Potential Mechanisms for Intussusception After Rotavirus Vaccine-Pilot Study
Phase: PHASE4
Conditions: Intussusception, Rotavirus Infections
Sponsor: Children's Hospital Medical Center, Cincinnati

Title:
Potential Mechanisms for Intussusception after Rotavirus Vaccine-Pilot Study
Cincinnati Children's Hospital Medical Center
Principal Investigator:
Mary Allen Staat, MD, MPH
Co-Investigators:
Steven Black, MD
Alexander J. Towbin, MD
Elizabeth P. Schlaudecker, MD, MPH
Jean A. Tkach, PhD
Ardythe L. Morrow, PhD
Monica M. McNeal, MS
Mekibib Altaye, PhD
Centers for Disease Control and Prevention (CDC)
Co-Principal Investigators:
Karen Broder, MD and Margaret Cortese, MD
Co-Investigators:
Umesh Parashar, MD, MBBS
Devindra Sharma, MSN, MPH
ClinicalTrials.gov ID: NCT02542462

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Contents
1
KEY ROLES.........................................................................................................................................4
2
BACKGROUND AND SCIENTIFIC RATIONALE ..........................................................................4
2.1
Rotavirus Infection and the History of Rotavirus Vaccines.
4
2.2
Intestinal Imaging
7
2.3
Potential Modifiers of Rotavirus Infection, Vaccine Response and Shedding and
Intussusception
9
3
Purpose and Objectives...................................................................................................................12
3.1      Purpose
12
3.2
Hypothesis
12
3.3
Primary and Secondary Objectives
12
3.4
Study Outcome Measures
12
4
Research Plan......................................................................................................................................13
4.1
Overview of Study
13
4.2
Selection of Study Population
14
4.3
Inclusion and Exclusion Criteria
14
4.4
Randomization and Study Withdrawal
16
5 STUDY SCHEDULE ( See Appendix 1) ................................................................................................16
5.1
Recruitment and Enrollment
16
5.2
Parent Education about Rotavirus Vaccines and the Study
17
5.3
Study Visits
18
6   STUDY PROCEDURES .......................................................................................................................20
6.1
Rotavirus Vaccine Randomization and Administration
20
6.2
Breast Milk Collection
20
6.3
Saliva Collection
20
6.4
Stool Collection
20
6.5
Blood Collection
20
6.6
MRI/Ultrasound Imaging
21
6.7
Rotavirus Vaccine Administration
21
6.8
Reactogenicity Assessment
21
6.9
Receipt of other Recommended Vaccines
22
6.10      Study Compensation
23

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7        STUDY VACCINE PRODUCT.......................................................................................................23
7.1
Study Product Description, Storage and Stability
23
7.2
Dosage, Preparation, and Administration of Study Product
23
7.3
Assessment of Subject Compliance With Study Product
24
8   LABORATORY EVALUATIONS AND METHODS .........................................................................24
8.1
Blood-Cytokine and Inflammatory Responses and Maternal Antibody (IgG) ...........................24
8.2
Stool-RV1 and RV5 Shedding and Cytokine Responses
25
8.3
Saliva-DNA Genotyping for Secretor Status
25
8.4
Breast Milk-IgA antibody and Secretor Status of the Mother
25
8.5
Future Testing
25
9   IMAGING METHODS .........................................................................................................................26
9.1
MR Imaging
26
9.12
Methods
26
9.13
Image Interpretation
26
9.2
Ultrasound Imaging
26
9.21
Methods
26
9.22
Image Interpretation
26
10
RANDOMIZATION, DATA MANAGEMENT, STATISTICAL PLAN .....................................27
10.1
Randomization Plan
27
10.2
Data Management Plan
27
10.3
Statistical Analysis Plan
27
11
ASSESSMENT OF SAFETY.........................................................................................................29
11.1
Adverse Events
29
11.2
Safety Reporting: For Adverse Events
30
12
POTENTIAL RISKS AND BENEFITS.........................................................................................30

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PROTOCOL SUMMARY
Title:
Potential Mechanisms for Intussusception after Rotavirus Vaccine
– Pilot Study
Phase:
Phase 4
Population:
Infants 6 weeks to 13 weeks of age
Site:
Cincinnati Children’s Hospital Medical Center
Subject Participation
Two weeks
Duration:
Description of Agent
Single oral dose of either of the licensed and recommended
Or Intervention:
rotavirus vaccines: Rotarix® (RV1) or RotaTeq® (RV5) given alone or
with other routinely given vaccines
Primary Objectives:
1) to examine the effects of RV1 and RV5 with or without other
routine immunizations on gastrointestinal anatomy
2) to assess the feasibility of conducting a larger scale study
Exploratory Objectives:
1) to examine the effects of RV1 and RV5 with or without other
routine immunizations on gastrointestinal motility
2) to examine the effects of RV1 and RV5 with or without other routine
immunizations on blood and stool cytokines and inflammatory
responses, including clinical reactogenicity events
3) to assess patterns and level of vaccine virus shedding after RV1 and
RV5
4) to provide preliminary data assessing associations with the pattern of
rotavirus vaccine strain (RV1 and RV5) shedding in the stool and
blood, stool cytokine responses, and the anatomy and/or functional
motility of the small intestine; and examine other factors such as
maternal antibody, breast milk antibody, and human blood group
antigens to see if these factors modify these responses
5) to compare MRI and ultrasound imaging
Description
Prospective randomized clinical study of the potential
of Study Designs:
effects of the first dose of RV1 or RV5 on gastrointestinal motility and
anatomy and blood and stool cytokine responses and assessment of
associations with the pattern of the shedding of vaccine strain rotavirus
in the stool with these outcomes

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1
KEY ROLES
Cincinnati Children’s Hospital Medical Center
Principal Investigator:
Mary Allen Staat, MD, MPH
Co-Investigators:
Steven Black, MD
Alexander J. Towbin, MD
Elizabeth P. Schlaudecker, MD, MPH
Jean A. Tkach, PhD
Ardythe L. Morrow, PhD
Monica M. McNeal, MS
Mekibib Altaye, PhD
Study Coordinator:
Nancy Back, RN, MPH
Study Manager:
Marilyn Rice, MS
Study Team:
Michelle Roth, RN
Krista Doerflein, LPN
Brittany Cassell, LPN
Jacqui DellaTorre, LPN
Centers for Disease Control and Prevention (CDC)
Co-Principal Investigators:
Karen Broder, MD and Margaret Cortese, MD
Co-Investigators:
Umesh Parashar, MD, MBBS
Devindra Sharma, MSN, MPH
Coordinator:
Oidda Museru, MSN, MPH
This study is funded by a CDC contract with Cincinnati Children’s Hospital Medical Center as a Task
Order in the CISA Project Contract. The Cincinnati Children’s Medical Center PI  (Mary Allen Staat)
will oversee the overall study and direct activities. CDC personnel will collaborate to develop the
protocol, conduct the study, ensure the study is aligned with CDC public health priorities, and analyze the
data and disseminate the results. CDC may receive access to coded data. This study will be registered at
ClinicalTrials.gov.
2
BACKGROUND AND SCIENTIFIC RATIONALE
2.1 Rotavirus Infection, the History of Rotavirus Vaccines and Intusscusception
Prior to the licensure of rotavirus vaccines, rotaviruses were the most common cause of severe acute
gastroenteritis (AGE) in children worldwide with nearly all children infected in the first five years of
life1,2. In the United States (US), rotaviruses were estimated to cause 55,000 to 70,000 hospitalizations,
205,000 to 272,000 emergency department visits, and 20 to 60 deaths in young children annually before
rotavirus vaccines were approved and universally recommended for use in infants3-6. Worldwide,
rotaviruses were estimated to be responsible each year for 2 million hospitalizations and 352,000 to
592,000 deaths in children younger than 5 years7-11. Due to the morbidity in the US and morbidity and
mortality around the world in resource poor countries there was a need to develop rotavirus vaccines.

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The first rotavirus vaccine licensed in 19982,12 was effective but was withdrawn within a year due to its
association with intussusception13,14. Years later, two new vaccines were licensed in the US, resulting in a
dramatic decline in rotavirus disease. While these two vaccines have been found to be effective in other
countries, in the resource-poor countries both vaccines have been far less efficacious compared to
resource-rich countries. Further, these two new vaccines have now been found to be associated with
intussusception, albeit at a lower attributable risk than the original vaccine. Rotavirus experts and those
responsible for rotavirus vaccine policy have proposed that the benefits of rotavirus vaccines in
preventing rotavirus disease and death, even at a lower efficacy in developing countries, far outweigh the
risks of intussusception. CDC’s Advisory Committee on Immunization Practices (ACIP) routinely
recommends rotavirus vaccine for infants2. It is however, important to have a level of certainty about this
balance of risk and benefit given that intussusception in resource poor countries has a higher likelihood of
a poor outcome since there are delays in evaluation and decreased access to standard treatment which can
lead to high surgery rates and death. A better understanding of the potential biological mechanisms of
rotavirus vaccines to cause intussusception as well as factors which may modify risk may provide
information to lessen the risk for intussusception or a poor outcome if a child gets intussusception. Below,
we summarize what is known with regard to rotavirus vaccines, rotavirus infection and intussusception to
date.
RRV-TV and Intussusception
On August 31, 1998, the tetravalent rhesus-based rotavirus vaccine (RRV-TV) (RotaShield®, Wyeth-
Lederle Vaccines and Pediatrics) was licensed by the Food and Drug Administration (FDA) for routine
use among infants15. RRV-TV was found to be safe and effective pre-licensure. However, less than a year
after licensing, 15 cases of intussusception which occurred shortly after receipt of RRV-TV were reported
to the Vaccine Adverse Event Reporting System (VAERS)16. Subsequent analyses led by the CDC found
an association between the receipt of RRV-TV and the development of intussusception. This led the
ACIP to withdraw the recommendation for use of RRV-TV14,17 and the manufacturer to voluntarily
withdraw RRV-TV from the market in the US14. The initial publication describing the association
reported an increased risk for development of intussusception from 3 to 14 days after receipt of the first
dose of vaccine (adjusted odds ratio, 21.7)18. A smaller risk also existed after receipt of the second dose of
vaccine. Researchers estimated that one case of intussusception attributable to vaccination with RRV-TV
would occur for every 4670 to 9474 infants vaccinated. Ecologic studies that followed reported a lower
vaccine-attributable risk19,20. Further analysis revealed that the risk was age related and substantially
increased in those older than 90 days21,22. Of note, in pre-licensure studies of this rotavirus vaccine,
intussusception was seen in 5 of 10,054 vaccine recipients (0.5 per 1,000) and 1 of 4,633 controls (0.2 per
1,000); a difference that the investigators concluded was not statistically significant23. The pathogenic
mechanism for this association has not been identified24.
Current Rotavirus Vaccines
After RRV-TV was withdrawn from the market, two new rotavirus vaccines were subsequently developed
and introduced into the pediatric immunization schedule. Large pre-licensure studies for both vaccine
candidates were required to determine if there was an increased risk of intussusception for each vaccine.
In these large phase 3 studies, no increased risk for intussusception was identified25,26. The ACIP and
American Academy of Pediatrics (AAP) recommended routine use of these two rotavirus vaccines in US
infants: RotaTeq® (RV5) which was licensed by the FDA in February 200627 and Rotarix® (RV1) which
was licensed in April 20082. Post-licensure programs to monitor the safety of these two vaccines were
established both in the US and abroad, and after years of use, post-marketing studies from different
countries have identified a low level of risk of intussusception with both rotavirus vaccines; however,
results have varied28-30.

