A Double-Blind, Randomized, Placebo-Controlled Study of Antibody Induction by Vitamin Supplementation at the Time of Influenza Virus Vaccinations in Children
Phase: PHASE2
Conditions: Healthy Participants
Sponsor: St. Jude Children's Research Hospital

St. Jude 
FLUVIT 
Initial version, dated:  10-02-2015, (IRB Approved: 12-02-2015) 
Revision 0.1, dated: 12-04-2015, (IRB Approved: 12-15-2015) 
Revision 0.2, dated: 01-05-2016, (IRB Approved: 01-08-2016) 
Amendment 1.0, dated: 07-13-2016, (IRB Approved: 08-16-2016) 
Revision 1.1, dated 08-30-2016, (IRB Approved: 9/30/2016) 
Revision 1.2, dated 01-05-2017, (IRB Approved: 02-01-2017) 
Activated: 02-07-2017 
Revision 1.3, dated 06-08-2018. (IRB Approved: 06-14-2018) 
Activated: 
 
A DOUBLE-BLIND, RANDOMIZED, PLACEBO-CONTROLLED STUDY OF 
ANTIBODY INDUCTION BY VITAMIN SUPPLEMENTATION AT THE TIME 
OF INFLUENZA VIRUS VACCINATIONS IN CHILDREN 
 
 
Principal Investigator 
Nehali Patel, MD 
Department of Infectious Diseases 
 
 
 
Faculty Sponsor 
Julia Hurwitz, PhD 
Department of Infectious Diseases 
 
 
 
Sub-Investigators 
Ronald Dallas, PhD1 
Li Tang, PhD2 
Yilun Sun, MS2 
Rhiannon Penkert, PhD1 
 
 
 
 
 
1Department of Infectious Diseases 
2Department of Biostatistics 
 
 
 
 
St. Jude Children's Research Hospital 
262 Danny Thomas Place 
Memphis, Tennessee  38105-3678 
Telephone:  (901) 595-3300 
 
 
Contents of this document may not be extracted without permission from the Principal Investigator. 

 
Revision 1.3, dated: 06/08/2018 
IRB Approval date: 06/14/2018 
Protocol document date: 06/08/2018 
 
 
 
Protocol MNEMONIC and Title: FLUVIT - A double-blind, randomized, placebo 
controlled study of antibody induction by vitamin supplementation at the time of 
influenza virus vaccinations in children 
Principal Investigator: Nehali Patel, MD 
Brief Overview: Respiratory virus infections are responsible for more than 4.5 million 
deaths each year worldwide. Among these, up to 0.5 million deaths are caused by 
influenza viruses. Children are particularly vulnerable to respiratory virus infections, and 
extensive campaigns now target their improved healthcare. Vaccination is the single best 
strategy for the prevention of infectious diseases. Despite the clear benefit of respiratory 
virus vaccines, efficacy rates can be disappointing.  
Vitamin A and D deficiencies are each associated with impaired innate and humoral 
immune responses and vulnerability to respiratory tract infectious diseases. Rates of 
vitamin A and D deficiencies or insufficiencies have skyrocketed in the United States, 
especially in the mid-south region. Animal studies have demonstrated that in vitamin 
deficient mice, antibody immune responses are poor toward influenza virus vaccine and 
are improved if a vitamin supplement is administered at the time of vaccination. The 
central hypothesis of this protocol is that vitamin supplements will enhance antibody 
responses toward an influenza virus vaccine in children. Children will be randomized 
using a stratified permuted block method to receive  an influenza virus vaccine with 
vitamin A+D supplement or placebo. Participants will be stratified based on retinol 
binding protein (RBP) levels at screening, using a cut-off  indicative of vitamin A 
insuffiency (<22,000 ng/ml). Co enrolled sibling participants will be first stratified by 
RBP levels, then siblings within a same stratum will be equally assigned to different arms 
to provide greater assurance of balanced treatment assignment. Children will be tested for 
vitamin levels and immune responses before and after influenza virus vaccinations to 
determine if vitamin supplementation improves the vaccine-induced antibody response.   
Intervention: Children will be randomized to receive  influenza virus vaccine plus 
vitamin A and D supplements (intervention) or Influenza virus vaccine plus matched 
placebo. 
Brief Outline of Treatment Plan: Children between the ages of 2 and 8 years will be 
enrolled. All will receive two doses of  an influenza virus vaccine administered at least 
28 days apart. Vitamin levels and antibody responses toward the vaccine will be 
measured on days 0 (baseline levels), 28, and 56. Vitamins A + D will be orally 
administered at the levels of 20,000 IU and 2,000 IU respectively on the days of 
vaccinations for the children randomized to receive vitamin supplements. Parents will be 
asked to fill out diary cards to indicate food intake for children (intervention & control) 
over the study period along with an optional food frequency questionnaire given on day 

 
Revision 1.3, dated: 06/08/2018 
IRB Approval date: 06/14/2018 
Protocol document date: 06/08/2018 
 
Protocol MNEMONIC and Title: FLUVIT - A double-blind, randomized, placebo 
controlled study of antibody induction by vitamin supplementation at the time of 
influenza virus vaccinations in children 
56. Specific measurements on days 28, and 56 will include analyses of vaccine-specific 
and total antibody, IgA, IgG, and IgA/IgG plus IgA/IgM ratios in sera. Functional 
activities of antibodies toward the vaccine will also be measured. Data analyses will 
indicate effect size for the design of a larger, clinical analysis. 
Study Design: Stratified permuted block, double-blind randomized controlled trial of 
influenza virus vaccine + vitamin A & D supplements, vs. influenza virus vaccine + 
matched placebo.  
Table. Subject Allocation by Intervention vs. Control 
Group 
Influenza virus 
vaccine Dose 
Vitamin A Dose 
 
Vitamin D Dose 
 N 
Intervention 
per 
manufacturer’s 
specification 
20,000 IU 
2,000 IU 
40 
Control 
 per 
manufacturer’s 
specification 
Placebo 
Placebo 
40 
 
 
Sample Size: Approximately 100 participants enrolled over 2 flu seasons. 
Data Management:  The Department of Infectious Diseases will design forms for data 
collection. The Department of Biostatistics will provide statistical analysis. 
Human Subjects:  The risks to human subjects are considered minimal. Possible risks 
to the participant include those associated with the licensed influenza virus vaccine 
product or when blood is drawn. Parents/guardians will be informed that the study is 
experimental.  

