potentially due to inhibitory factors in the infant gut), we suspect 
that infants in this study will “see” a lower inoculum of virus when administered similar doses, compared 
to children in the U.S. and Canada. 
 
Blood draw: Venipuncture and heelstick will be used to obtain the necessary blood specimens for this study. 
As both primary and secondary outcome measures rely on plasma antibody measurement, there is no 
alternative to blood collection in the performance of this study. Potential risks of venipuncture or heelstick 
include local pain and discomfort, bleeding or bruising at the site of puncture, and infection, but these risks 
are minimal.  
 
Confidentiality: Breach of confidentiality is another potential risk when collecting and recording personal 
identifying information.   
 
Adequacy of protection against risks 
Recruitment and informed consent. Mothers of potential study participants will be recruited during the 
prenatal period using community-based field research assistants. Extensive discussion will take place at 
this time regarding the study design, rationale, risks and benefits. Mothers who express interest in 
participating will be identified and coordination will take place to ensure that a field research assistant visits 
the household within one week following the birth of the infant. At that time, the study will be reviewed 
again, and after confirmation that the family desires to participate and has had all questions answered to 
their satisfaction, informed consent will be obtained from the mother and the child will be enrolled in the 
study. Because all study participants will be infants, assent will not be obtained. Consent will be obtained 
in consideration of the literacy level of the family, and in cases where the mother cannot write, a witnessed 
thumbprint will be obtained. Witnesses may not be associated with the study. Only after written informed 
consent is obtained will any study-specific procedures be performed.  
 
Informed consent forms will be revised to reflect any significant amendment(s) which directly affects 
volunteers, and must also be reviewed and approved by the IRB with the amendment. Study participants 
will be consented using the most recently-approved informed consent form. Participants already enrolled 
in the study will be informed about revisions to the protocol and, depending on the impact of the 
amendment, may be asked to re-consent. Re-consenting may be accomplished by repeating the informed 
consent process using the approved revised informed consent form, with attention given to the changes, or 
it may be done using an approved addendum to the informed consent form, clearly stating any revisions 
or new information and summarizing the potential effect upon participation in the study. The new 
informed consent form or addendum must be signed and placed into the study record. A signed copy is 
given to the volunteer. 
 
Protections against risk. 
Vaccine safety: To monitor for any potential adverse events and safety concerns during the study period, 
extensive safety surveillance will be solicited (for 7 days following each vaccine dose and at least weekly 
thereafter), and mothers will be educated and encouraged to report unsolicited events and concerns 
whenever they occur. The Mirpur study clinic is capable of providing basic primary care and acute care 
needs, and these services will be available to all participating infants. If warranted, the study team will 

43 
 
coordinate referral to appropriate facilities for AEs requiring more extensive medical evaluation or 
hospitalization. 
 
Study withdrawal: Mothers can choose to withdraw their child from the study at any time. Further, pausing 
criteria have been established as described above in “Pausing criteria.”  
 
Blood draw: Blood collection will be performed by trained personnel to minimize risk of pain, discomfort, 
bruising, bleeding, and infection. The blood volumes proposed for this study are within published standards 
for safe phlebotomy in children, which is up to 5% of total blood volume per day estimated at 80 mL/kg 
body weight (i.e. 4 mL/kg/day) and up to 10% of total blood volume over 8 weeks (8 mL/kg) (45).  
 
Confidentiality: Study participant confidentiality is strictly held in trust by the participating investigators, 
their staff, and their agents.  This confidentiality includes documentation, investigation data, participants’ 
clinical information, and all other information generated during participation in the study. To ensure 
confidentiality and prevent unauthorized breath of confidentiality, study participants will be assigned a 
unique SID to be used on study forms and in the study database. Whenever feasible, study participants will 
be identified only via their SID. Each study participant will have a study chart that is labelled only with the 
SID. This study chart will contain complete source documentation and CRFs. Neither the study forms nor 
the study database will contain participants’ names or other information that could be used to identify them. 
Any identifying information will be kept in a separate study binder for all study participants. The study 
binder and study charts will all be kept in a secure, locked office that will not be accessible to unauthorized 
personnel. In addition, the following will be kept in a locked office and will not be accessible to 
unauthorized personnel: 1) Informed consent documents, forms, lists, logbooks, appointment books, or any 
other document linking the SID to participants’ names or identifying information; and 2) other study forms, 
including laboratory reports and administrative forms. All computers containing the study database or 
electronic records that contain any identifying information will be password-protected will not be accessible 
to unauthorized personnel. The key linking SIDs to personal identifiers will be kept on a password-protected 
computer file that will only be available to key personnel. Chart information and information from study 
records will not be released without written permission of the participant.  However, records may be 
reviewed by representatives from the icddr,b ERC, DSMB, UVM IRB, and DGDA. No identifying 
information will be disclosed in the process of analysing or publishing results from this study.   
 