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RV1 and Intussusception
In a post-licensure study using a self-controlled case series and case-control methods in Mexico and
Brazil, cases of children with intussusception were identified through active hospital-based surveillance30.
In Mexico, there was an increased risk of intussusception 1-7 days after dose one with either method
accounting for 1 additional case of intussusception for every 52,000 children vaccinated with RV1.
However, no increased risk was found after the first RV1 dose in Brazil.  They did, however find a
smaller attributable risk (AR) of 1 case of intussusception for every 76,000 children vaccinated after dose
2. In Australia, active surveillance data from a self-controlled case series analysis estimated the AR for
intussusception to be 4.3 (95% CI, 0.8–23.3) cases per 100,000 infants vaccinated in the 1–21 days after
dose 1 and the 1–7 days after dose 2 29. Similarly, in US infants, an increased risk for intussusception after
RV1 was identified in the Vaccine Safety Datalink (VSD) with an elevated risk during the first 1-7 days
after vaccination with dose 1 and dose 231. In this study, there were 207,995 doses of RV1 which included
115,908 first doses administered. The AR after the RV1 series was 5.3 per 100,000 infants vaccinated
compared to historical background rates when no rotavirus vaccine was in use31. In another US study,
preliminary data from an FDA-sponsored study through the Post-licensure Rapid Immunization Safety
Monitoring System (PRISM) suggested an increased risk after RV1, although the study was
underpowered32.
RotaTeq (RV5) and Intussusception
Since RV5 was licensed and recommended for use in the US prior to RV1, there have been more doses
given and therefore a larger amount of data for monitoring post-licensure safety.  Four studies in the US
from 2010 to 2012 found no increased risk of intussusception with RV533-36. However, more recently, in
an Australian study, active surveillance data from a self-controlled case series analysis estimated an AR
for intussusception in the first 21 days after dose 1 and the first 7 days after dose 2, to be 7.0 (95% CI,
1.5–33.1) cases per 100,000 for RV529. Data from PRISM identified an increased risk of intussusception
after RV5. In the first 7 days after dose 1 of RV5 the AR was 1.1 (95% CI 0.3, 2.7) additional
intussusception cases per 100,000 infants vaccinated and in the first 21 days after dose 1 of RV5 the AR
was 1.5 (95% CI 0.2, 3.2) additional cases of intussusception per 100,000 infants vaccinated32.
Gaps in Knowledge-Intussusception and Rotavirus Vaccines
In summary, consistent levels of risk for intussusception have not been observed in these post-licensure
studies. This could be due to the low level of risk, small sample sizes for a rare outcome and/or
differences in vaccine responses in different populations. Even though there are inconsistencies in these
studies, overall there is evidence that RRV-TV, RV1 and RV5 are associated with an increased risk of
intussusception. The reasons for the increased risk are unknown and it is not clear if there are product-
specific differences that are real or if factors specific to the populations studied differ. Factors such as
transplacental maternal antibody and breast milk antibody can influence vaccine-virus replication37, and
human blood group antigens (FUT-2) can affect susceptibility to rotavirus disease38. Other factors such as
nutritional status, micronutrient deficiencies and interfering gut flora could also impact viral replication39.
It is also possible that oral polio vaccine could influence risk for intussusception.  In the post-licensure
safety studies in Brazil and Mexico30, Brazilian children routinely received oral polio vaccine (OPV)
while Mexican children are given inactivated polio vaccine as part of their routine immunizations. Since
replication of polio virus is highest with dose one, and has been shown to lower the take of RV140,41, this
may be one factor that influences risk of intussusception where OPV is used. While OPV is not used in
the US, as we examine the association of intussusception and rotavirus in resource poor countries,
concomitant use of OPV with rotavirus vaccines will need to continue to be studied. In considering
factors associated with the risk of intussusception from rotavirus vaccines, it will be important to examine
these potential modifiers whenever possible. Understanding the pathophysiology for such possible
associations is important not only in terms of performing risk-benefit analyses for existing vaccines but
also to facilitate introduction of new vaccines for which large pre-licensure assessments have not been

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done. This information could also be used in determining ways to monitor for intussusception in resource
poor countries, which could aid in evaluating new rotavirus vaccines such as those nearing licensure in
Viet Nam and India.
Intussusception-Still Not Well Understood
Intussusception is a type of bowel obstruction where one segment of bowel becomes enfolded within
another segment. While intussusception can occur at any age, it is most commonly seen among young
children, especially infants 4 to 10 months of age. The diagnosis is considered in young children with
severe abdominal pain. In the US, the use of ultrasound allows for a rapid, non-invasive diagnosis of
intussusception which is sensitive and somewhat specific. Children with findings of intussusception on
ultrasound are usually promptly sent for an air or contrast enema for treatment and simultaneous
confirmation of the intussusception. In the case that the intussusception cannot be reduced, the child is
then taken to surgery for manual reduction with some children requiring partial resection of their intestine
due to ischemic damage from the intussusception or because of a pathologic lead point.
The causes of intussusception are not known; however, prevailing theories include: 1) edema/thickening
of the bowel wall (resulting from inflammation and lymphoid hyperplasia),  2) prominent mesenteric
lymph nodes acting as lead points42 and 3) alterations in gut motility43. A small fraction of cases have an
anatomical or pathologic “lead point”, such as a polyp, or Meckel’s diverticulum, that increases a child’s
susceptibility to intussusception. However in most children there is no clear explanation. Some studies
have suggested infectious agents as causes of intussusception, including adenoviruses24,44-48, rotavirus49,
HHV-648, HHV-748, Epstein-Barr virus48, Escherichia coli50, Salmonella50, Shigella50, and
Campylobacter50; however, the implications of these findings are unclear since most studies do not
include a comparison group. Evidence of rotavirus infection has been found in 5-37% of infants with
intussusception in case series49; however, the association between natural rotavirus infection and
intussusception has never been confirmed with rigorous epidemiologic studies11,42,45,51. Prior to the
widespread use of rotavirus vaccines in the US, when rotavirus disease was distinctly seasonal,
hospitalizations for intussusception were not found to be seasonal, suggesting that either rotavirus did not
play a major role in the etiology of intussusception or that multiple infectious agents with different or less
distinct seasonal patterns made the association between rotavirus infection and intussusception difficult to
detect51,52. Thus, while wild-type rotavirus infection has not been linked to increased risk for
intussusception, a small increased risk for intussusception after wild-type rotavirus cannot be ruled out11.
One study suggested that rotavirus infection induced anatomical changes in the intestine (hyperplasia of
Peyer’s patches and mesenteric lymph node enlargement) compatible with those that could cause
intussusception42 however another examining intussusception in RRV-TV recipients did not find these
changes24, suggesting there may be different pathophysiologic mechanisms that lead to intussusception.
2.2
Intestinal Imaging
Imaging Studies in Children with Rotavirus Infection
Two previous studies have been done in children to explore possible mechanisms for the cause of
intussusception in children with wild-type rotavirus infection42,43. In the first study, 13 children with
confirmed rotavirus infection were age- and sex-matched to healthy children and ultrasound
measurements were done to assess ileum wall thickness and mesenteric lymph node diameters at
enrollment and one month later; motility was not assessed42. The average age at enrollment was
comparable (7.2 months for cases and 7.3 months for controls). They found children with rotavirus
infection to have significantly thicker ileum wall thickness, greater lymph node size and number of nodal
aggregates and greater free fluid compared to controls. While distal ileum loops were visualized in all
study subjects, the terminal ileum was only visualized in 46% of patients with rotavirus infection and in
62% of controls. They concluded that these changes suggested a mechanism by which rotavirus could
cause intussusception. Since most intussusception occurs at the terminal ileum, it is critical that an

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imaging study evaluating risk for intussusception can effectively image this area.  With MRI, we will be
able to visualize and measure the wall of the terminal ileum for all patients as well as assess motility.
In a second study, investigators enrolled five children with symptomatic rotavirus infection and did
imaging with ultrasound and MRI within 5 days of the onset of symptoms and repeated imaging 5-9
weeks later with an age range of 8-20 months (mean age of 14 months)43. They found three of the five
infants had a significantly thicker ileal wall thickness in the acute period compared to the convalescent
period but no differences in the number and size of mesenteric lymph nodes. They found no difference in
peristaltic activity between the two time periods using MRI imaging without sedation. The authors did not
appear to have a period where the infants were made NPO.  A small sample size (4 infants for the MRI)
and lack of a specific protocol for feeding may have impacted their ability to assess motility patterns.
While these studies provide useful preliminary data, none were done in the age group in question or with
rotavirus vaccine exposure. In addition, radiographic techniques and assessments of motility have
improved since 2004 allowing for more standardized measures of normal anatomy and motility (pre-
immunization) and more sensitive measures post-immunization with the use of MRI.
Intestinal Motility Imaging
The standard imaging modality for clinical evaluation of intestinal motility is fluoroscopy where opaque
contrast material is administered orally or via a nasogastric tube into the bowel lumen, followed by serial
radiographs to determine bowel anatomy and successful movement of the contrast through the bowel. A
significant drawback is that fluoroscopy results in a relatively large radiation exposure. While abdominal
ultrasound avoids radiation exposure, technical challenges of ultrasound limit the ability to assess
intestinal motility. Ultrasound can only focus on one bowel loop at any given time, making assessment of
the entire bowel quite time intensive. Assessment of a single bowel loop over several minutes in order to
qualitatively and quantitatively assess motility is challenging. Any air within the bowel lumen
significantly decreases image quality. In addition, ultrasound is very dependent on the skill and
experience of the ultrasonographer and there is a high degree of inter-operator variability. Alternatively,
MRI is extremely effective in providing detailed information about anatomy and function including
motion53. MRI uses magnetic fields and radio waves to create detailed images of the organs and tissues;
there is no radiation exposure with this procedure. Recently, MRI has been used in the evaluation of in-
utero suspected congenital malformations of the intestine54, as well as evaluation of ongoing disease
activity in pediatric patients with inflammatory bowel disease55. Several recent studies have demonstrated
the ability of dynamic MRI, using ultrafast acquisitions (< 1 sec), to evaluate intestinal motility56-61.
2.3 Inflammatory Mediators and Intussusception-Use of Blood and Stool Cytokine
Analyses
While we know that both infectious agents and receipt of vaccines induce immune responses and
inflammatory mediators, little is known about specific patterns of inflammatory mediator expression
which might be associated with an adverse event from a vaccine such as rotavirus vaccine and
intussusception. We know that specific cytokine responses can alter intestinal motility and lymph node
enlargement. Therefore we postulate that specific patterns of expression could predispose to
intussusception. While limited animal and human studies have examined serum cytokine responses, to our
knowledge, no studies of stool cytokines have been undertaken in evaluating rotavirus vaccines or
intussusception.
Animal Studies
Early studies in mice demonstrated that lipopolysaccharide (LPS) can induce intussusception62.
Inflammatory mediators such as tumor necrosis factor, platelet-aggregating factor, and other cytokines

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were thought to disrupt gastrointestinal tract motility and contribute to the etiology of intussusception63-65.
In a more recent model, both homologous murine and heterologous simian rotavirus strains signiﬁcantly
enhanced the rate of LPS-induced intussusception in mice66. This occurred despite there being no
measured effect of rotavirus infection on gastrointestinal transit. TNFα and IL-6 are also known to
significantly affect gastrointestinal motility and have been associated with LPS-induced intussusception
in mice67. In one study, indomethacin (a non-steroidal anti-inflammatory drug) treatment of mice after
LPS, prevented intussusception67. However, in another study, no correlation was demonstrated between
serum TNFα levels and the development of intussusception in mice68.
Human Studies
Several studies have evaluated serum cytokines in children at the time of acute ileocolic
intussusception69,70. Levels of IL-6, neopterin, and C-reactive protein (CRP) were significantly elevated in
children with intussusception compared to normal laboratory ranges and convalescent samples69. Another
study demonstrated elevated IL-6 levels in 22 infants with intussusception compared to 20 healthy control
infants70. Levels were significantly increased in infants requiring surgery compared to those reduced by
enema. In another report, in two children with adenovirus and intussusception, the investigator identified
an inflammatory neuropathy of a network of nerves in the intestines and proposed that inflammation of
the intestine caused disruption of gastrointestinal motility that could lead to intussusception.
Stool Cytokine Responses
As compared to serum cytokine levels described above, stool cytokine levels are more likely to reflect gut
mucosal immune responses to infection or other inflammatory changes. Studies have examined fecal
cytokine responses to norovirus71, E. coli72,73, and other etiologic agents of travelers’ diarrhea using
enzyme immunoassays (EIA)74. Dr. Haslam, our co-investigator, enrolled children with symptomatic C.
difficile (cases), symptomatic controls (without C. difficile) and asymptomatic controls with and without
C. difficile. Stool samples were tested using EIA for fecal IL-8, lactoferrin, and phosphorylated-p38
protein concentrations, as well as the novel quantitative reverse transcriptase polymerase chain reaction (q
RT-PCR) to determine IL-8 and chemokine ligand (CXCL)-5 RNA relative transcript abundances75. Fecal
inflammatory cytokines were found to be useful in distinguishing C. difficile colonization from disease
and identifying children with C. difficile infection likely to have prolonged diarrhea. In addition, fecal
cytokines were higher in samples from symptomatic children (with and without C. difficile) than in
samples from asymptomatic children with C. difficile. In a study in adults, persistent diarrhea was also
shown to be correlated more with intestinal inflammation than fecal pathogen burden. The RT-PCR
method was more sensitive than ELISA for IL-8 measurement, and the mRNA levels measured by RT-
PCR correlated with disease severity. Cytokine levels measured by ELISA did not correlate with disease
severity, as the cytokines were frequently undetectable via ELISA76.
Cytokines Associated with Lymphocyte Proliferation and Systemic Inflammation
Lymphoid hyperplasia in the terminal ileum is a recognized cause of intussusception77,78. Cytokines
specifically associated with lymphocyte proliferation and development include IL-279, IL-780, IL-1581, and
various members of the TNF superfamily82. Elevated CRP and IL-6 have also been associated specifically
with acute intussusception69,70. Measuring these cytokines that drive lymphocyte proliferation, as well as
those associated with acute systemic inflammation, may identify inflammatory markers in both stool and
serum that may be associated with the pathophysiology seen in intussusception. This project will be the
first to investigate novel fecal measures of inflammation and lymphoid hypertrophy during immunization.
2.3
Potential Modifiers of Rotavirus Infection, Vaccine Response and Shedding
and Intussusception