 
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TABLE OF CONTENTS 
1.0 
OBJECTIVES ...................................................................................................................3 
1.1. Primary Objective ................................................................................................... 3 
1.2. Secondary Objectives.............................................................................................. 3 
1.3. Exploratory Objectives ........................................................................................... 3 
2.0 
BACKGROUND AND RATIONALE .............................................................................3 
2.1. Background ............................................................................................................. 3 
2.2. Rationale ................................................................................................................. 4 
3.0 
RESEARCH PARTICIPANT ELIGIBILITY CRITERIA AND STUDY 
ENROLLMENT .............................................................................................................12 
3.1. Inclusion Criteria .................................................................................................. 13 
3.2. Exclusion Criteria ................................................................................................. 13 
3.3. Research Participant Recruitment and Screening ................................................. 14 
3.4. Enrollment on Study at St. Jude ............................................................................ 14 
3.5. Procedures for Identifying and Randomizing Research Participants ................... 15 
4.0 
TREATMENT PLAN .....................................................................................................15 
4.1. Treatment .............................................................................................................. 15 
4.2. Contraindications to an influenza virus vaccine ................................................... 17 
4.3. Concomitant Therapy............................................................................................ 17 
4.4. Study Visit Flow Diagram .................................................................................... 18 
5.0 
DRUG/DEVICE/BIOLOGIC AGENT INFORMATION ..............................................18 
5.1. Influenza Virus Vaccine ....................................................................................... 18 
5.2. Vitamin A and D3 Nutritional Supplements, Oral ................................................ 18 
5.3. Matched Placebo ................................................................................................... 19 
6.0 
REQUIRED EVALUATIONS, TESTS, AND OBSERVATIONS ...............................19 
6.1. Screening Visit ...................................................................................................... 20 
6.2. Day 0 (Vaccine dose #1) Visit .............................................................................. 20 
6.3. Day 28 (Vaccine dose #2) Visit ............................................................................ 21 
6.4. Day 56 Follow-Up Visit........................................................................................ 21 
6.5. Special Instructions and Definitions of Evaluations ............................................. 22 
7.0 
EVALUATION CRITERIA ...........................................................................................23 
7.1. Toxicity Evaluation Criteria ................................................................................. 23 
7.2. Acceptable Percentage of Missed Doses for Research and Standard of Care Drugs ...... 23 
8.0 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF-STUDY 
CRITERIA ......................................................................................................................23 
8.1. Off-study criteria ................................................................................................... 23 
9.0 
SAFETY AND ADVERSE EVENT REPORTING REQUIREMENTS .......................24 
9.1. Reporting Adverse Experiences and Deaths to St. Jude IRB ............................... 24 
9.2. Recording Adverse Events and Serious Adverse Events ...................................... 27 
9.3. Un-blinding Procedures ........................................................................................ 28 
10.0 
DATA COLLECTION, STUDY MONITORING, AND CONFIDENTIALITY..........30 
10.1. Data Collection ..................................................................................................... 30 
10.2. Study Monitoring .................................................................................................. 30 
10.3. Confidentiality ...................................................................................................... 31 
11.0 
STATISTICAL CONSIDERATIONS............................................................................31 
11.1. Anticipated Completion Dates .............................................................................. 31 
11.2. Sample Size ........................................................................................................... 32 

 
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11.3. Participant Accrual, Follow-up and Retention...................................................... 32 
11.4. Summary of Primary and Secondary Objectives .................................................. 33 
12.0 
OBTAINING INFORMED CONSENT .........................................................................35 
12.1. Informed Consent Prior to Research Interventions ............................................... 35 
12.2. Consent at Age of Majority................................................................................... 35 
APPENDIX I: SCHEDULE OF EVALUATIONS ....................................................................41 
APPENDIX II: CLINICAL AND RESEARCH TESTS ............................................................42 
APPENDIX III: TOXICITY GRADING TABLES ...................................................................43 
 
 
 
 

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1.0 OBJECTIVES 
1.1. Primary Objective 
To assess the vaccine-induced and total antibody (including IgG and IgA) 
response after influenza virus vaccine administration and IgA/IgG plus IgA/IgM 
ratios at 28 and 56 days in sera 
1.2. Secondary Objectives 
To assess the functional antibody response toward influenza virus vaccine in the 
sera 
1.3. Exploratory Objectives 
To describe the socio-demographic characteristics and diet among children 
participating in the trial.  
 
To assess the feasibility of the food diary among healthy volunteers as measured 
by completion of the diary. 
 
To monitor serum Vitamin A and D levels at various time points. 
2.0  BACKGROUND AND RATIONALE 
2.1. Background 
Respiratory virus infections are the leading causes of morbidity and mortality in 
children (1). The risk of death by respiratory disease is approximately 2:1 in 
vitamin A deficient children compared to controls (2). Low Vitamin D is similarly 
associated with increased morbidity and mortality due to respiratory disease (3-6). 
Vaccination is the best way to prevent childhood infections, but many respiratory 
virus vaccine candidates have failed in clinical trials. Furthermore, the currently 
licensed influenza vaccines are not fully protective in children. A recent study by 
MedImmune reported that approximately 10% to  50% of children between the 
ages of 2 to 17 years who received the quadrivalent FluMist vaccine had evidence 
of immune responses towards any one of the influenza virus A and B components, 
respectively (7).  

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Nutrition and the immune response. Basic studies have shown that vitamins A 
and D each play a key role in the differentiation of T cell subsets, the proper 
migration (“homing”) of cells into tissues, and the development of protective 
antibody responses (3, 8-10). Antibodies, a first line of defense against influenza 
virus at its point-of-entry (11), is impaired when individuals are vitamin deficient 
(12). Epidemiologic studies confirm that deficiencies of vitamin A and vitamin D 
impact the immune response (3-6;13-15).  
While vitamins A and D are often studied separately, their activities are cross-
regulated. They differentially bind heterodimeric receptor complexes (RAR-RXR, 
VDR-RXR), which in turn bind DNA sequence motifs in or near promoter 
regions of immune response genes (3; 16-17). The cross-regulatory capacities of 
vitamins A and D are often overlooked, perhaps explaining in part why past 
clinical supplementation studies with one vitamin or the other have yielded 
conflicting results (15;18-24). No clinical study has comprehensively evaluated 
the influences of vitamins A and D, independently or together, on responses 
toward influenza virus vaccines.  
This project will test the central hypothesis that vitamin A+D supplementation in 
children will improve systemic antibody responses toward an influenza virus 
vaccine. Our studies are expected to demonstrate a logistically feasible method to 
improve  influenza virus vaccine efficacy. Ultimately, our data may instruct new 
healthcare initiatives to improve vaccination against numerous respiratory 
pathogens, and thereby reduce morbidity and mortality in children. 
2.2. Rationale 
Vitamin deficiencies and insufficiencies are widespread among children in the 
United States (3;25-27). Based on results from our mouse model (please see 
preliminary data), we hypothesize that children with low vitamin levels will 
exhibit poor antibody responses toward the influenza virus vaccine, but that 
antibodies (including IgG and IgA),  IgA/IgM, and IgA/IgG1 ratio measurements 
will be improved among children who receive vitamin A and D supplements at 
the time of vaccination. To test our hypothesis, we will randomize children who 
are receiving the influenza virus vaccine into two groups. One group will receive 
oral vitamin A+D supplements on the days of vaccination while the other group 
will only receive the influenza virus vaccine. If we find that supplements enhance 
antibody responses, we will (i) provide an explanation for the low efficacy of 
respiratory virus vaccines in humans, and (ii) provide evidence that vaccine 
efficacy can be easily improved. By optimizing the influenza virus vaccine and 

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other respiratory virus vaccine programs, we may ultimately reduce the morbidity 
and mortality caused by respiratory infections in children. 
Vitamin deficiencies and insufficiencies. Memphis, TN, the site of St. Jude 
Children’s Research Hospital (St. Jude), is a mid-south city with many families of 
low socioeconomic status and high vulnerability to infectious disease. A 
published study of adults in a general internal medicine practice at the University 
of Tennessee demonstrated very low vitamin levels in Memphis (25).  We also 
examined (i) individuals from Memphian families who had at least one influenza 
virus-infected member, and (ii) individuals from the Tennessee Blood Services 
for levels of vitamin A using the surrogate retinol-binding protein [RBP, (28)]. 
RBP is generally present at a 1:1 molar ratio with retinol. We also measured 
vitamin D levels. As shown in Figure 1, we found that approximately ½ 
individuals exhibited deficiencies or insufficiencies in vitamin A based on IOM 
standards (vitamin A deficiencies= ≤0.7μM or ≤15,400 ng/ml RBP; 
insufficiencies = >0.7 to ≤1.05 μM  or >15,400 to <22,000 ng/ml RBP). Most 
individuals were deficient in vitamin D [25(OH)D] based on IOM standards (<20 
ng/ml (http://ods.od.nih.gov/ factsheets/VitaminD-HealthPorfessional)).  No one 
in the study exhibited sufficiency for vitamin D, based on reports that certain 
tissues require >30 ng/ml, an opinion reflected by the practice guidelines of the 
US Endocrine Society (30-31).   
 
 
Figure 1, Low vitamin levels in Memphis, TN. Samples from households in 
which at least one individual was influenza virus infected (32) and from the 
Tennessee Blood Services were tested for human RBP4 (Quantikine ELISA Kit, 
Panel) and for vitamin D in the Pathology Department at St. Jude using a Roche 
Elecsys Vitamin D assay that measures 25-hydroxylated metabolites of 
cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). Black and dotted 
lines indicate cut-offs for vitamin insufficiencies and deficiencies respectively.  
 