Potential benefits of the proposed research to human subjects and others. RV1 vaccine is an FDA-
approved, WHO pre-qualified vaccine but has not been incorporated into the Bangladesh EPI and is 
therefore generally unavailable to this study population. All participants may potentially benefit from 
participation in this study by receiving RV vaccine and subsequent protection against severe gastroenteritis, 
dehydration, and/or death. This vaccine is given nearly universally in settings where it is available, and 
since global introduction, has prevented hundreds of thousands of deaths due to prevention of dehydrating 
diarrhoea due to RV(4). Importantly, the WHO has recommended that RV vaccine be administered to all 
infants in spite of the known risks of the vaccine, as the potential risks described above are vastly 
outweighed by the potential benefits to infants receiving vaccination. 
 
Importance of the knowledge to be gained. This study has the potential to identify a strategy that may 
improve oral RV vaccine performance in the developing world, and identify candidates for more reliable 
COPs for RV, which would vastly increase the efficiency of clinical RV research. Either of these outcomes 
would improve the health of children by leading to improvements in strategies to prevent and study RV 
infection, which remains the most common cause of diarrhoeal disease in children worldwide. Therefore, 
the importance of the knowledge to be gained regarding improvement in child health is substantial. 
 

44 
 
Use of Animals 
Describe if and the type and species of animals to be used in the study. Justify with reasons the use of particular animal species in the research 
and the compliance of the animal ethical guidelines for conducting the proposed procedures. 
 
No animals will be used in this study. 
 
 
Collaborative Arrangements 
Describe if this study involves any scientific, administrative, fiscal, or programmatic arrangements with other national or international 
organizations or individuals. Indicate the nature and extent of collaboration and include a letter of agreement between the applicant or his/her 
organization and the collaborating organization. 
 
This study will be carried out in collaboration with the icddr,b and the University of Vermont.  The 
investigative teams for this project have a long-standing collaborative relationship. All necessary 
arrangements to conduct this study at the icddr,b have been established with the investigators of the 
collaborating institutions. Any requirements related to financial reporting and scientific progress required 
by sources of funding for the UVM investigator(s) will be the sole responsibility of the UVM 
investigator(s).  
 
Facilities Available 
Describe the availability of physical facilities at site of conduction of the study. If applicable, describe the use of Biosafety Level 2 and/or 3 
laboratory facilities. For clinical and laboratory-based studies, indicate the provision of hospital and other types of adequate patient care and 
laboratory support services. Identify the laboratory facilities and major equipment that will be required for the study. For field studies, describe 
the field area including its size, population, and means of communications plus field management plans specifying gender considerations for 
community and for research team members. 
 
The icddr,b has a large multi-disciplinary international and national scientific research staff. Existing 
field, hospital, laboratory, and office facilities will be used for this study. icddr,b scientists have 
conducted a variety of observational field studies and clinical trials in the Mirpur community where the 
field site is located.  
 
Local clinical research study personnel from icddr,b will be used to execute the clinical aspects of the 
study at the Mirpur field site, an outpatient clinical research clinic with a full complement of support staff. 
The clinic building includes office and clinic spaces, storage capacity, and appropriate facilities for 
conducting the study. The catchment area for this site is approximately 100,000 residents.  
 
Laboratory assays will be performed at both UVM and at icddr,b using existing equipment. Personnel at 
both sites will be experienced in all site-specific procedures proposed herein. All initial specimen 
processing will take place at icddr,b. 
 
 
 
 
 
 
 
 

45 
 
 
 
 
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the text. For unpublished references, provide complete information in the text and do not include them in the list of Literature Cited. There is 
no page limit for this section, however, exercise judgment in assessing the “standard” length. 
 
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