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Age-Dependent Susceptibility to Rotavirus Disease and Intussusception
Severe rotavirus disease occurs most commonly in infants younger than 24 months. The onset of severe
rotavirus disease has been reported to coincide with the decline of maternal IgG antibody titers83. In
addition, we have found a decreased antibody response to the live rotavirus vaccine RV1 in pre-licensure
studies in infants with higher levels of transplacental neutralizing antibody to the vaccine strain
(unpublished results). Therefore, early protection from rotavirus disease including asymptomatic neonatal
rotavirus infections84,85 may be due, at least partially, to protection by transplacental antibody that may
persist for the first months of life86. The risk of intussusception increases with age and many felt the risk
of RRV-TV-associated intussusception also increased with age18. Therefore, in order to best understand
potential mechanisms associated with intussusception and rotavirus vaccines, ideally, it would be
important to examine responses in children with the broadest age range possible.  Since the age
recommendations for the first dose of rotavirus vaccine is 6 weeks to 14 weeks, 6 days, the prospective
study will be limited to that age group. We have selected the age range of 6 week to 13 weeks (12 weeks,
6 days) in order to provide assurances that the other 2 month old vaccines can be given on schedule.
Breast Milk-Rotavirus Susceptibility and Rotavirus Vaccine Response
The role of breast milk and rotavirus susceptibility and rotavirus vaccine response is unclear.  Two studies
found that the highest incidence of rotavirus-associated diarrhea is in children aged 4 to 6 months,
suggesting that risk for rotavirus infection increases at the age when weaning from breast-feeding
began87,88. Other studies have shown breast-feeding to be associated with protection against symptomatic
rotavirus infections89-92. Unfortunately, there are limitations in study design, as other factors such as the
presence of maternal antibody or lack of opportunity for exposure were not examined. However, one
study specifically supported the role of human milk and factors other than breast milk antibody in
protection against rotavirus infection, demonstrating that infants who received human milk with higher
concentrations of the glycoprotein lactadherin were more likely to have asymptomatic rotavirus infections
compared with those receiving milk with lower concentrations90. Since both licensed vaccines are live-
attenuated vaccines, there is also a concern that breast milk could play a role in limiting the response to
rotavirus vaccination which would impact protection but also the pattern of cytokine response after
vaccination37,93,94. While sub analyses in studies of RV1 and RV5 did not show a decreased efficacy in
breastfed infants, the immune response and protection provided by live oral rotavirus vaccines are less in
resource poor countries, where nearly all infants were breastfed, than in developed countries 95,96. It has
been speculated that this could at least in part be due to breast milk antibody and other constituents
limiting the replication of the vaccine strains as the levels of antibody are higher in women in resource
poor countries. However, more recent data have not found this to be true. To our knowledge there are no
data describing differences in vaccine virus shedding, cytokine responses or intussusception in breastfed
and non-breastfed infants. In the case-control study evaluating intussusception and RRV-TV, data were
re-analyzed to assess risk factors for intussusception unrelated to RRV-TV97. Cases were infants who had
intussusception between November 1998 and June 1999. Controls were matched by age and hospital of
birth. Sociodemographic and feeding practice data were collected through parent and provider interviews.
Risk factors for intussusception were identified while controlling for exposure to RRV-TV. Of the 429
cases enrolled, 87% had not received RRV-TV. There were 1763 controls. In addition to finding male
sex, Hispanic or black race/ethnicity, and Medicaid enrollment to be factors associated with
intussusception, an interaction was found between introduction of solid food and type of formula
consumption.  Using breast milk as the referent group, infants with introduction of solid food for at least 5
weeks who consumed soy milk-based formula had a lower risk (OR 0.26; 95% CI 0.1-0.7) and infants
without introduction of solid food who consumed cow's-milk formula had an increased risk (OR 2.33;
95% CI 1.4-3.9).

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Another case control study which studied idiopathic intussusception found that, compared with non-
breast-fed infants, breast-fed infants had a relative risk of intussusception of 6.0 (95% CI, 1.8 to 20.4)
when breast-feeding at admission was exclusive and of 2.3 (95% CI, 0.8 to 6.6) when it was partial98.
They concluded that exclusive breast-feeding may be a risk factor for intussusception in infancy.
FUT2 Secretor Gene and Rotavirus Susceptibility
Recently, we and others have reported that, similar to norovirus, risk of rotavirus infection is determined
in part by an individual’s FUT2 (fucosyltransferase 2) genotype38,99,100. The FUT2 (secretor) gene is
responsible for producing an enzyme that makes carbohydrate histo-blood group antigens (HBGAs)
available on the surface of the gut. The major human rotavirus genotypes (P4, P6, and P8) bind to HBGA
that are synthesized by FUT2 gene enzymes, specifically H type 1, Lewis B or A antigens101,102.
Individuals who are homozygous recessive for the FUT2 (secretor) gene, and are thus known as “non-
secretors,” lack these HBGA on their mucosal surfaces, thereby reducing the ability of rotaviruses to bind
and infect intestinal epithelial cells. From a collaborative multi-site surveillance study with the CDC New
Vaccine Surveillance Network (CDC NVSN), we identified 56 rotavirus-positive cases of acute
gastroenteritis (AGE). None of these rotavirus AGE cases had the non-secretor FUT2 mutation, compared
with 23% of healthy controls (p<0.01). Additional surveillance by the same network identified another
120 rotavirus positive cases, and similarly, found that only 2% of the rotavirus AGE cases were FUT2
non-secretors compared to 23% of healthy controls (p<0.01).
FUT2 non-secretors are approximately one-quarter of the population and appear to be largely protected
not only against rotavirus but also against norovirus, while the majority of the population with FUT2
secretor genotype appears to convey risk. Thus, the question arises, why is the FUT2 genotype maintained
– what is the benefit? Several lines of investigation suggest that the carbohydrates synthesized by the
FUT2 enzyme have an anti-inflammatory impact on the intestinal tract. The FUT2 secretor genotype and
oligosaccharide made by FUT2 enzyme favors microbial diversity103, and the growth of beneficial,
immune-regulatory intestinal microbiota104-106. Conversely, the lack of these carbohydrates in the FUT2
non-secretor genotype is a risk factor for inflammatory bowel disease (IBD), and functional changes in
the microbiota that are accompanied by sub-clinical levels of inflammation in the local intestinal
mucosa103.
Other Routine Immunizations
Young infants receive multiple immunizations that are associated with elevated blood cytokines and other
inflammatory markers. Routine childhood immunizations have been found to be associated with pro-
inflammatory cytokine responses, including INF-γ23,24, IL-525, IL-626, and IL-827. These routine
immunizations may therefore contribute to or interfere with the inflammatory response following
rotavirus immunization, potentially altering the risk for intussusception.
Therefore it will be important that in two arms of the study infants will receive routine 2 month old
immunizations with one of the two recommended rotavirus vaccines (RV5 or RV1) to evaluate the
contribution of blood cytokine responses associated with the addition of routine immunizations to
rotavirus vaccine and whether there are stool cytokine responses that are associated with routine
immunizations with rotavirus vaccine given concurrently. This will provide two arms that reflect “real
world” immunization schedule, allowing for the determination of any changes in the outcomes from
receiving all the recommended vaccines.  The outcomes found in these two arms will be compared to the
RV1 and RV5 alone arms.

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3
Purpose and Objectives
3.1
Purpose
The purpose of this project is to conduct a pilot study to explore potential biological mechanisms for an
association between rotavirus vaccines and intussusception.
3.2
Hypothesis
Our hypothesis is that rotavirus vaccines may increase the risk for intussusception by inducing cytokines
or other inflammatory responses that affect the anatomy or functional motility of the small intestine and
that there will be a relationship between the pattern of the shedding of vaccine strain rotavirus and the
anatomy and or functional motility of the small intestine. We also hypothesize that potential factors such
as maternal antibody, human blood group antigens and breast milk in breast-fed infants could modify
rotavirus vaccine virus shedding and/or inflammation following vaccination.
3.3
Primary and Exploratory Objectives
Primary Objectives:
1) to examine the effects of RV1 and RV5 with or without other routine immunizations on
gastrointestinal anatomy
2) to assess the feasibility of conducting a larger scale study
Exploratory Objectives:
1) to examine the effects of RV1 and RV5 with or without other
routine immunizations on gastrointestinal motility
2) to examine the effects of RV1 and RV5 with or without other routine immunizations on blood and
stool cytokines and inflammatory responses, including clinical reactogenicity events
3) to assess patterns and level of vaccine virus shedding after RV1 and RV5
4) to provide preliminary data assessing associations with the pattern of
rotavirus vaccine strain (RV1
and RV5) shedding in the stool and blood, stool cytokine responses, and the anatomy and/or functional
motility of the small intestine; and examine other factors such as maternal antibody, breast milk antibody,
and human blood group antigens to see if these factors modify these responses
5) to compare MRI and ultrasound imaging
3.4 Study Outcome Measures
Intestinal Anatomy and Motility-Use of Magnetic Resonance Imaging (MRI) and Ultrasound (US)
We have selected magnetic resonance imaging (MRI) for our main imaging study outcome. Ultrasound
will also be done and the results obtained will be compared to the MRI results.   This will be done
because ultrasound is the standard clinical mode of imaging for intussusception and is more readily
available in both the US and abroad compared to MRI. In addition, ultrasound was used in previous
studies. While ultrasound is a useful modality to assess the presence or absence of intussusception
clinically, it has several limitations which make it difficult to assess the bowel serially. Some of the
limitations of ultrasound include its lack of standard planes of imaging, lack of reproducibility, operator
dependence, and inability to accurately assess motility and to image the entire abdomen. Alternatively,
MRI is ideally suited to evaluate both intestinal anatomy and motility to assess for changes pre- and post-
immunization with rotavirus vaccines and this is why MRI will be our main imaging study outcome.
Inflammatory Mediators-Blood and Stool Cytokines
We will measure both blood and stool cytokines pre- and post-rotavirus vaccination. The cytokines
chosen include those shown in past studies to be associated with intussusception and others were chosen
because of the association with lymphoid hyperplasia. For blood, we will test C-reactive protein as it is