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Pre-clinical studies of vitamin A and D deficiencies and the immune response 
toward viral vaccines. 
 
To study the impact of vitamin deficiencies on respiratory virus vaccine 
immunogenicity, we established vitamin deficient mouse models. Briefly, 
C57BL/6 pregnant mice were placed on vitamin deficient or control diets at 4-5 
days gestation (Harlan diets). Pups were born and maintained on the diets until 
they reached adulthood and throughout the course of experiments. Vitamin D 
deficient mice were housed under LED lights. 
 
 
Analyses were first conducted to monitor CD8+ T cell recruitment to the 
respiratory tract airway following immunization with low dose (250 EID50) 
Sendai virus (SeV, a parainfluenza virus type I of mice) in vitamin deficient mice. 
The frequencies of virus-specific CD8+ T cells in the airways were significantly 
reduced compared to controls when there were deficiencies in vitamins A or D 
[(33) and unpublished data].  
 
Experiments were then focused on the study of respiratory virus vaccines and 
antibody responses in vitamin deficient mice. We found that vaccine-specific IgA 
antibody forming cells and IgA responses in the upper respiratory tract were 
significantly reduced in animals with vitamin A deficiencies (34), and were 
further reduced in animals with vitamin A+D deficiencies. This demonstrated a 
failed first line of immune defense at the pathogen’s point-of-entry (11-12). 
 
Figure 2. Vitamin A+D double deficiency impairs IgA more than a single vitamin 
deficiency. Mice were reared on a diet deficient in vitamin A (VAD) or both 
vitamins A+D (VAD+VDD). As adults, mice were vaccinated with a cold-
adapted influenza virus vaccine. After one month, mice were evaluated for IgA-
producing AFCs (panel A) or IgA in nasal washes (NW) diluted 1:10, 1:50 and 
1:250 prior to assay (panel B). Averages are shown.   
 
A.                           B. 

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To determine if failed immune responses could be corrected, we vaccinated mice 
with influenza virus vaccine and supplemented mice with vitamins on days 0, 3, 
and 7 relative to vaccination (35). As shown in Figure 3, the IgA responses were 
extremely low in animals that were deficient for vitamins A+D (clear bars; VAD 
+ VDD) compared to controls (black bars). IgA responses were partially corrected 
in double deficient animals when they received either vitamin A or vitamin D. 
However, the best correction was when animals received both vitamins A and D 
(hatched bars; +Vit A and D).  
 
 
Figure 3. Oral vitamin supplements correct IgA responses in double deficient 
mice. Vitamin A (retinyl palmitate, 600 IU) and vitamin D (cholecalciferol, 40 
IU) were administered to mice deficient in vitamins A and D on days 0, 3, and 7 
relative to vaccination with a cold-adapted influenza virus vaccine. Mucosal IgA 
responses were examined 1 month later. IgA responses were examined in nasal 
washes. Shown are averages for each mouse set, with sample dilutions of 1:10. 
1:50, and 1:250.  
 
To answer the question of benefit of vitamin supplementation with intramuscular 
influenza vaccine, we injected mice i.m. with an inactive H1N1 influenza virus 
vaccine. Each mouse received two i.m. injections with a 4 week interval. Half of 
the mice received 600 IU retinyl palmitate on Days 0, 3, and 7 relative to both the 
priming and boosting doses of vaccine.  Animal sera were evaluated for function 
with a hemagglutination inhibition assay (HAI) against influenza virus. Samples 
were tested before vaccination, and after both prime and booster injections. As 
demonstrated in Figure 4, animals that received vitamin supplements generated 
significantly improved influenza virus-specific antibody responses compared to 

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controls. Results were striking in that no mice responded to the priming vaccine 
dose, except for mice that received vitamin supplements. After the booster 
vaccine, vitamin supplemented mice exhibited significantly improved antibody 
titers compared to controls. Altogether, data demonstrate that vitamin 
supplements improve antibody responses when animals are vaccinated either with 
a replication-competent or inactive influenza virus vaccine, and either by the i.n. 
or i.m. route. 
 
1 2 3 4 5
1 2 3 4 5
1 2 3 4 5
1 2 3 4 5
0
20
40
60
80
Control-No Vac
Vitamin Supplemented-No Vac
Control-Vac
Vitamin Supplemented-Vac
p<.05
Mouse Number
Hemagglutination Inhibition Titer
1 2 3 4 5
1 2 3 4 5
1 2 3 4 5
1 2 3 4 5
0
50
100
150
200
250
Control-No Vac
Vitamin Supplemented-No Vac
Control-Vac
Vitamin Supplemented-Vac
ND
p<.05
Mouse Number
Hemagglutination Inhibition Titer
 A.  Mice primed i.m. with flu vaccine                      B. Mice boosted i.m. with flu vaccine
 
Figure 4. Vitamin supplements improve immune responses to an i.m. 
influenza virus vaccine. HAI titers are shown for unvaccinated and vaccinated 
mice. Results from vaccinated, control mice are shown as hatched bars and results 
from vaccinated, vitamin-supplemented mice are shown as solid bars. Responses 
are shown after the priming vaccine dose (A), and after the booster (B).  In both 
cases, the vitamin supplements significantly improved antibody activities (Mann 
Whitney Tests). ND = not done 
 
Human samples were next examined to determine if vitamins A and D associated 
with antibodies. Samples were available for this study from the Memphian 
families affected by influenza virus infections and from the Tennessee Blood 
Service (Memphis, TN). Strong associations were revealed between RBP and 
isotype patterns. There were significant associations between RBP and IgA, 
IgA/IgM, IgA/IgG1, and IgG4 (Figure 5). There were also associations between 
RBP and neutralizing antibody titers toward influenza virus (data not shown). 
These results are exciting and supportive of our previous findings in vitro and in 
mice. 
 
 

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Figure 5. RBP associates with isotypes represented by antibody heavy chain 
genes located at the 3’ side of the immunoglobulin heavy chain locus. Blood 
samples were examined for RBP levels by ELISA and for total immunoglobulin 
isotypes using Luminex technology (HGAMMAG-301K Map human isotyping 
magnetic bead panel, Milliplex, Millipore, St. Charles, MO).  
 
Taken together, our preliminary experiments demonstrate that vitamin A (based 
on RBP levels) and vitamin D deficiencies are common in Memphis and that 
vitamin A levels correlate with IgA levels in mice and humans. In the mouse 
model, the vitamin A and D deficient animals exhibited extremely low IgA levels, 
which could be corrected when mice received vitamin supplements at the time of 
vaccination.  
 
Results strongly encourage a test of vitamin supplementation in Memphis residing 
children who have low vitamin levels and who will receive  an influenza virus 
vaccine. Improvements in  influenza virus vaccine efficacy may ultimately protect 

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children and adults from the morbidity and mortality caused by influenza virus 
disease. Thus, efforts are underway to examine the efficacy of Vitamin A & D 
supplementation in conjunction with children receiving the  influenza virus 
vaccine.  
 
 
Rationale for Two Doses of influenza virus vaccine 
 
Current guidelines  are based on the fact that many children younger than 9 years 
of age have not been infected with influenza viruses previously, and a booster 
dose is needed for them to produce a protective immune response (36). Previous 
research among healthy children has also demonstrated the efficacy of two doses 
of influenza vaccine in preventing laboratory-confirmed cases of influenza illness 
(37-38). Based on previous evidence and the current guidelines supporting the 
administration of two doses of influenza vaccine among young children, this 
study will require two doses of influenza virus vaccine for all participants.   
 