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readily available and used clinically.  For both blood and stool, we will measure: IL-2, IL-6, IL-7, IL-8,
IL-15, INF-γ and TNF-α.
Rotavirus Vaccine Virus Shedding
We will use RT-PCR to identify rotavirus vaccine strains, to examine the duration of shedding and to
quantitate rotavirus shedding 5 days post-vaccination. We will then assess the correlation of the amount
of virus shedding with intestinal anatomy and motility as well as cytokine and inflammatory responses.
The Ct values will be examined and categorized as none, low or high for analysis. We chose 14 days to
assess the duration of shedding since studies have shown that most vaccine-associated events occur in the
first 14 days and shedding becomes less frequent after 14 days. Vaccine virus shedding (qualitative) will
be done on all stools collected. In addition, since we will be collecting reactogenicity data for Days 0-14,
stool from other days could be evaluated for stool cytokines should the infant have an adverse event either
before or beyond the Visit 2 stool evaluation.
Potential Modifiers of Rotavirus Infection, Vaccine Response and Shedding and Intussusception
Maternal Serum Antibody
In order to examine the possible role of maternal antibody to rotavirus, we will test the initial blood
sample for maternal antibody to see if maternal antibody affects shedding of vaccine virus, blood and
stool cytokine responses and intestinal motility and anatomy. In addition, we will measure the infant’s
IgA antibody to assure that the child has not been infected with rotavirus.
Breast Milk Antibody
We will obtain a breast milk sample from breastfeeding mothers of infants enrolled in the study in order
to examine the potential effect of rotavirus IgA in breast milk on vaccine virus shedding, cytokine
responses, and intestinal anatomy and motility. The analysis will be exploratory given the small sample
size.
Secretor Status
Thus, in an exploratory analysis, we will examine whether there is an association with secretor status and
inflammatory response.
4
Research Plan
4.1
Overview of Study
This study is a prospective clinical study designed to 1) examine the effects of RV1 and RV5 alone or
with other routine immunizations on gastrointestinal anatomy 2) to assess the feasibility of conducting a
larger scale study. Exploratory objectives include 1) to examine the effects of RV1 and RV5 with or
without other routine immunizations on gastrointestinal motility 2)to examine the effects of RV1 and
RV5 with or without other routine immunizations on blood and stool cytokines and inflammatory
responses, including clinical reactogenicity events 3) to assess patterns and level of vaccine virus
shedding after RV1 and RV5 4) to provide preliminary data assessing associations with the pattern of
rotavirus vaccine strain (RV1 and RV5)shedding in the stool and blood, stool cytokine responses, and the
anatomy and/or functional motility of the small intestine; and examine other factors such as maternal
antibody, breast milk antibody, and human blood group antigens to see if these factors modify these
responses and 5)to compare MRI and ultrasound imaging.
Healthy infants 6-13 weeks of age will be randomized (1:1:1:1) to receive either, RV1 alone, RV5 alone,
RV1 with ACIP routinely recommended immunizations (Diphtheria, Tetanus and Pertussis (DTaP),
Haemophilus influenza type b ( Hib), pneumococcal conjugate (PCV13), Hepatitis B (HBV) and

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inactivated polio (IPV)) or RV5 with routine immunzations. Imaging study personnel and parents will be
blinded to the rotavirus vaccine type; parents will be informed about the rotavirus vaccine type at the
completion of the study. Up to 150 infants will be enrolled (see inclusion/exclusion below).  The age-
group of infants 6-13 weeks of age was chosen so enrolled infants can receive their first dose of rotavirus
vaccine at the AAP/ACIP recommended age of 2 months (minimum age of 6 weeks and maximum age of
14 weeks and 6 days)2 and, to assure that other ACIP recommended vaccines can be administered at the
recommended age.
Recruitment and enrollment will occur prior to the first study visit. There will be three scheduled study
visits after recruitment/enrollment. . Infants will be randomized to either RV1, RV1 plus other
immunizations, RV5 alone, or RV5 plus other immunizations. Visit 1 (day 0) will include blood, saliva,
stool and breast milk collection . The MRI and ultrasound will be performed prior to vaccination.
Children will receive the immunizations to which they are randomized. Imaging personnel and parents
will be blinded to rotavirus vaccine type; they will be informed about other vaccines administered.  A
second MRI and ultrasound will be performed at Visit 2 (day 5) and blood and stool samples collected.
Parents will be unblinded at the completion of Visit 3 (day 14). Arrangements will be made to get
remaining doses of same rotavirus vaccine. Daily stool samples will be collected at home during the 15
day study period (on vaccination day 0 and for next 14 days). A memory aid will be completed to collect
reactogenicity data on days 0 and for the next 14 days. Remaining stools and reactogenicity data from
parents will be collected at Visit 3. See Appendix 1 for detailed description of visits and timeline.
We will assess blood and stool cytokine responses and intestinal anatomy and motility after rotavirus
vaccination by comparing pre-vaccination with post-vaccination responses in the study infants. Cytokines
and intestinal anatomy and motility will be assessed at baseline (Visit 1, the day of vaccine receipt) and 5
days after vaccination (Visit 2). For both blood and stool, the following cytokines will be tested: IL-2, IL-
6, IL-7, IL-8, IL-15, INF-γ and TNF-α. Additional biomarkers may be studied. The 5 day period post-
vaccination was chosen because this time period corresponds with the peak period for intestinal
replication of rotavirus vaccine virus for both vaccines107.
In order to protect the health of infants, we will provide procedures to facilitate and assure that infants
enrolled in the study will receive the routinely recommended vaccines for 2 month old infants which are
DTaP, Hib, PCV13, HBV and IPV that will also allow them to get their 4 month vaccines on schedule108.
4.2
Selection of Study Population
Normal healthy full term infants (>37 weeks gestational age) will be recruited. Subjects will be recruited
without regard to race, gender, or ethnicity. Up to 150 subjects will be recruited from established patient
populations at CCHMC. These include our CCHMC Pediatric Primary Care Clinic, employees at
CCHMC, and our CCHMC study database. Parental permission /informed consent will be obtained from
the parents/legal guardians before any procedures are performed. Their parents/legal guardians will be
interviewed to assure that the infants meet all the eligibility criteria and will be available for the duration
of the study.
Geographically, our proposed study population will be from our primary service area at CCHMC. We do
not anticipate targeted recruitment based on minority status, but no minority will be excluded from the
study.
4.3
Inclusion and Exclusion Criteria
Subject Inclusion Criteria
Infants who meet the following criteria will be eligible to participate in this study:

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1.
healthy infant 6 to 13 weeks (12 weeks and 6 days) of age at day of rotavirus vaccine administration
2.
free of obvious health problems as established by medical history and confirmed with infant’s primary
physician prior to Visit 1
3.
parent/legal guardian willing to have infant feed from a bottle for contrast
4.
parent/legal guardian willing and capable of signing informed consent
5.
parent/legal guardian and infant expected to be available for entire study
6.
parent/legal guardian can be reached by telephone
7.
parent/legal guardian expresses willingness to complete study procedures and receive 2 month
immunizations, according to recommended schedule
Subject Exclusion Criteria
Infants will not be eligible if any of the below conditions apply:
1.
gestational age of <37 weeks
2.
infant unable to fast for 4 hours prior to MRI procedure
3.
receipt of any vaccine except initial HBV (must have at least 28 days between HBV and Visit 1 to be
included)
4.
history of severe allergic reaction to HBV vaccine
5.
contraindications for any of the routine vaccines
a.
Severe Combined Immune Deficiency
b.
history of intussusception
6.
precautions for either RV1 or RV5 (may interfere with study outcomes)
a.
altered immunocompetence
i.
infants with primary and acquired immunodeficiency states, cellular
immunodeficiency, hypogammaglobulinemic and dysgammaglobulinemic states
ii.
infants with blood dyscrasias, leukemia, lymphomas, or other malignant neoplasms
affecting the bone marrow or lymphatic system
iii.
infants on immunosuppressive therapy (including high-dose systemic corticosteroids)
iv.
infants who are HIV-exposed or infected
b.
acute gastroenteritis
c.
moderate or severe acute illness with or without fever
d.
pre-existing chronic gastrointestinal diseases (e.g., congenital malabsorption syndromes,
Hirschsprung's disease, or short-gut syndrome)
e.
infants with spina bifida or bladder exstrophy (latex rubber is contained in the RV1 oral
applicator)
7.
sensitivity to latex (latex rubber is contained in the RV1 oral applicator)
8.
febrile illness within previous 14 days (axillary temperature of 100.4◦F or higher)
9.
history of vomiting (forceful expulsion of partially digested milk/food) within 24 hours prior to Visit
1 and/or diarrhea (3 watery stools) within 14 days of Visit 1
10. receipt of any steroids, immunoglobulins, other blood products/transfusion
11. receipt of non-steroidal anti-inflammatory drugs in previous 72 hours (may affect cytokine response)
12. receipt of an antipyretic medication (acetaminophen or ibuprofen) within 72 hours prior to the first
dose of rotavirus vaccine or is already planning to administer a prophylactic antipyretic medication on
the day of and the day following vaccination (this exclusion does not apply if the caretaker indicates
he/she might administer antipyretics after vaccination to reduce a fever)
13. is enrolled or plans to enroll in another clinical trial while participating in this study (observational
studies are allowed)
14. any condition which, in the opinion of the investigators, may post a health risk to the subject or
interfere with the MRI or vaccine evaluation

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15. currently receiving medication for gastroesophageal reflux (GERD) or any other gastrointestinal
condition including colic
16. infant who is a relative of any research study personnel
17. allergy to barium
18. failed newborn hearing screening
4.4 Randomization and Study Withdrawal
Randomization Procedures
The randomization code will be prepared by statisticians in the Epidemiology and Biostatistics Core at
CCHMC. The randomization code will link to the vaccine assignment. The subject will be assigned a
randomization code. Randomization will take place prior to the vaccine administration, but after the
parent/legal guardian has given permission/informed consent , signed the IRB-approved consent form and
met all of the inclusion and none of the exclusion criteria. Infants will be randomized 1:1:1:1 to one of
four groups: 1) RV1 alone, 2) RV5 alone, 3) RV1 with routine immunizations (DTaP, Hib, PCV13, HBV
and IPV or 4) RV5 with routine immunzations.
Reasons for Withdrawal
The study personnel may withdraw a subject from the study at any time for the following reasons:
1.
unable to comply with the study protocol
2.
receipt of blood or blood products (including immunoglobulin) during the study period
3.
malignancy or confirmed or suspected immunodeficiency such as HIV infection or severe combined
immunodeficiency
4.
receipt of or history of any medications or treatments that affect the immune system, such as immune
globulin, interferon, immunomodulators, cytotoxic drugs, or other drugs known to be frequently
associated with significant major organ toxicity since enrollment
5.
receipt of long-term (> 2 weeks) potentially immunosuppressive corticosteroid medication during the
study period
6.
parent/legal guardian request
7.
infants who vomit within 15 minutes of rotavirus vaccination will not be withdrawn but will be
replaced for analysis purposes
8.
allergic reaction to barium
Handling of Withdrawals
Every effort will be made to undertake protocol-specific safety follow-up procedures for subjects who are
withdrawn by their parents/legal guardians.  The subject’s parents/legal guardians will be encouraged to
participate in the final follow-up contact to occur 14 days after the initial visit.
Subjects who are withdrawn may be replaced if deemed necessary by the study team to meet study
enrollment goals.
5 STUDY SCHEDULE ( See Appendix 1)
5.1 Recruitment and Enrollment
Recruitment will be done by our study personnel through the Pediatric Primary Care (PPC) Clinic at
CCHMC and through study advertisement at CCHMC targeted towards our employee e-mail system and
database of past research participants who have agreed to be e-mailed for future studies. Any written
communication provided to the potential subjects' parents/legal guardians about the study will be
submitted to the IRB for their approval before study initiation.
We routinely conduct studies through the PPC and our research personnel are present in the clinic. We
will review the patients scheduled and will identify those who are age eligible for the study.  The medical

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record for those age-eligible will be screened to identify children who would not be eligible for the study
(exclusion criteria). Parents of potentially eligible infants will be approached to discuss participation in
the study. If interested, informed consent will be obtained before giving instructions and scheduling Visit
1. After consent is obtained, infant will be randomized into one of four study groups.
For recruitment of infants of CCHMC employees and through our research database, the interested parent
will call our study line. Relatives of study personnel will be excluded from participation as is standard
procedure. Study personnel will discuss the study by phone. Subjects will be screened by medical
history from the parent. If eligible and interested, a meeting time will be arranged prior to Visit 1 to
obtain informed consent. The infant’s primary care provider will be contacted to determine if the infant is
healthy and to inform the primary care provider of the study after informed consent and  prior to Visit 1.
Parents will be informed that their decision to participate or not to participate in the study will not have
any effect on them as an employee.
Because the infants to be enrolled will be healthy and will be receiving a licensed vaccine that is approved
for use in this population, no additional screening will be necessary. No baseline laboratory screening will
be performed prior to enrollment. Screening records will be kept to document the reason why a subject
was screened but failed entrance criteria. If a parent chooses not to agree to the study, we will ask for a
reason and it will be documented.
Infants must be 6 weeks to 13 weeks of age (12 weeks, 6 days) for Visit 1, the day of immunization.
Inclusion and exclusion criteria will be reviewed. All  medications taken within the 30 days prior to
enrollment will be recorded. Demographic and contact information will be obtained. The parent/legal
guardian will be given containers for stool and breast milk collection at home. Instructions will be given
to the parent with the date, time and location for Visit 1 and the need for their infant to be NPO for 3
hours prior to appointment and to return with a stool collected the day of Visit 1 (note for MRI must be 4
hours NPO, but will ask to be 3 hours NPO since it will take ~one hour from arrival until feed for MRI).
5.2
Parent Education about Rotavirus Vaccines and the Study
Parents approached for this study will be educated about the two licensed rotavirus vaccines (RV1 and
RV5) and informed that the AAP and ACIP routinely recommend rotavirus vaccines for infants and do
not express a preference for RV1 or RV52. We will explain that their infant may be randomized to a
vaccine type different from the vaccine given at their primary care practice. The parent will also be
informed that RV1 is a two dose vaccine series and RV5 is a three dose vaccine series and that if they
choose to have their infant participate in the study, their infant will need to complete the series with the
same vaccine type (all RV1 or all RV5). RV1 is to be administered orally in a 2-dose series, with doses
administered at ages 2 and 4 months. RV5 is to be administered orally in a 3-dose series, with doses
administered at ages 2, 4, and 6 months. The minimum age for dose 1 of rotavirus vaccine is 6 weeks; the
maximum age for dose 1 is 14 weeks and 6 days. The minimum interval between doses of rotavirus
vaccine is 4 weeks and there is no maximum interval set. All doses should be administered by age 8
months and 0 days. The parent of infants who are randomized to receive rotavirus vaccines without other
childhood vaccines will be informed that since the purpose of the study is to study the effects of rotavirus
vaccine, their infant will wait to receive the rotavirus vaccine until two weeks or more after other
routinely administered immunizations or will not receive their other recommended vaccines until two
weeks or more after their rotavirus vaccine. If the child is receiving routine immunizations after the
rotavirus vaccine, every effort will be made for the infant to receive these vaccines within the
recommended ACIP schedule for vaccines routinely recommended at 2 months of age (DTaP, Hib,
Prevnar, Hepatitis B and IPV vaccine)108. The parents of infants who are randomized to receive rotavirus
vaccines with other recommended vaccines will be informed of this for their planning and provided with a

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letter for their pediatrician. These infants will still be recommended to have a 2-month well child check-
up, but will be asked not to receive 2-month vaccines at this visit.  Children receiving other childhood
vaccines as part of the study will receive the following vaccines Pentacel (DTaP, Hib and IPV
combination), Prevnar (PCV13)and HBV, in addition to the rotavirus vaccine. If one of these is not
available we will replace it with another equivalent licensed vaccine.
All vaccines administered as part
of the study will be documented for the parent and child’s health care provider. This information will be
faxed to the provider.  The vaccines administered will be documented in EPIC, in IMPACT (the Ohio
State Registry) and in letter form with copies of the EPIC documentation to the parent and provider of the
enrolled child.
5.3 Study Visits
Enrollment/Recruitment Visit

review inclusion/exclusion criteria and medical history

obtain informed consent

obtain a release to get medical information from the primary care provider

collect contact information including the primary care provider of infant and the date of 2 month
check up if known

if refuses study, record refusal reason on screening log

sample collection instructions

distribute sample collection supplies

review MRI and ultrasound prep

randomize to study group and inform parent of result of all routine vaccines or rotavirus vaccine
alone (not specific rotavirus vaccine type)

schedule visit 1
give written instructions for Visit 1 to parent (Appendix 5) Reminder Call  1 (day prior to Visit 1)

review sample collection instuctions

review MRI/ultrasound prep
review time, date and location of Visit 1Visit 1 (Day 0-Vaccine Administration)

contact information will be reviewed and updated if necessary

inclusion and exclusion criteria will be reviewed to determine if child still meets criteria for study

temperature will be recorded prior to immunization; axillary temperature must be <100.4◦F prior to
administration of vaccines

breast milk specimen brought from home if mother is breast feeding or can be collected at the visit if
needed

saliva specimen collected for human blood group antigen testing

baseline stool specimen (brought from home) before receiving the rotavirus vaccine

MRI to assess intestinal anatomy and motility

Ultrasound of abdomen to assess intestinal anatomy

baseline blood specimen for cytokine testing and maternal antibody testing (IgG)

The Vaccine Information Sheet (VIS) will be provided to parents/guardians for all vaccines
administered.

The study product administrator, who is licensed to administer the vaccine (e.g., MA, LPN, RN, NP,
PA, or MD), will orally administer the rotavirus vaccine as indicated in the randomization code
o
if the infant vomits after receiving the vaccine, in accordance with AAP and ACIP
recommendations, the vaccine will not be readministered but time of vaccine
administration and  vomiting will both be documented.

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
For infants randomized to receive other vaccines as part of the study, the study product administrator,
who is licensed to administer the vaccine (e.g.,MA, LPN, RN, NP, PA, or MD), will administer the
vaccines, according to standard practice: Pentacel (DTaP, Hib and IPV combination), Prevnar
(PCV13)and  HBV), in addition to the rotavirus vaccine.  If one of these is not available we will
replace it with another equivalent licensed vaccine.

Memory aide will be reviewed with the parent and the parent will demonstrate ability to take
temperature and will be instructed in the collection of reactogenicity, adverse event, fever and
concomitant medication administration data for 14 days after vaccination. Parent/gurardian will be
given a thermometer to use for daily axillary temperature recording

distribution of supplies for daily stool collection for 14 days with instructions for storage and return

written instructions (Appendix 6) will be given to the parent with the date, time and location for Visit
2 and need for infant to be NPO (nothing to eat or drink) for 3 hours prior to appointment and to
return with daily stools collected

payment for time and travel will be provided for the study procedures

study personnel will complete the case report form CRF
Reminder Call 2 (Day 3 +2 days)

review sample collection instuctions

review MRI/ultrasound prep

review time, date and location of Visit 2

review memory aide with parent/guardian
Visit 2 (Day 5 +/- 1 day) Post-Vaccination)

contact information will be reviewed and updated if necessary

inclusion and exclusion criteria will be reviewed to determine if child still meets criteria for study (no
vaccines given since last visit)

MRI to assess intestinal anatomy and motility

ultrasound to assess intestinal anatomy

submission of daily stool specimens collected

blood collected for cytokine testing

memory aide will be reviewed with the parent and the parent will demonstrate ability to take
temperature and will be instructed in the collection of reactogenicity, adverse event, fever and
concomitant medication admiration data for remaining study period

distribution of supplies for daily stool collection with instructions for storage and return

payment for time and travel for study procedures

written instructions (Appendix 7) given to the parent with the date, time and location for Visit 3 and
need to return with the collected daily stool specimens and memory aide

study personnel will complete the Case Report Form (CRF)
Reminder Call 3 (Day 11 +/-2 days)

review sample collection instuctions

review time, date and location of Visit 3

review memory aide with parent/guardian
Visit 3 (Day 14 +/- 4 days)

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
inclusion and exclusion criteria will be reviewed to determine if child still meets criteria for study
(has not had any vaccines since Visit 1)

submission of all remaining daily stool specimens collected from the infant

memory aide reviewed with the parent and collected

payment for time and travel provided for the study procedures

ensure parent has written plan on how to receive next dose(s) of rotavirus vaccine

administer parent/guardian satisfaction survey
6 STUDY PROCEDURES
6.1
Rotavirus Vaccine Randomization and Administration
Infants will be randomized to receive 1) RV1 alone, 2) RV5 alone, 3) RV1 with routine immunizations
(DTaP,  Hib, PCV13, HBV and IPV or 4) RV5 with routine immunzations prior to Visit 1. They will
receive the vaccination(s) after the MRI/Ultrasound is completed at Visit 1.
6.2
Breast Milk Collection
Breastfeeding mothers will be asked to collect one breast milk sample to bring to Visit 1 to assess anti-
rotavirus IgA antibody ELISA and the secretor status of the mother. Approximately 30 mL of breast milk
will be collected at home prior to the study visit. Collection via hand expression or breast pump is
acceptable. The breast milk sample should be collected up to 24 hours prior to the clinic visit and stored
and transported with an ice pack. Once delivered to the lab, the whey will be separated from the sample,
and the breast milk will be frozen at a minimum of -20°C. If unable to collect this sample at Visit 1, it can
be collected at Visit 2 or 3.
6.3
Saliva Collection
At Visit 1, a saliva specimen will be collected for secretor genotyping by placing sterile cotton-tipped
swabs in the infant’s mouth, saturating it with saliva and placing it in the Oragene (DNA Genotek)
solution. Once mixed with Oragene, the DNA in saliva is stabilized and can be stored indefinitely at room
temperature. If unable to collect this sample at Visit 1 , it can be collected at Visit 2 or 3.
6.4
Stool Collection
Parents will be asked to collect bulk stool samples daily (Day 0-Day 14) to assess rotavirus vaccine virus
shedding patterns and stool cytokines. Cytokines will be tested for stools from Visit 1 and Visit 2 (stool
collected on the days of the MRI).  Vaccine virus shedding (qualitative) will be done on stools from Visit
1 through Visit 2 (stool collected on the day of the MRI). If the Visit 2 stool is positive then the additional
stools will be tested to determine the duration of shedding. If the Visit 2 stool is positive, a quantitative
PCR will be done on that stool.
Only bulk specimens will be collected. Stools may be collected in diapers and placed in collection bag.
Rectal swabs will not be used for stool collection because we have found that they are not a reliable
source for detection of rotavirus109. Specimens will be refrigerated or frozen within 8 hours of collection.
Parents who collect the specimen at home will be asked to freeze the specimen within 8 hours of
collection. Once delivered to the lab, bulk stool and extracts will be frozen at a minimum of -20°C.
6.5
Blood Collection
Experienced nurses will obtain a venous blood specimen (approximately 2-3 mL of blood) by
venipuncture (preferably) or by heel stick if venipuncture unsuccessful at Visit 1(pre-vaccination and
Visit 2 (post-vaccination) for cytokine and biomarker assays.

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6.6
MRI/Ultrasound Imaging
Using MRI, a non-invasive imaging method that does not use radiation, we will 1) assess intestinal wall
thickness, 2) lymph nodes and 3) intestinal motility patterns that may be associated with an increased risk
for intussusception. The MRI will be done at Visit 1 just prior to the ultrasound and rotavirus vaccination
and similarly at Visit 2 to ascertain for any changes in these three parameters between the two exams. A
trained MRI technician will conduct the procedures in accordance with standard practice.
At recruitment/enrollment, parents will be informed that to be included in the study, they will be asked to
keep their infant NPO for approximately 3 hours prior to Visit 1 and Visit 2 appointment time. Parents
will have a reminder call the day prior to each visit to confirm the study time and remind them to keep the
infant NPO. Research staff will meet the parent at the Welcome Center and escort the parent and child to
the CCHMC Imaging Research Center (IRC). The infant will not be sedated for the MRI; rather, the
infant will be fed and swaddled110,111. All images will be obtained on a 1.5T Philips MRI scanner located
in the IRC after appropriate metal screening and ear protection (ear plugs and MiniMuffs™ (Natus
Medical Incorporated, San Carlos, California).
The infants will not receive intravenous contrast. However, they will receive enteric contrast by mouth to
optimally distend the small bowel and therefore improve image quality. Thirty minutes prior to imaging,
each subject will be fed 20 mL/kg of a 3:1 mixture of VoLumen (Bracco Diagnostics) slowly over about
25 minutes. The infants will then be imaged supine in an appropriately sized coil such as a cardiac coil.
The approximate time in the MRI to obtain the images needed will be 30-60 minutes. Once started the
MRI procedure may be stopped if the baby becomes upset.  Once calm, the scan can be repeated as
necessary. If a clinically concerning finding is found on the images, the radiologist will notify the PI who
will contact the parents and primary care physician.
After completion of the MRI, an ultrasound of the abdomen will be done.  The non-invasive ultrasound
will be done to 1) assess intestinal wall thickness and 2) lymph nodes that may be associated with an
increased risk for intussusception. The ultrasound will be done at Visit 1 just after the MRI and prior to
rotavirus vaccination and similarly at Visit 2 to ascertain for any changes in these two parameters between
the two exams. A trained ultrasound technician will conduct the procedures in accordance with standard
practice. The ultrasound will take up to 30 minutes to complete
6.7
Rotavirus Vaccine Administration
No restrictions are placed on the infant's feeding before or after receipt of rotavirus vaccine (except as
needed for the MRI). Documentation of whether the child is being breastfed and/or type of formula will
be collected. While the efficacy of the rotavirus vaccine series is similar among breastfed and
nonbreastfed infants, breastfeeding could potentially be related to outcome so we will document feeding
practice at each visit and daily on the memory aid. Even though rotavirus vaccine can be administered to
infants with minor acute illness (e.g., mild gastroenteritis or mild upper-respiratory tract infection, with or
without fever), both minor and major acute illnesses will be exclusion criteria, as these conditions could
interfere with our outcome measures (gastrointestinal motility and anatomy and cytokine responses).
6.8
Reactogenicity Assessment
Reactogenicity will be assessed for both groups for fourteen days after vaccine receipt (Days 0-14) and
recorded by the parent/legal guardian on a provided memory aid. The events will include fever, fussiness
and gastrointestinal symptoms such as vomiting and diarrhea (number of stools per day and looser than
normal). Parents will be instructed not to take temperature after bundling and how to distinguish vomiting
from normal spitting. Data will be collected using review of memory cards with the parent. We have
found this to be accurate, simple and acceptable to the parents of young infants. The following
reactogenicity signs/symptoms will be collected:

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•
Diarrhea:  defined as 3 or more episodes of looser-than-normal stools in 1 day
•
Vomiting: 1 episode of vomiting (forceful expulsion of partially digested milk/food) in 1 day
•
Fever:  axillary temperature ≥ 99.5 degrees F (measured once daily or more frequently if perceived
fever)
•
Hematochezia: maroon stools or any evidence of blood in the stool-black and tarry stools or frank red
blood
•
Irritability
The following grading system will be used in evaluating the fever and systemic events during Days1-14
following vaccination:
Reaction
Mild (Grade 1)
Moderate (Grade 2)
Severe (Grade 3)
Fever (axillary)
≥99.5°F to ≤100.4°F
>100.4°F to ≤102.2
>102.2°F and higher
Diarrhea
3 looser than normal stools/
day
4-5 loose stools/ day
> 6 loose stools/ day
Vomiting
1 episode of vomiting/ day
2 episodes of vomiting/ day
≥ 3 episodes of vomiting/ day
Irritability
Crying more than usual
Some interference with normal
activity
Significant interference with
normal activity
Hematochezia
Any stools that are black and tarry; maroon in color; or, frank red blood-each episode will be reported
to infant’s medical provider immediately
6.9
Receipt of other Recommended Vaccines
At the final visit, Day 14, arrangements will be made to facilitate receipt of initial ACIP recommended
vaccines for 2 month olds108 if they have not already received them prior to Visit 1. See tables below to
reference the recommended ages and time intervals between vaccine doses:
Study Visit 1
Study Visit 3
Primary MD Visit
(not part of study)
RV Immunization
“2 month” Immunizations
“4 month” Immunizations*
6 weeks
8 weeks
12 weeks
7 weeks
9 weeks
13 weeks
8 weeks
10 weeks
14 weeks
9 weeks
11 weeks
15 weeks
10 weeks
12 weeks
16 weeks
11 weeks
13 weeks
17 weeks
12 weeks
14 weeks
18 weeks
*minimum of 4 week interval between immunizations
Recommended and minimum ages and intervals between vaccine doses
Vaccine and
dose number
Recommended age for
this dose
Minimum age for
this dose
Recommended interval to
next dose
Minimum interval
to next dose
HepB-1
Birth
Birth
1-4 months
4 weeks
HepB-2
1-2 months
4 weeks
2-17 months
8 weeks
DTaP-1
2 months
6 weeks
2 months
4 weeks
DTaP-2
4 months
10 weeks
2 months
4 weeks
Hib-1
2 months
6 weeks
2 months
4 weeks
Hib-2
4 months
10 weeks
2 months
4 weeks
IPV-1
2 months
6 weeks
2 months
4 weeks
IPV-2
4 months
10 weeks
2-14 months
4 weeks
PCV13-1
2 months
6 weeks
8 weeks
4 weeks

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PCV13-2
4 months
10 weeks
8 weeks
4 weeks
Assurance of Rotavirus Vaccine Series Completion with Same Brand
The ACIP recommends that the rotavirus vaccine series be completed with the same product whenever
possible (all RV1 or all RV5). We have established procedures to make every effort for children in the
study to complete the rotavirus vaccine series with the same brand used for the first dose in the study. We
will provide the parent and their infant’s health care provider with written and electronic/fax
documentation respectively of the dates, types, route, dose and manufacturer of vaccines given and will
enter these into the CCHMC electronic medical record (immunization section and encounter note) as well
as the Ohio State Registry IMPACT system. We will offer the parents to return to receive the same brand
dose if they do not receive their care at CCHMC and their primary care provider does not offer the same
brand. For children receiving their care at CCHMC, we will provide their primary care provider with the
same brand dose. These additional doses of rotavirus vaccines will not be considered a study
procedure.7
STUDY VACCINE PRODUCT
7.1
Study Product Description, Storage and Stability
See the following links for study product package inserts. Since the focus of the study is rotavirus
vaccine, we have provided details about dosage preparation and administration of RV1 and RV5. Other
standardvaccines will be administered according to standard practice.
https://www.gsksource.com/gskprm/htdocs/.../ROTARIX-PI-PIL.PDF
https://www.merck.com/product/usa/pi.../r/rotateq/rotateq_pi.pdf
7.2
Dosage, Preparation, and Administration of Study Product
RotaTeq®
Each 2-mL dose is supplied in a container consisting of a squeezable plastic, latex-free dosing tube with a
twist-off cap, allowing for direct oral administration (RotaTeq® 2009).
6.10  Study Compensation Parents will be compensated for their time,effort and travel while
in the study. The compensation provided will be based on the standard amounts decided on by CCHMC
for research subjects who undergo the procedures outlined in this study and the time involved in the clinic
visits and collection of samples and reactogenicity data at home.  Payments will be given in the form of a
debit card (ClinCard). We will load payment onto the ClinCard after each clinic visit that is completed.

$150 at 1st Clinic Visit

$150 at 2nd Clinic Visit

$100 at 3rd Clinic Visit

$50 if asked to come in for an unscheduled visit
If parent comes in for a clinic visit and the child is not eligible, they will receive $25. If unable to
complete the study, they will be paid for each study visit completed.

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To administer the vaccine:
1.
Tear open the pouch and remove the dosing tube.
2.
Clear the fluid from the dispensing tip by holding the tube vertically and tapping the cap.
3.
Open the dosing tube in 2 easy motions:
a.
Puncture the dispensing tip by screwing the cap clockwise until it becomes tight.
b.
Remove the cap by turning it counterclockwise
4.
Administer dose by gently squeezing the liquid into the infant's mouth toward the inner cheek
until the dosing tube is empty. (A residual drop may remain in the tip of the tube.)
If for any reason an incomplete dose is administered (e.g., infant spits or regurgitates the vaccine), a
replacement dose will not be administered. The infant should continue to receive any remaining doses in
the recommended series. There are no restrictions on the infant’s consumption of food or liquid, including
breast milk, either before or after vaccination with RotaTeq® (RotaTeq® 2009).
Rotarix®
Each 1-mL dose consists of a vial of lyophilized vaccine to be reconstituted with a liquid diluent in a
prefilled oral applicator, allowing for direct oral administration (Rotarix® 2009).
To administer the vaccine:
1.
Remove the vial cap and push the transfer adapter onto the vial (lyophilized vaccine).
2.
Shake the diluent in the oral applicator (white, turbid suspension). Connect the oral applicator to
the transfer adapter.
3.
Push the plunger of the oral applicator to transfer the diluent into the vial. The suspension will
appear white and turbid.
4.
Withdraw the vaccine into oral applicator.
5.
Twist and remove the oral applicator.
6.
Administer Rotarix® orally.
In the event that the infant spits out or regurgitates most of the vaccine dose, a replacement dose will not
be administered. There are no restrictions on the infant’s food or liquid consumption, including breast
milk, either before or after vaccination with Rotarix® (Rotarix® 2009).
7.3 Assessment of Subject Compliance With Study Product
The study vaccines, RotaTeq® and Rotarix®, will be administered by the study product administrator,
who is licensed to administer the vaccine (e.g., LPN [licensed practical nurse], RN [registered nurse], NP
[nurse practitioner], PA [physician assistant], or medical doctor [MD]), in the clinic. Study personnel will
record the administration time on the CRF and include whether it was a complete dose or a partial dose
due to regurgitation.
8 LABORATORY EVALUATIONS AND METHODS
8.1
Blood-Cytokine and Inflammatory Responses and Antibody Testing (IgG and
IgA)
Blood specimens from Visit 1 will be used for assessment of anti-rotavirus IgG antibody (maternal) and
anti-rotavirus IgA antibody to rotavirus and measurement of serum cytokine levels and on the day of the
second MRI (Visit 2) for measurement of serum cytokine levels and anti-rotavirus IgA. Blood for the
antibody assays will be drawn into a serum separator tube by venipuncture (2-3 mL). After collection, the
tubes will be kept at room temperature until the blood has clotted. The tubes will be centrifuged at room
temperature at approximately 1000-1300g (2,400-2,700 RPM) for 10-15 minutes to ensure the sample is
sufficiently separated. The serum will be removed using sterile glass or plastic pipets and aliquoted into

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appropriately labeled cryovials (approximately 3 x 0.5 mL). The serum aliquots will be stored at -20 °C or
colder until testing. The cytokines of interest will be assayed in a multiplex Luminex assay using the
serum samples (Nefertiti C. DuPont et al 2005). The following cytokines will be measured: IL-2, IL-6,
IL-7, IL-8, IL-15, INF-γ and TNF-α. In addition, a C-reactive protein (pre- and post-vaccination) will also
be done. Additional cytokines or biomarkers may be done should funding become available.
Maternal antibodies to rotavirus will be measured using an anti-rotavirus IgG ELISA assay developed and
qualified in the Laboratory for Specialized Clinical Studies (LSCS). The antibody enzyme immunoassay
will be used to detect and quantify rotavirus IgG antibody concentrations in human serum112. Each assay
will include a serially diluted 8-point standard curve generated by a reference standard consisting of a
human serum pool arbitrarily assigned as having 1000 Units/mL. The curve will be modeled using the 4-
parameter logistic fit regression function.  Samples will be tested at 4 different concentrations. Dilution-
corrected titers will be reported for the sample using the quantifiable range of the standard curve. Serum
anti-rotavirus IgA will be determined using the same assay but using an anti-human IgA conjugate.
8.2
Stool-RV1 and RV5 Shedding and Cytokine Responses
Stool specimens will be utilized for both assessment of rotavirus vaccine shedding patterns and
measurement of stool cytokines. For stool cytokine testing, stool specimens from Visit 1 and Visit 2 will
be used. For assessment of rotavirus vaccine virus shedding patterns, all collected stools will be
analyzed. Cytokine testing will be done on Visit 1and Visit 2 specimens only. All stool samples will be
kept frozen at -20 °C or colder until testing. For determination of rotavirus shedding and cytokine
responses, stools will be thawed and nucleic acids will be extracted.For cytokine determination, PCR will
be performed using Taqman gene expression assays from Life Technology for each of the cytokines of
interest. The following cytokines will be done: IL-2, IL-6, IL-7, IL-8, IL-15, INF-γ and TNF-α.
Additional biomarkers or cytokines may be done should funding become available.
8.3
Saliva-DNA Genotyping for Secretor Status
Extracted DNA from the saliva will be tested to determine the child’s secretor status by genotyping.
Assay plates can accommodate up to 96 subjects each. We anticipate a yield of 25-30 ug of DNA per
child using Oragene solution, which is more than sufficient for the purposes of this analysis. Secretor
genotype will be determined at CCHMC through testing for known single nucleotide polymorphisms of
the secretor gene (FUT2), 428G>A114.
8.4
Breast Milk-IgA Antibody and Secretor Status of the Mother
Breast milk samples will be used to assess anti-rotavirus IgA antibody and secretor status of the mother.
The anti-rotavirus IgA assay is a validated, standard ELISA assay that has been used for both RV1 and
RV5 clinical trials by our lab (LSCS)112. We will also evaluate the mother’s secretor status from breast
milk using the genotyping method described above114.
8.5
Future Testing
The informed consent form includes information about specimen storage and future use of the stored
specimens. Samples will be retained for possible future testing with a linkage to identifiers. The samples
will be stored locally at the site and local site investigators will have access to the samples. However,
before the use of these samples by any investigator(s), request for use of these samples will require
project approval by the CDC. The stool, saliva, serum, breast milk and DNA samples will be banked for
future testing beyond the defined scope of this project; the consent forms will specifically include consent
for banking samples for future use. Potential testing that could be done if funding were available includes
(but is not limited to) testing of additional biomarkers or cytokines in the stool and blood as well as
examining changes to the stool microbiome pre- and post-vaccination.