Rationale for Vitamin A and Vitamin D Dose 
 
Consistent with previous literature and our pre-clinical data (as detailed above), 
high dose vitamin supplementation will be utilized as part of this study (20,000 IU 
per dose of vitamin A and 2,000 IU per dose of vitamin D). A dose that is 10 
times the recommended daily allowance (RDA) at the tolerable upper intake level 
(UL) will be used for vitamin A. The chosen vitamin A dose is unlikely to cause 
adverse effects as it is well below that recommended by the WHO for children in 
developing countries. According to UNICEF’s guidelines “the World Health 
Organization (WHO) has established dosage guidelines; each child between 6 and 
11 months is to receive 100,000 international units (IU) of vitamin A. This 
increases to 200,000 IU every six months from 12 to 59 months of age” (40). 
Vitamin D levels will be maintained at the UL (41), because although high-dose 
vitamin D has been successfully studied in humans, and a combination of vitamin 
A and D supplements support IgA production in research animals deficient in 
both vitamins, the correlation between vitamin D and IgA is not convincing in our 
human study. In addition, high-dose vitamin D can yield paradoxical effects, and 
vitamins A and D can act as antagonists to one another (42-44) 
 
Additionally, we have conducted vitamin studies in vitamin A deficient mice 
(VAD) and in mice deficient for both vitamins A and D (VAD+VDD).  Caveats 
to the direct translation of results from these mice to humans are: (i) the vitamin A 
content of a normal mouse diet is approximately 15X the RDA of humans based 

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on weight, (ii) vitamin A was completely withheld from our test mice throughout 
their development prior to vaccination, (iii) the vaccinations in mice were either 
with influenza virus vaccine, a mouse parainfluenza virus, or a cold-adapted, 
mouse-adapted influenza virus (PR8), (iv) supplements were administered to 
VAD mice by either the intranasal or oral route, and (v) mice were anesthetized 
when vaccinated, a condition conducive to vaccine transmission to the lower 
airway. We found that a dose of 600 IU retinyl palmitate (high dose) in a ~20 
gram mouse (approximately 30,000 IU/kg), corrected an influenza virus vaccine-
induced IgA response when administered on Days 0, 3, and 7 relative to 
vaccination. The same dose corrected responses when administered intranasally 
on Day 0 to mice that received a parainfluenza virus vaccine. In the latter case, 
significant, but incomplete improvements were also observed with doses of 60 IU 
(3,000 IU/kg, medium dose) and 6 IU (300 IU/kg, low dose), the latter being 
comparatively lower than the vitamin A dose proposed here for the clinical study 
(45).  
 
More recently, we conducted additional experiments in which mice deficient in 
both vitamins A and D were given a cold-adapted PR8-based influenza virus 
vaccine and supplemented orally. When mice received 600 IU retinyl palmitate 
and 40 IU vitamin D orally on Days 0, 3, and 7 relative to vaccination, the IgA 
response was rescued. Responses were less well improved when 600 IU retinyl 
palmitate or 40 IU vitamin D were used alone. In another study, mice received 
600 IU retinyl palmitate (~30,000 IU/kg) and 40 IU vitamin D, or 60 IU retinyl 
palmitate and 40 IU vitamin D, or 6 IU retinyl palmitate and 4 IU vitamin D 
orally on Day 0 relative to vaccination with the cold-adapted PR8 influenza virus 
vaccine. The highest dose was most effective at correcting the vaccine-induced 
IgA response. The medium and lower doses were less effective. To balance safety 
and efficacy and using the 15X correction factor for human versus mouse diets, 
we chose a vitamin A dose for young children that converts to ~450 IU in a 
mouse based on weight, falling between the medium and high doses described 
above. 
      
Our long-term goal is to identify a logistically-feasible public health strategy to 
provide all children a vitamin supplement in conjunction with the influenza virus 
vaccine to enhance their immune protection against influenza virus disease. A 
dose of 10X the RDA UL given once per month should not pose risk to children 
who have sufficient levels of vitamins. As stated above, the vitamin A dose by 
weight is well below that recommended by the WHO for children in developing 
countries (each child between 6 and 11 months may receive 100,000 international 

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units (IU) of vitamin A; from 12-59 months of age, children may receive 200,000 
IU every six months). If a child of 1 year of age and 10 kg receives a single dose 
of 200,000 IU vitamin, the dose/kg is 20,000 IU/kg, as opposed to the 1500 IU/kg 
proposed here.  Despite previous indications of vitamin safety, there are also 
studies that have indicated risk in the context of high vitamin A or D intake. 
Penniston and Tanumihardjo describe acute toxicities when children ingest >20X 
the vitamin A RDA over a period of hours or a few days (46). Tolerance to high 
vitamin intake may be variable among children (47). A dose of 25,000 IU 
increases the incidence of transient bulging of the fontanelle in infants and a dose 
of greater than 25,000 IU in pregnant females is discouraged at certain periods of 
gestation due to a risk of teratogenicity (48). These and other studies were likely 
considered by the IOM when defining UL RDA guidelines. Vitamins A and D 
bind nuclear receptors (e.g. RAR-RXR, VDR-RXR, PPAR-RXR, LXR-RXR, and 
TR-RXR) that in turn bind DNA, impacting transcription at a plethora of loci and 
influencing a wide variety of cell functions. As stated above, the vitamins and 
receptors can be agonists or antagonists of other receptors and of one another (42-
44).  The vitamin A dose proposed here (20,000 IU) is below doses previously 
associated with toxicities. 
 
The proposed pilot study led by the Department of Infectious Diseases will be a double-
blind, randomized, placebo-controlled trial in healthy volunteers. The primary objective 
will be to assess the vaccine-induced and total antibody (including IgG and IgA) response 
and IgA/IgG plus IgA/IgM ratios in sera. The secondary objective will be to assess the 
functional antibody response toward Influenza virus vaccine influenza vaccine in the 
sera. Participants will be randomly assigned to receive either two doses of an influenza 
virus vaccine plus Vitamin A + D (intervention group), or two doses of  an influenza 
virus vaccine plus the placebo (control group). The targeted accrual total is 100 with a 
targeted evaluable 80 participants recruited over 2 influenza seasons (approximately 2 
years).  
3.0 RESEARCH PARTICIPANT ELIGIBILITY CRITERIA AND STUDY 
ENROLLMENT  
 
According to institutional and NIH policy, the study will access research participants 
regardless of gender and ethnic background.  Institutional experience confirms broad 
representation in this regard. 

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3.1. Inclusion Criteria  
Male or female child, age 2 to 8 years (inclusive) at the time of enrollment. 
 
Parent or legal guardian willing and able to give informed consent and comply 
with study requirements. 
3.2. Exclusion Criteria 
Current use of investigational or immunosuppressive drugs (e.g., steroids) at the 
time of enrollment 
 
Currently taking a daily (routine) vitamin A, D, or multivitamin. Note: 
participants who report occasional or sporadic vitamin use will be allowed to 
enroll.  
 
History of lung disease, asthma, immunodeficiency, sickle cell disease, or any 
other serious underlying condition or disease in the opinion of the principal 
investigator.  
 
Evidence of developmental delay or evolving neurological disorders at screening. 
 
Current use of antibiotics or antivirals at enrollment. 
 