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9 IMAGING METHODS
9.1
MR Imaging
9.12
Methods
Anatomic imaging will be acquired with axial and coronal T2-weighted sequences. Motility will be
evaluated using dynamic high spatial resolution 2D balanced steady state free precession (bSSFP)
imaging. Coronal and axial balanced steady state images will be acquired during free breathing with an
acquired temporal resolution of approximately 0.6s/image over 85 seconds. Diffusion weighted imaging
will also be acquired using multiple b-values in order to evaluate any changes in diffusion restriction
within the bowel wall or mesenteric lymph nodes between the two MRI exams.
9.13
Image Interpretation
Anatomic imaging will be assessed by a pediatric radiologist with expertise in abdominal imaging (Dr.
Towbin). The well-distended distal/terminal ileum bowel wall thickness will be measured in 3 locations,
and a mean wall thickness value will be obtained on each exam. The largest visible right lower quadrant
mesenteric lymph nodes will be measured in short axis on each exam. Any secondary signs of
inflammation (perienteric fat stranding, engorged vasa recta, free fluid) will be documented on each
exam. After completion of the primary anatomic review by Dr. Towbin, a second pediatric radiologist
with expertise in abdominal imaging will perform the same review on a random subset of subjects in
order to determine inter-reader reliability.  The second reviewer will be blinded to the intial assessment as
well as to the time point of imaging.
Motility imaging will be assessed as follows: First, a quantitiative assessment will be performed. A
well-distended segment of distal/terminal ileum will be chosen by the first reviewer (Dr. Towbin).
Novel, semi-automated software, will be utilized to assess the coronal cine sequences in this
investigation. No pre-market submission to the FDA has been made for the software nor are there any
current plans to do so in the future. The semi-automated method computes a cross sectional area across
time and the creates a best fit curve to determine the frequency or frequencies of contraction.  After
reviewer 1 identifies the bowel segment of interest, a post processing assistant will draw a region of
interest on the bowel lumen on every image of the sequence. The application then creates a map of
luminal cross-sectional area over time. The area versus time waveforms can be submitted to secondary
analysis from which contraction frequency and other quantitative metrics of interest can be extracted.
Next a qualitative assessment will be performend.  The research assistant will create a movie file for each
subject, randomize the timepoints, and then insert the movie file into a PowerPoint presentation.  The two
reviewers, along with a third reviewer(a pediatric gastroenterologist with expertise in gut motility) will
review the images to determine which movie has faster motility, and for each movie, if the motility is
organized or disorganized. Finally, each of the three reviewers will assess the focal segment of the
distal/terminal ileum in the same manner.
9.2
Ultrasound Imaging
9.21
Methods
Anatomic imaging of the terminal ileum will be done by ultrasound by an ultrasound technologist.
9.22
Image Interpretation
Anatomic imaging will be assessed by a pediatric radiologist with expertise in abdominal imaging (Dr.
Towbin). The well-distended distal/terminal ileum bowel and cecum wall thickness will be measured in 3

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locations, and a mean wall thickness value will be obtained on each exam. The largest right lower
quadrant mesenteric lymph nodes will be measured in short axis on each exam. Any secondary signs of
inflammation (perienteric fat stranding, engorged vasa recta, free fluid) will be documented on each
exam.
After completion of the primary anatomic review, a second pediatric radiologist with expertise in
abdominal imaging will perform the same review on a random subset of subjects in order to determine
inter-reader reliability.  The second reviewer will be blinded to the intial assessment as well as to the time
point of imaging.
10
RANDOMIZATION, DATA MANAGEMENT, STATISTICAL PLAN
10.1
Randomization Plan
Upon meeting the inclusion and exclusion criteria, subjects will be randomized to one of the four vaccine
groups: 1) RV1 alone, 2) RV5 alone, 3) RV1 with routine immunizations (DTaP, Hib, PCV13, HBV and
IPV) or 4) RV5 with routine immunzations This will be a simple randomization using SAS® PROC
PLAN, which uses a random number generator to make assignments. Study statisticians will generate the
assignments in advance of any enrollment. After obtaining informed consent , an unblinded research
personnel will contact the statistician to obtain a specific subject’s assignment. Parents and all study
radiologists will be blinded as to which arm the child is randomized to until completion of the study.
Parents will learn which rotavirus the child received at the completion of Visit 3.  Parents will be
informed about the specific other vaccines used in the study. If there is an urgent medical need to unblind
during the study,  then this can be done by calling the on call study staff.
10.2
Data Management Plan
The Data Management Center (DMC) of the Division of Biostatistics and Epidemiology (DBE) will
provide full data management support for this study. In collaboration with the study team and the CDC,
case report forms will be developed. The DMC will develop the data capture system using a web-based
data collection system, REDCap, as the primary source of data entry and storage. REDCap is a software
toolset and workflow methodology for electronic collection and management of research and clinical trial
data developed by Vanderbilt University, with collaboration from a consortium of institutional partners
including the University of Cincinnati Academic Health Center. REDCap provides HIPAA-compliant and
21 CFR Part 11-ready audit trails for tracking page views, data manipulation and export procedures. A
copy of coded data with the unique study ID number but without personal identifiers will be sent to CDC
upon request; patient identifiers will be kept at CCHMC; no identifying information will be included on
any data sent to the CDC.
REDCap is hosted on a network specially designed to support the rigorous security and compliance
requirements of basic, clinical and translational research projects. Administered by the Division of
Biomedical Informatics (BMI), this network features multiple firewalls as well as a central facility for
managing hosted systems and users. The result is another layer of access control and audit capability on
top of what REDCap already provides.
10.3
Data Analysis Plan
Primary objectives - Statistical Analysis.
a) Descriptive statistics:

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Descriptive statistics will be calculated for all demographic, anthropometric, reactogenicity, serum, image
and fecal variables. For continuous variables, five-number (mean, SD, minimum, median, maximum)
summaries will be produced. For class (categorical) variables, frequencies and percentages will be
reported. Descriptive statistics will be calculated for the entire study population, as well as for each
vaccine group at each time point. Investigators will be advised by study statisticians about potential
outliers, or data entry issues, based on these summary statistics. To determine whether continuous serum,
image and fecal outcomes meet the assumption of a Gaussian (normal) distribution, the Shapiro-Wilk test
will be used. Where a significant departure from normality exists, an appropriate transformation of the
data will be employed prior to inferential analyses. If transformations are not successful, non-parametric
analyses will be used.
b) Inferential statistics
The primary analysis for this study is the comparison of anatomical outcome as measured by MRI between
(RV1, RV5 with and without other vaccines: 4 groups) and within (pre and post) study groups. The primary
anatomical outcome will be the terminal ileum thickness measured in mm. The change in this outcome
before and after vaccination will be compared for each study group (within group test) using a paired t-test,
while the between group difference at pre, post and on the difference between pre and post (change score)
will be initially analyzed using general linear model (GLM) where the ileum thickness (at pre, post and
change score) is modeled as a function of group (4-groups). Depending on the result of the F-test from the
GLM analysis, this will be followed by pairwise comparison between groups (6-comparisons). This will be
examined using a two sample t-test. If the difference between groups with and without other vaccines for a
given vaccine (RV1 or RV5) is not significant we might combine the categories and conduct a comparison
test for the outcome between the two vaccine types. If the primary outcome is not normal before or after
transformation we will use the non-parametric Kruskal-Wallis test to compare the groups. Although the
primary between group comparisons is based on the pre-post change score, we are conducting additional
analysis based on the pre and post data to get more insight about the distribution of the primary outcome
variable.
We will also examine the distribution of socio-demographic and other potential covariates among the
groups using a GLM analysis. If some covariates are differentially distributed among the groups, those
covariates will be controlled for in the primary analysis described above. Potential modifiers such as breast
milk or secretor status will be examined by modeling the primary outcome as a function of group, potential
modifier and the interaction between group and modifier. In this case the parameter of interest is the
coefficient of the interaction term, however it is possible that the interaction test might not be sensitive due
to sample size. Regardless we will examine and report the observed change between groups for each
potential modifier as a subgroup. For example we will report difference between groups in the primary
outcome for secretors and non-secretors separately.
To assess how vaccine shedding is related with the primary outcome by looking at the correlation between
the imaging outcome and the fecal shedding for each group (using sample obtained close in time with the
imaging). This will be examined using Pearson or Spearman correlation coefficients based on the normality
of the data. The resulting correlation coefficient from each group can be compared with each other
(pairwise) using Fisher’s transformation and compare the resulting z-scores. If the correlation are not
different we may then combine the groups and run the correlation across group to get an insight on the
relationship between shedding and imaging parameter.

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For all the other imaging parameters (cecum thickness, wave pattern and amplitude) similar analysis
described for the primary outcome (ileum thickness) will be conducted. For Lymph node presence a logistic
regression instead of GLM, and for the lymph node number, and wave frequency a Poisson regression will
be used.
For the cytokines outcome (IL2, IL6, IL7, IL8, IL15, TNF) similar analysis described for the primary
imaging outcome including examination of modifying variables and potential subsequent stratified analysis
will be conducted.
Power
Since sample-sizes were predetermined and since this is a pilot study, no power calculations were
conducted.  Effect sizes will be reported from the results, to aid in the development of future studies.
Feasibility Analysis
The proportion of children meeting the outlined feasibility benchmarks (Appendix 3) will be determined.
11
ASSESSMENT OF SAFETY
11.1
Adverse Events
RV1 and RV5 are recommended routinely for infants in the US and is consistent with normal standard of
care The risks associated with vaccination are not greater than those encountered in the performance of
routine medical care.
RV1 Adverse Events
Anticipated common (>5%) solicited adverse events related to the administration of the RV1 rotavirus
vaccine include (as per Package Insert) fussiness/irritability, cough/runny nose, fever, loss of appetite, and
vomiting115. There is also a small risk for intussusception as shown in recent studies31,32.
RV5 Adverse Events
Anticipated common adverse events related to the administration of the RV5 rotavirus vaccine include (as
per Package Insert) include diarrhea, vomiting, irritability, otitis media, nasopharyngitis, and
bronchospasm116. There is also a small risk for intussusception as shown in recent studies31,32.
Other vaccines – All vaccines used will be licensed and recommended for routine use by the ACIP.
Possible risks  are explained in the Vaccine Information Statements.
(http://www.cdc.gov/vaccines/hcp/vis/).
Events meeting Vaccine Adverse Event Reporting System (VAERS) reporting requirements will be
reported as indicated below.
As previously mentioned, there are minimal risks associated with venipuncture for obtaining research
related labs. Foreseeable adverse events associated with venipuncture area: mild, temporary discomfort at
the venipuncture site, bruising, and phlebitis.
MRI /Ultrasound Adverse Events
Subjects may have some discomfort due to the noise from the MRI scanner. Earplugs and
headphones and/or MiniMuffs will be used to decrease sound levels to <+99dB(dBA). Infants may

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become fussy/irritable from being NPO 4 hours prior to the MRI (NPO three hours prior to visit
and one hour for intake and preparation for the MRI).
Low density barium sulfate (VoLumen) will be used as an oral contrast agent for the MRI
enterography.  Barium sulfate is an inert substance and has an excellent safety profile with well
over 60 years of use in patients.  Barium is biologically inert and is not absorbed or metabolized by
the body and is excreted unchanged. The risk of an allergic reaction is 1 in 1 million and the risk of
a fatal reaction is 1 in 10 million (Appendix 3-Package Insert). There are no known risks from
ultrasound.
11.2
Safety Reporting: For Adverse Events
The investigator must report the following events to VAERS with a notification to the CDC Project
Manager by e-mail, including a copy of the report (VAERS form). In addition, these events will be
reported on the VAERS/AESI case report form:

Any adverse event listed by the vaccine manufacturer as a contraindication to further doses of the
vaccine such as a severe allergic reaction

Any adverse event listed in the VAERS Table of Reportable Events Following Vaccination found
using the following link:
https://vaers.hhs.gov/.../VAERS_Table_of_Reportable_Events_Following_ Vaccination.pdf

In the judgment of the PI, any unexpected reaction to the vaccine will report to VAERS as well as
the CDC Project Manager.
The PI and the CDC project manager will report adverse events to their respective IRBs in
accordance with institutional policies
12
POTENTIAL RISKS AND BENEFITS
Potential Benefits
There is not likely to be a direct benefit to the infants participating in this study. The information learned
from this research study may benefit other children receiving rotavirus vaccines and increase our
knowledge about why some babies get intussusception.
Potential Risks, Discomforts, Inconveniences and Precautions
This study poses no greater than minimal risk to the subjects. As defined in 45 US Code of Federal
Regulations (CFR) 46.102 (i), “Minimal risk means that the probability and magnitude of harm or
discomfort anticipated in the research are not greater in and of themselves than those ordinarily
encountered in daily life or during the performance of routine physical or psychological examinations or
tests.” The risks associated with rotavirus vaccination and other recommended vaccines are the same as
those encountered in the performance of routine medical care and are described in section 11.1.
Subjects will have their saliva specimen collected using a sponge provided by an Oragene collection
kit. The sponge may cause temporary dryness of the mouth that should not last longer than 5-10
minutes. Subject will also be asked to provide stool specimens by collection of soiled diapers.
There are minimal risks associated with venipuncture for obtaining research related labs. Foreseeable
adverse events associated with venipuncture area: mild, temporary discomfort at the venipuncture site,
bruising, and phlebitis.
The MRI uses a large magnet and a computer to take detailed pictures of the intestine. There is no
radiation in an MRI scan. There are no known risks to an intestinal MRI in babies when appropriate
procedures are used. MRI is routinely used in neonates and infants for certain conditions such as brain

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anomalies and gastroschisis. Subjects may have some discomfort due to the noise from the MRI scanner.
Earplugs and headphones and/or MiniMuffs will be used to decrease sound levels to <+99dB(dBA).
Infants may become fussy/irritable from being NPO prior to the MRI. There is a very small chance of
identifying clinically significant findings from MRI/Ultrasound imaging.   If this occurs, the parents and
the primary care physician will be notified.
Low density barium sulfate (VoLumen) will be used as an oral contrast agent for the MR
enterography.  Barium sulfate is an inert substance and has an excellent safety profile with well
over 60 years of use in patients.  Barium is biologically inert and is not absorbed or metabolized by
the body and is excreted unchanged. The risk of an allergic reaction is 1 in 1 million and the risk of
a fatal reaction is 1 in 10 million (Appendix package insert). There are no known risks for
ultrasound imaging.
The primary goals of this study are to evaluate potential mechanisms for intussusception by
randomizing children to receive either of the two licensed rotavirus vaccines. No serious (or
severe) adverse events are anticipated using these licensed products but we will collect adverse
event data as outline in section 11.
There is a potential risk of loss of confidentiality of data.

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APPENDICES
APPENDIX 1.  STUDY VISITS AND PROCEDURES
Study Days
Recruitment/
Enrollment
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Visit
Recruitment
1
2
3
Age
<11wks
6 wks to-
12 wks, 6 days
Informed Consent
X
Demographics
X
X
X
X
Medical History
X
X
X
X
Inclusion/Exclusion
Criteria
X
X
X
X
X
Explain & Distribute
Memory Aide
X
X
Axillary Temperature
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Reactogenicity
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Stool Instructions
X
X
X
X
Supplies Distributed
X
X
Review MRI Prep
X
X
X
Schedule
Appointment
X
X
X
Reminder Call
X
X
X
Infant Stool
Collection
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Randomized
X
X
MRI
X
X
Ultrasound
X
X
Stool Submission
X
X
X
Memory Aid Review
X
X
X
X
Blood Obtained
X
X
Breast Milk Sample
X
Saliva Sample
X
Vaccination
One of Four
Groups
Case Report Form
X
X
X
X
X
Vaccination Record
X
X
Parent Satisfaction
X

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APPENDIX 2. FEASIBILITY BENCHMARKS
Item
Goal
Willingness to enroll
10% of subjects who are screened and found eligible will enroll in the
study
Reason for non-enrollment
90% of those not enrolled will be asked for reason for refusal
Temperature monitoring
At least 70% of parents will check and record  their baby’s axillary
temperature on  at least 70% of the study days
Reactogenicity
At least 70% of the parents will complete the reactogenicity assessment for
70% of the days
Study visits
At least 70% of subjects will complete all study visits on time (per
protocol) and remain in the study until completion
Blood draws
At least 70% of the blood draws will be successfully completed per
protocol
MRI imaging
At least 70% of subjects will successfully complete both MRIs
Stool collection
At least 70% of parents will collect 70% of the 14 stool samples
Breast milk collection
At least 50% of breastfeeding moms will collect a breast milk sample
Saliva collection
At least 70% of subjects will successfully have saliva obtained
Receipt of 2 month
vaccinations
90% of infants will receive remaining immunizations within ACIP
guidelines
Remaining rotavirus doses
75% of infants will receive the second RV immunization with the same
RV vaccine given as the first dose

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APPENDIX 3.
VoLumen Package Insert

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APPENDIX 4.
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and neutralizing activity. J Pediatr Gastroenterol Nutr 2006;42:560-7.
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Jiang B, Patel M, Parashar U. Rotavirus vaccines for global use: what are the remaining issues
and challenges? Hum Vaccin 2010;6:425-7.
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Jiang V, Jiang B, Tate J, Parashar UD, Patel MM. Performance of rotavirus vaccines in developed
and developing countries. Hum Vaccin 2010;6:532-42.
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factors for natural infant intussusception in the United States. J Pediatr Gastroenterol Nutr 2010;51:458-
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Payne DC, Baggs J, Zerr DM, et al. Protective association between rotavirus vaccination and
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Imbert-Marcille BM, Barbe L, Dupe M, et al. A FUT2 gene common polymorphism determines
resistance to rotavirus A of the P[8] genotype. J Infect Dis 2014;209:1227-30.
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Huang P, Xia M, Tan M, et al. Spike protein VP8* of human rotavirus recognizes histo-blood
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Liu Y, Huang P, Tan M, et al. Rotavirus VP8*: phylogeny, host range, and interaction with histo-
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Tong M, McHardy I, Ruegger P, et al. Reprograming of gut microbiome energy metabolism by
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Wacklin P, Makivuokko H, Alakulppi N, et al. Secretor genotype (FUT2 gene) is strongly
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Rausch P, Rehman A, Kunzel S, et al. Colonic mucosa-associated microbiota is influenced by an
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Haber P, Patel M, Pan Y, et al. Intussusception after rotavirus vaccines reported to US VAERS,
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Mathur AM, Neil JJ, McKinstry RC, Inder TE. Transport, monitoring, and successful brain MR
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Windram J, Grosse-Wortmann L, Shariat M, Greer ML, Crawford MW, Yoo SJ. Cardiovascular
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Gautam R, Esona MD, Mijatovic-Rustempasic S, Ian Tam K, Gentsch JR, Bowen MD. Real-time
RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix and RotaTeq vaccine
strains in stool samples. Hum Vaccin Immunother 2013;10.
114.
Morrow AL, Meinzen-Derr J, Huang P, et al. Fucosyltransferase 2 non-secretor and low secretor
status predicts severe outcomes in premature infants. J Pediatr 2011;158:745-51.
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APPENDIX 5.
First Visit Instruction
Thank you so much for participating in our study. We will be calling you in
the next few days to schedule your baby’s study visits.  Below are the
instructions for the first visit. We will call you before the visit to go over all of
this with you. Do not hesitate to call us with any questions between now and
your scheduled visit.
Day before first study visit

We will call you the day before your first scheduled visit. If your contact information changes
please call us at 513-636-2479/513-303-8800 so we have the most up to date information. You
may also email us at IDstudies@cchmc.org

Put the cold pack you were given at enrollment in your freezer so it will be ready to use

If you are breastfeeding, collect about an ounce of breastmilk within 24 hours of your
appointment and keep in refrigerator.

We want you to bring a fresh refrigerated (not frozen) stool sample with you to the first visit.
Save a poopy diaper within 48 hours before the visit. Ideally we would like a diaper within 24
hours of the visit, but all babies do not have bowel movements every day! If baby has another
poopy diaper after you have saved one, you should throw away the first and bring the most recent
diaper to the visit. Place diaper in clear labeled bag and put in silver bag you were given at
enrollment. Label with date and time of collection and keep in refrigerator.
Day of first study visit

Do not feed baby for 3 hours before your scheduled visit time

Put the cold pack in the silver bag with the stool sample and bring with you, along with the breast
milk sample, if breastfeeding

Bring the baby’s social security number (if available), pacifier, empty bottle/nipple, formula or
breast milk and other items you may need. The visit will last approximately 3.5 hours

We will meet you at your appointment time in front of the gift shop, main hospital near the
location C welcome desk. If you are driving, park near the C elevators in the garage.  If you are
dropped off or coming by bus, enter thru C location doors in the main entrance circle on Burnet
Avenue

Any questions, do not hesitate to call us at 513-636-2479 or 513-303-8800 or email us
IDstudies@cchmc.org

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APPENDIX 6.
Second Visit Instruction
Thank you so much for completing your first study visit. We will be calling
you the day before your next visit to remind you of the appointment.  Below
are the instructions for what to do between now and your next visit.  Do not
hesitate to call us with any questions between now and your scheduled visit.
Beginning today until next visit

Fill out the memory aid everyday as instructed starting today. Take the temperature under your
baby’s arm every evening and record on the memory aid. Remember to document any
medications or doctor visits on the memory aid.

Save all poopy diapers in the dated labeled clear bags and keep all in silver bag in your
refrigerator.

We will call you the day before your next scheduled visit. If your contact information changes
please call us at 513-636-2479/513-303-8800 so we have the most up to date information. You
may also email us at IDstudies@cchmc.org

Put the cold pack you were given at enrollment back in your freezer so it will be ready to use
Day of second study visit

Do not feed baby for 3 hours before your scheduled visit time

Put the cold pack in the silver bag with  all of the poopy diapers and bring with you, along with
your memory aid

Bring the baby’s pacifier, empty bottle/nipple, formula or breast milk and other items you may
need. The visit will last approximately 3.5 hours

We will meet you at your appointment time in front of the gift shop, main hospital near the
location C welcome desk. If you are driving, park near the C elevators in the garage.  If you are
dropped off or coming by bus, enter thru C location doors in the main entrance circle on Burnet
Avenue

Any questions, do not hesitate to call us at 513-636-2479 or 513-303-8800 or email us at
IDstudies@cchmc.org

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APPENDIX 7.
Third Visit Instruction
Thank you so much for completing your second study visit. We will be calling
you the day before your next visit to remind you of the appointment.  Below
are the instructions for what to do between now and your next visit.  Do not
hesitate to call us with any questions between now and your scheduled visit.
Beginning today until next visit

Continue to fill out the memory aid everyday as instructed. Take the temperature under your
baby’s arm every evening and record on the memory aid. Remember to document any
medications or doctor visits on the memory aid.

Save all poopy diapers in the dated labeled clear bags and keep all in silver bag in your freezer.

We will call you the day before your next scheduled visit. If your contact information changes
please call us at 513-636-2479/513-303-8800 so we have the most up to date information. You
may also email us at IDstudies@cchmc.org

Put the cold pack you were given at enrollment back in your freezer so it will be ready to use
Day of third study visit

Put the cold pack in the silver bag with all of the poopy diapers and bring with you, along with
your memory aid. We must have both in order to complete the visit.

If your baby is a Pediatric Primary Care Clinic (PPC) patient, we will meet you at the PPC. IF
baby is not a PPC patient the appointment will be scheduled at the T1 research clinic and we will
meet you at the gift shop.

The study visit will take about 15 minutes. If scheduled in the PPC this will be followed by your
child’s regularly scheduled well check up with PPC staff.

Any questions, do not hesitate to call us at 513-636-2479 or 513-303-8800 or email us at
IDstudies@cchmc.org