History of having a severe allergy to eggs or to any inactive ingredient in the 
Influenza virus vaccine 
 
History of  a life-threatening reaction to influenza vaccinations 
 
Currently wheezing at the time of enrollment   
 
History of Guillain-Barre Syndrome 
 
History of heart, kidney, or lung conditions 
 
History of diabetes 
 
Use of an anti-influenza medication (including amantadine, rimantadine, 
oseltamavir, and zanamivir) within 14 days prior to enrollment  
 

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Acute febrile [>100.0oF (37.8oC) oral] illness or acute respiratory illness (e.g., 
cough or sore throat) within 3 days prior to enrollment 
 
Previous receipt of current seasonal influenza vaccine 
 
3.3. Research Participant Recruitment and Screening 
Children ages 2 to 8 years old will be sought in the Memphis community from a 
variety of both private and public venues through active recruitment by study staff 
as well as self-referral in response to advertisements. Types of advertisements that 
may be used include flyers, postcards, brochures, and newspaper ads. 
Advertisements may be distributed in many ways including email, mass mailings, 
and in person by study staff. Verbal permission will be sought at private locations 
where advertisements will be posted by study staff and a signed letter of 
authorization will be obtained from any performance sites where recruitment 
activities take place. All recruitment materials will be approved by the IRB before 
use. Advertisements will indicate the nature of the study and provide a phone 
number for further information. 
Study investigators or their designees will explain the study to potential research 
participants, including eligibility and exclusion criteria. Persons who indicate they 
are interested in participating in the study and potentially eligible will be offered a 
screening visit.  
During the screening visit, study investigators or their designees will assign a 
participant ID and provide individuals with information about the study and an 
opportunity to ask questions. Written informed consent for study participation 
will be obtained from the parent or legal guardian. Participants who fail the 
screening process due to the time sensitive criteria (e.g., recent febrile illness) will 
be allowed to rescreen.  
3.4. Enrollment on Study at St. Jude 
A member of the study team will confirm potential participant eligibility as 
defined in Section 3.1-3.2, complete and sign the ‘Participant Eligibility 
Checklist’.  The study team will enter the eligibility checklist information into the 
Patient Protocol Manager (PPM) system. Eligibility will be reviewed, and a 
research participant-specific consent form and assent document (where 
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be faxed or emailed to the CPDMO at 595-6265 to complete the enrollment 
process. 
The CPDMO is staffed 7:30 am-5:00 pm CST, Monday through Friday. A staff 
member is on call Saturday, Sunday, and holidays from 8:00 am to 5:00 pm.  
Enrollments may be requested during weekends or holidays by calling the 
CPDMO “On Call” cell phone (901-413-8591) or referencing the “On Call 
Schedule” on the intranet). 
3.5. Procedures for Identifying and Randomizing Research Participants 
Eligibility of research participants will be confirmed by the PI or study team. A 
study team member will then approach the participant and his or her legal 
authorized representative regarding the study. If the participant and/or caregiver 
agree to participate and meet eligibility criteria, the participant will be 
randomized.  
Once informed consent has been obtained and eligibility confirmed, participants 
will be randomized to receive influenza virus vaccine + Vitamins A & D (n=40) 
or influenza virus vaccine + placebo (n=40). Randomization will be performed in 
the SJCRH pharmacy by an unblinded pharmacist, using the randomization 
program developed by the Department of Biostatistics. In order to ensure similar 
enrollment numbers within each group, randomization will be restricted with a 
permuted block randomization design. All eligible participants who are enrolled 
and receive at least one administration of influenza virus vaccine will be included 
in the analyses. 
4.0 TREATMENT PLAN 
4.1. Treatment 
This is a single site, pilot, randomized controlled trial in healthy children. The 
primary and secondary endpoints assessing vaccine-induced and total antibody 
(IgG and IgA)  response and IgA/IgG plus IgA/IgM ratios in the sera as well as 
the functional antibody response toward influenza virus vaccine in the sera will be 
measured at Day 28, and 56. Approximately 100 total and 80 evaluable 
participants will be enrolled equally into two groups over 2 flu seasons, one of 
which will receive oral vitamins A and D (intervention), while the other group 
will receive the  influenza virus vaccine and placebo gummy (control).  
 

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Potential participants will be approached by study staff. Participants who wish to 
participate in the study will complete the informed consent process prior to 
conducting the screening evaluations.  
 
Allocation will be stratified by levels of Vitamin A using the surrogate retinol-
binding protein (RBP). Vitamin A insufficiency (inclusive of deficiency) will be 
defined as  ≤1.05 μM  or  <22,000 ng/ml RBP. RBP will be used to stratify 
participants based on previous mouse and human study demonstrating that 
vitamin A (for which RBP is a surrogate) is well correlated with IgA. RBP 
measurements are variable in humans, distributed somewhat equally above and 
below sufficiency cut-off. In contrast, our clinical work has indicated that study 
participants in Memphis are largely homogenous for vitamin D measurements in 
that most of the serum values fall below the sufficiency cut off. After the 
screening evaluations are obtained, the participant without co-enrolled siblings 
will be allocated to the intervention or placebo arm , in the pharmacy department, 
by means of a computerized stratified permuted block method (block size 4: 
allocation ratio 1:1) provided by the department of biostatistics.  Co-enrolled 
siblings will first be stratified by their screening RBP levels, and then siblings 
within the same RBP level stratum will be equally  assigned to intervention or 
control to avoid the chance of treatment imbalance, with the use of a 
computerized stratified permuted block method (block size 2: allocation ratio 
1:1).  
 
Participants will be randomized to receive either influenza virus vaccine plus 
vitamins A and D supplements (n=40) or influenza virus vaccine plus matched 
placebo. Gummies for the intervention and control arm (placebo) will be provided 
in the same container by an unblinded pharmacist to maintain blinding. For the 
purposes of this study, Study Day 0 is defined for each participant as the day that 
the first influenza vaccine dose  is administered. All participants will receive 2 
doses of influenza virus vaccine at least 28 days apart.   
 
Children will have the first influenza virus vaccine dose (dose 1) administered on 
day 0 of the trial, and the second dose (dose 2) administered 28 days later. 
Vaccines will be administered by a technique consistent with the manufacturer’s 
specification.  
Participants will be observed for 30 minutes following vaccination. Participants 
will be given a food diary card to record their diet between the Day 0 and Day 56 
study visits.  
 

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Blood serum samples will be collected from participants at Screening and prior to 
receiving the influenza virus vaccine on Day 28 as well as during their Day 56 
follow-up visit. 
 
Chewable Vitamin or PlaceboAdministration 
 
Chewable Vitamin A & D3 or Placebo Gummy 
The chewable gummy contains either Vitamin A (20,000 International 
Units)  and Vitamin D3 (2,000 International Units) or matched placebo, 
which  should be fully chewed under supervision of study staff with 
documention in the participants research record and eMAR (electronic 
medical administration record). The chewable gummy should be 
administered prior to receiving influenza virus vaccine vaccination.   
 
If participants have difficulty  chewing the gummy, another option is to cut 
the chewable vitamin in half along score line for easier administration.   
4.2. Contraindications to an influenza virus vaccine 
Influenza virus vaccinewill not be administered to anyone with previously known 
allergic reaction (e.g., anaphylaxis) to any component of  the vaccine, or after a 
previous dose of any influenza vaccine during the same season prior to study 
participation.  
4.3. Concomitant Therapy 
Information (including name and indication) regarding medication use by 
participants will be collected at the time of enrollment. All medications taken 
from enrollment through Day 56 will also be collected. Concomitant medications 
reported throughout the course of the study will be recorded including: all 
prescription medications, over-the-counter preparations, vitamins, nutritional 
supplements, and herbal preparations. 
 
The use of anti-influenza medications for the prevention of influenza is prohibited 
during the trial as use of these agents prophylactically may affect culture results. 
Participants caregivers will be asked to inform study staff should their child 
require anti-influenza medications while on study.  Use of all other concomitant 
therapies will be allowed.  

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4.4. Study Visit Flow Diagram 
  
5.0 DRUG/DEVICE/BIOLOGIC AGENT INFORMATION  
 
5.1. Influenza Virus Vaccine 
The influenza virus vaccine can be of any licensed brand, preferably Fluzone. 
 
5.2. Vitamin A and D3 Nutritional Supplements, Oral 
Vitamin A  palmitate 20,000 International Units  and Vitamin D3 2,000 
International Units liquid formulations will be compounded  with gelatin base, 
tangerine oil, polysorbate 20F, citric acid anhydrous USP, and steviol glycosides 
95% into a chewable gummy.  The gummies will be compounded for participants 
by Regal PharmaLab (a local compounding pharmacy) following U.S. 
Pharmacopeial Convention  (USP) 795 good compounding practices for 
pharmaceutical compounding of nonsterile preparations.   
 

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The expiration date will be listed on the product label for storage at controlled 
room temperature. Individual doses will be dispensed with participant name, 
medical record number, dosing instructions, and date.  St. Jude Children’s 
Research Hospital Pharmacy will dispense the gummies and keep accountability 
records as per randomized assignment. 
 
Certificates of Analysis (CoA) are available for the Vitamin A palmitate liquid, 
Vitamin D3 liquid, gelatin base, tangerine oil, polysorbate 20 NF, citric acid 
anhydrous USP, and steviol glycosides 95%. As vitamin supplements, these 
products are not FDA approved for prevention, diagnosis or treatment of disease.  
5.3. Matched Placebo 
Placebo gummies will be compounded for participants by Regal PharmaLab 
(local compounding pharmacy) following U.S. Pharmacopeial Convention  (USP) 
795 good compounding practices for pharmaceutical compounding of nonsterile 
preparations.  The placebo gummies will be formulated with gelatin base, 
tangerine oil, polysorbate 20 NF, citric acid anhydrous USP, and steviol 
glycosides 95% and will match the Vitamin A and D3 gummies in shape, taste, 
texture, and appearance.   
The expiration date will be listed on the product label for storage at controlled 
room temperature. Individual doses will be dispensed with participant name, 
medical record number, dosing instructions, and date.  St. Jude Children’s 
Research  Hospital Pharmacy will dispense the gummies and keep accountability 
records as per randomized assignment. 
    Certificates of Analysis (CoA) are available for the gelatin base, tangerine oil, 
polysorbate 20 NF, citric acid anhydrous USP, and steviol glycosides 95%. 
6.0 REQUIRED EVALUATIONS, TESTS, AND OBSERVATIONS 
 
A summary of the evaluations described below and their schedule for completion can 
be found in Appendix I. Protocol related procedures, evaluations, and labs not 
performed as scheduled due to malfunctioning equipment, participant non-compliance, 
participant need or preference (e.g., vacation), participant condition or due to falling 
on a weekend or holiday will be noted in the participant study record but not be 
reported as deviations. Adverse events related to commercially available influenza 
virus vaccine will also not be collected as part of this study.  

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6.1. Screening Visit 
At the screening visit, after informed consent is obtained the following will be 
completed: 
 
Medical History to include: 
• History of a severe allergy to eggs or to any inactive ingredient in the influenza 
virus vaccine 
• History of a life-threatening reaction to influenza vaccinations 
• History of Guillain-Barre syndrome 
• Currently wheezing or history of wheezing if under 5 years old 
• History of immunodeficiency  
• History of heart, kidney, or lung problems 
• History of diabetes  
• Currently taking Tamiflu, Relenza, Amantadine, and/or Rimantadine 
• Currently taking daily vitamin or multivitamin 
 
Laboratory Evaluations 
• Serum for vitamin studies 
• Serum for immunologic studies 
 
Questionnaires 
• Socio-demographic questionnaire 
 
Once screening criteria is confirmed and vitamin A retinol protein levels have 
been resulted, the participant will be randomized.  
6.2. Day 0 (Vaccine dose #1) Visit 
For the purposes of this study, Study Day 0 is defined for each participant as the 
day that the first vaccine (Influenza virus vaccine) is administered. 
 
Day 0 must be completed within 10 days of the screening assessment. If greater 
than 10 days passes, the participant must repeat the screening visit to confirm 
eligibility.  
 
All Screening and Entry visit data must be collected prior to the receipt of 
vaccination and vitamin (A and D) dose #1. Vitamin or placebo chewable 

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gummy must be administered prior to influenza virus vaccine dose 
administration. 
 
The following will be collected as a part of the Entry Visit: 
• Chewable gummy (vitamin supplements or placebo depending on 
randomization) dose administration   
• Administration of influenza virus vaccine Dose #1 for intervention and 
control group and observation in clinic 30 minutes post-vaccine 
administration 
• Dispense food diary to control and intervention participants.  
• Serum for vitamin studies 
• Serum for immunologic studies 
6.3. Day 28 (Vaccine dose #2) Visit  
The following will be completed: 
• Interim medical history and medication use 
• Laboratory evaluations 
o Serum for vitamin studies 
o Serum for immunologic studies 
• Collection of food diary from participants 
• Administration of influenza virus Vaccine Dose #2  to Intervention and 
Control group participants  
• Administration of chewable gummy dose #2  
• Dispense food diary to control and intervention participants.  
6.4. Day 56 Follow-Up Visit  
The following will be completed:  
• Interim Medical History and medication use 
• Collect food  diary from participants 
• Laboratory evaluations: 
o Serum for vitamin studies 
o Serum for immunological studies 
• At day 56, participants enrolled after approval of Revision 1.1, will be 
given the option to complete the Block food frequency questionnaire 
(FFQ).  This questionnaire includes about 90 questions and asks about a 
child's "usual eating habits in the past 6 months." It takes a caregiver or 
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developed from NHANES III dietary recall data. The nutrient database 
was developed from the USDA Nutrient Database for Standard Reference. 
Individual portion size is asked for beverages but not other foods. Dietary 
information obtained from this questionnaire is for exploratory purposes 
only. 
6.5. Special Instructions and Definitions of Evaluations 
Study Visit Windows 
 
Day 0 (Vaccine dose #1) visit must occur within 10 days of screening.  
 
Day 28 (Vaccine dose #2) visit must be performed within +3 days of the protocol 
specified date 
 
Day 56 (follow-up)visit must be performed within +/- 7 days of the scheduled 
protocol visit date.  
 
Vaccination 
Influenza virus vaccines will be administered per manufacturer’s 
recommendationsby trained study or clinic staff.  
Participant caregivers will be informed to contact their child’s PCP should any 
influenza virus vaccine related reactions occur over the course of the study.  
 
Vitamins A and D3 
Chewable vitamin supplements or placebo gummies will be administered orally to 
participants prior to receiving  the influenza virus vaccine on Day 0 and Day 28 
by study staff. The gummy will be administered and documented in the 
participants research record prior to  the influenza virus vaccine vaccination..  
 
Laboratory Evaluations 
Immune responses (inclusive of IgM, IgG, IgA, and cytokines) will be measured 
in blood serum collections at Screening, Day 28, and Day 56. ELISAs and 
functional assays (neutralization or HAI assays) will examine virus-specific and 
total antibodies. Serum vitamins A and D will be tested by HPLC and/or tests 
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7.0 EVALUATION CRITERIA 
7.1. Toxicity Evaluation Criteria 
All adverse events will be graded by the Pediatric Toxicity Grading Tables from 
the Division of Microbiology and Infectious Diseases (DMID). See Appendix III 
for details.  
7.2. Acceptable Percentage of Missed Doses for Research and Standard of 
Care Drugs 
All eligible participants who receive at least one dose of vaccine will be included 
in the data analyses.  
8.0 CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND 
OFF-STUDY CRITERIA 
8.1. Off-study criteria 
Completion of study (completion of the final follow-up visit) 
Development of Influenza infection and/or receipt of anti-influenza medications 
during the study period 
Death 
Lost to follow-up 
Request of the Participant/Parent 
Discretion of the Study PI, such as the following  
• The researcher decides that continuing in the study would be harmful  
• A treatment is needed that is not allowed on this study 
• The participant misses so many appointments that the data cannot be 
used in the study 
• New information is learned that a better treatment is available, or that 
the study is not in the participant’s best interest 
• Study evaluations are complete 

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9.0 SAFETY AND ADVERSE EVENT REPORTING REQUIREMENTS 
9.1. Reporting Adverse Experiences and Deaths to St. Jude IRB 
Only “unanticipated problems involving risks to participants or others” referred to 
hereafter as “unanticipated problems” are required to be reported to the St. Jude 
IRB promptly, but in no event later than 10 working days after the investigator 
first learns of the unanticipated problem. Regardless of whether the event is 
internal or external (for example, an IND safety report by the sponsor pursuant to 
21 CFR 312.32), only adverse events that constitute unanticipated problems are 
reportable to the St. Jude IRB.  As further described in the definition of 
unanticipated problem, this includes any event that in the PI’s opinion was: 
• Unexpected (in terms of nature, severity, or frequency) given (1) the 
research procedures that are described in the protocol-related documents, 
such as the IRB-approved research protocol and informed consent 
document, as well as other relevant information available about the 
research; (2) the observed rate of occurrence (compared to a credible 
baseline for comparison); and (3) the characteristics of the subject 
population being studied; and 
• Related or possibly related to participation in the research; and 
• Suggests that the research places subjects or others at a greater risk of 
harm (including physical, psychological, economic, or social harm) than 
was previously known or recognized. An unanticipated problem involving 
risk to subjects or others may exist even when actual harm does not occur 
to any participant. 
 
Unrelated, expected deaths do not require reporting to the IRB.  Though death is 
“serious”, the event must meet the other two requirements of “related or possibly 
related” and “unexpected/unanticipated” to be considered reportable. 
Deaths meeting reporting requirements are to be reported immediately to the St. 
Jude IRB, but in no event later than 48 hours after the investigator first learns of 
the death.  
The following definitions apply with respect to reporting adverse 
experiences: 
 
Serious Adverse Event: 
Any adverse event temporally associated with the subject’s participation in 
research that meets any of the following criteria: 

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• 
results in death; 
• 
is life-threatening (places the subject at immediate risk of death from the 
event as it occurred); 
• 
requires inpatient hospitalization or prolongation of existing 
hospitalization; 
• 
results in a persistent or significant disability/incapacity; 
• 
results in a congenital anomaly/birth defect; or 
• 
any other adverse event that, based upon appropriate medical judgment, 
may jeopardize the subject’s health and may require medical or surgical 
intervention to prevent one of the other outcomes listed in this definition 
(examples of such events include:  any substantial disruption of the ability 
to conduct normal life functions, allergic bronchospasm requiring 
intensive treatment in the emergency room or at home, blood dyscrasias or 
convulsions that do not result in inpatient hospitalization, or the 
development of drug dependency or drug abuse), a congenital 
anomaly/birth defect, secondary or concurrent cancer, medication 
overdose, or is any medical event which requires treatment to prevent any 
of the medical outcomes previously listed.   
Unexpected Adverse Event:  
• 
Any adverse event for which the specificity or severity is not consistent 
with the protocol-related documents, including the applicable investigator 
brochure, IRB approved consent form, Investigational New Drug (IND) or 
Investigational Device Exemption (IDE) application, or other relevant 
sources of information, such as product labeling and package inserts; or if 
it does appear in such documents, an event in which the specificity, 
severity or duration is not consistent with the risk information included 
therein; or  
• 
The observed rate of occurrence is a clinically significant increase in the 
expected rate (based on a credible baseline rate for comparison); or  
• 
The occurrence is not consistent with the expected natural progression of 
any underlying disease, disorder, or condition of the subject(s) 
experiencing the adverse event and the subject’s predisposing risk factor 
profile for the adverse event.  
 
Internal Events: 
Events experienced by a research participant enrolled at a site under the 
jurisdiction of St. Jude IRB for either multicenter or single-center research 
projects. 

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Unanticipated Problem Involving Risks to Subjects or Others:  
An unanticipated problem involving risks to subjects or others is an event which 
was not expected to occur and which increases the degree of risk posed to 
research participants.  Such events, in general, meet all of the following criteria:  
• 
unexpected;  
• 
related or possibly related to participation in the research; and  
• 
suggests that the research places subjects or others at a greater risk of 
harm (including physical, psychological, economic, or social harm) than 
was previously known or recognized.  An unanticipated problem involving 
risk to subjects or others may exist even when actual harm does not occur 
to any participant.  
Consistent with FDA and OHRP guidance on reporting unanticipated problems 
and adverse events to IRBs, the St. Jude IRB does not require the submission of 
external events, for example IND safety reports, nor is a summary of such 
events/reports required; however, if an event giving rise to an IND safety or other 
external event report constitutes an “unanticipated problem involving risks to 
subjects or others” it must be reported in accordance with this policy.  In general, 
to be reportable external events need to have implications for the conduct of the 
study (for example, requiring a significant and usually safety-related change in the 
protocol and/or informed consent form). 
 
Although some adverse events will qualify as unanticipated problems involving 
risks to subjects or others, some will not; and there may be other unanticipated 
problems that go beyond the definitions of serious and/or unexpected adverse 
events.  Examples of unanticipated problems involving risks to subjects or others 
include: 
 
• Improperly staging a participant’s tumor resulting in the participant 
being assigned to an incorrect arm of the research study; 
• The theft of a research computer containing confidential subject 
information (breach of confidentiality); and  
• The contamination of a study drug.  Unanticipated problems generally 
will warrant consideration of substantive changes in the research 
protocol or informed consent process/document or other corrective 
actions in order to protect the safety, welfare, or rights of subjects or 
others.  

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9.2. Recording Adverse Events and Serious Adverse Events 
Adverse events associated with the influenza virus vaccine will not be recorded 
for this study. Other adverse events will be graded using the Division of 
Microbiology and Infectious Diseases (DMID) Pediatric Toxicity Tables 
(Appendix III). All grades of these events will be recorded. 
 
Adverse events will be collected from enrollment through the participant’s last 
visit. Any AE that has not had a stop date recorded by the participant’s last visit 
will continue to be followed-up until an outcome or resolution has occurred. 
Study participants will be instructed to notify study personnel at any time during 
the study if they are hospitalized or have any unusual, alarming or unexpected 
events. 
 
Adverse events will be assessed by the investigator for severity, relationship to 
the study product, possible etiologies and whether the event meets criteria as a 
serious adverse event.  
 
The following guidelines will be used by investigators to assess the relationship of 
an AE to study product administration: 
 
• Definite: Unquestionable relationship. The event was directly attributable 
to the study product. 
• Probable: Relationship is likely. It is likely that the study product caused 
the event. 
• Possible: Relationship may exist. There is at least a reasonable possibility 
that the event was caused by the study product, although the likelihood is 
low. 
• 
Unlikely: Relationship is not likely. Although it is unlikely that the 
study product caused this event, causality cannot be entirely ruled out. 
• 
Unrelated: No relationship. The study product did not cause the event. 
 
Factors considered in assessing the relationship of an AE to study product 
include:  
• 
the temporal relationship of the event to the administration of study 
product;  
• 
whether an alternative etiology has been identified; and  
• 
biological plausibility.  

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9.3. Un-blinding Procedures 
The investigator should follow the trial's randomization procedures and should 
ensure that the code is broken only in accordance with the protocol. The treating 
investigator should promptly document and explain to the St. Jude Principal 
Investigator (PI) any premature un-blinding of the investigational agent (e.g., 
accidental un-blinding, or un-blinding due to a serious adverse event). The study 
PI determines the attribution and grading of Adverse Events as well as decides, 
in sponsor-investigator trials, if breaking of a study blind is necessary.  In 
deciding, the St. Jude PI may consider: 
• Are there any specific antidotes for the investigational agent or blinded 
chemotherapy agent(s),  
• Are drug-drug interactions expected that may preclude medical 
intervention,  
• Would discontinuation of IND or blinded agent(s) be likely to have an 
immediate effect?  
 
- Participant and data management 
It is recommended that, when possible and appropriate, the blind be maintained 
for those persons responsible for analysis and interpretation of results at the 
study's conclusion. 
 
Any toxicities associated or possibly associated with study treatment should be 
managed according to standard medical practice. 
 
- Accidental Un-blinding 
In the event of accidental un-blinding by study personnel, the following should 
occur: 
• Information on accidental un-blinding (including subject number, 
person(s) involved, information shared, whether the un-blinded person 
has patient contact) is sent to the St. Jude site Principal Investigator (PI). 
• The St. Jude PI provides a summary of the incident to the Biostatistician 
and the un-blinded study clinical research monitor. 
• The protocol deviation will be recorded in a blinded manner and 
communicated to the primary blinded monitor. 
• Blinded site staff will be re-trained by the blinded MONITOR, and un-
blinded site staff will be retrained by the un-blinded MONITOR on 
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training will be documented in both the blinded monitoring visit report 
and the un-blinded monitoring visit report.  
• Monitors will ensure appropriate actions are taken (e.g., that the affected 
study participant is treated according to protocol specifications 
concerning un-blinding, that a corrective action to prevent unintentional 
un-blinding is implemented, that the event is recorded in the deviation 
log in a blinded manner, and that the SMC or other appropriate parties 
were notified or consulted as applicable) 
 
- Emergency Un-blinding 
In cases of patient safety (medical emergency or serious medical condition), the 
treating physician may request un-blinding a participant randomization code, if 
knowledge of the treatment assignment is essential for the clinical management 
or welfare of the participant. Un-blinding of treatment assignment prior to 
documented disease progression is permitted only for a serious, un-expected 
adverse event suspected to be study drug-related (as part of the safety reporting 
process) and if necessary for patient management.  
 
Procedure for Requesting Un-blinding of a Unique Participant Identifier or 
Randomization Code: 
 
• The study St. Jude PI is the only one who has authority to approve for un-
blinding a participant randomization code or identification code.  
• In a medical emergency, every effort should be made to consult with the 
St. Jude PI (insert PI emergency contact phone number) to discuss other 
options prior to requesting un-blinding of a participant randomization 
code. 
• The treating physician at St. Jude shall make a request to the St. Jude PI. 
• The treating physician may hold administration of the investigational 
agent while waiting for a decision on un-blinding to be made. 
• The St. Jude PI may consult with Investigational Pharmacy for un-
blinding, if applicable. 
• The St. Jude PI will contact the investigational pharmacist to provide the 
randomization code for the individual participant.  
• The un-blinding information for the participant should be limited to as few 
individuals as possible. The blinded study team and the participant 
should remain blinded. 
• Documentation of the incident will be maintained in the pharmacy binder. 
All documents associated with the incident should be recorded in a 

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blinded manner in the EMR and/or other documents, such as, emails, if 
the participant remains on study and the data will be included in study 
data analysis. 
• If the un-blinding was due to an adverse event, confirm the adverse event 
was reported and documented in accordance with applicable reporting 
procedures and timelines. 
10.0 DATA COLLECTION, STUDY MONITORING, AND 
CONFIDENTIALITY 
10.1. 
Data Collection  
All study data will be collected by the study staff using designated source 
documents. Data will be entered and maintained in an institutional relational 
database with separate backup and monthly off site backup. Standard GCP 
practices will be followed to ensure accurate, reliable and consistent data 
collection. 
10.2. 
Study Monitoring 
The study team will hold monthly meetings and review case histories or quality 
summaries on participants.  
Source document verification of eligibility and informed consent for 100% of St. 
Jude participants will be performed by the Eligibility Coordinators within 5 
working days of completion of enrollment. 
The Clinical Research Monitor will perform monitoring of applicable essential 
regulatory documentation.  Also, reviewing for the timeliness of serious adverse 
event reporting (type, grade, attribution, duration, timeliness and appropriateness) 
for selected study participants semi-annually and track accrual continuously.  The 
monitor will verify those data points relating to the primary study objective for a 
certain number of study enrollees as specified in the Moderate Risk monitoring 
plan checklist for this study.  Protocol compliance monitoring will include 
participant status, safety assessments, eligibility, the informed consent process, 
participant protocol status, off-study, and off-therapy criteria.  The Monitor will 
generate a formal report which is shared with the Principal Investigator (PI), study 
team and the Internal Monitoring Committee (IMC). 

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Monitoring may be conducted more frequently if deemed necessary by the 
CPDMO or the IMC.   
Continuing reviews by the IRB and CT-SRC will occur at least annually.  In 
addition, SAE reports in TRACKS (Total Research and Knowledge System) are 
reviewed in a timely manner by the IRB/ OHSP. 
10.3. 
Confidentiality 
All study procedures will be conducted in private, and every effort will be made 
to protect participant privacy and confidentiality to the extent possible.  
Data collection, process, and administrative forms, laboratory specimens, and 
other reports will be identified by a coded number only to maintain participant 
confidentiality.  The study database will identify study participants only by the 
study identification number and will not contain personal health or other 
identifiers. The list containing the study number and personal identifiers will be 
maintained in a secured file indefinitely. All study-related information will be 
stored securely. All participant information will be stored in locked file cabinets 
in areas with access limited to study staff. All records that contain names or other 
personal identifiers, such as locator forms and informed consent forms, will be 
stored separately from study records identified by code number.  Databases will 
be secured with password-protected access systems. Participants’ study 
information will not be released without their written permission, except as 
necessary for monitoring. Personal identifiers will not be included in any study 
reports. 
All study records will be kept confidential to the extent provided by national and 
local laws.  Authorized representatives of the sponsor(s), and regulatory agencies 
may examine (and when required by applicable law, to copy) study records for the 
purposes of quality assurance reviews, audits and evaluation of the study safety 
and progress. 
11.0 STATISTICAL CONSIDERATIONS 
11.1. 
Anticipated Completion Dates 
Anticipated Primary Completion Date: 24 months following enrollment of 
the first participant.    
Anticipated Study Completion Date 5 years following approval of the 

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protocol  
11.2. 
Sample Size 
The primary goal of the study is to estimate the effect size of vaccination when 
incorporating vitamin A/D supplements and standard care. The primary 
endpoint of interest will be the proportion of 4X increases or conversion from 
an undetectable to a detectable response in virus-specific antibody  after two 
immunizations (compared to the starting titer) in intervention and control 
groups. We plan to enroll 40 participants per group in total. Assuming an 
attrition rate of about 5%, approximately 38 vaccine plus vitamin and 38 
vaccine plus placebo participants will provide data for the primary objectives. 
Because this is a pilot study and there is very limited information about vitamin 
supplementation with influenza vaccination among children, the sample size 
was chosen based on past experience with clinical trials of other respiratory 
virus vaccines among a pediatric population at SJCRH and published literature 
(39). The sample size proposed will suffice for capturing the response rate 
difference between groups with a power of over 80% at a significance level of 
5%, if the true average response rate overall with the Vitamin A/D supplements 
is over 62% (EAST 6) while the response rate in the vaccine only group is 30% 
on average, the majority of the enrolled participants are either vitamin A or D or 
both deficient as suggested by our experience. If the effect size is smaller, we 
still believe the estimated effect size with the proposed 80 participants will 
inform future larger studies.  
11.3. 
Participant Accrual, Follow-up and Retention 
Based on previous studies of influenza vaccination among children, the accrual 
of 100 total and 80 evaluable participants will take approximately 2 years (or 2 
flu seasons). Participants lost to follow-up and/or discontinued early from the 
study will not be replaced. The study team will consider a participant “lost to 
follow-up” for this trial if he or she has missed 2 consecutive study visits and is 
unreachable after multiple contact attempts by study staff. However, every effort 
will be made to complete their regularly scheduled study evaluations. Study staff 
will target a 95% retention  of enrolled participants over the 56 day follow-up 
period.  

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11.4. 
Summary of Primary and Secondary Objectives  
For all analyses, all continuous titer measures will be log transformed unless 
otherwise specified 
 
Analysis of Primary Objectives 
 
In Section 4.1, we have described a way to equally assign enrolled siblings into 
two arms. By doing this, ignoring the possible nonindependence in siblings when 
obtaining group difference should still provide a consistent estimate of the group 
difference. The precision (i.e., the width of the 95% confidence interval) of the 
estimate may be inflated, while the degree of inflation of the precision depends on 
the number of co-enrolled siblings, which is not predictable at this stage. Given 
the fact that this study is a pilot study, we believe in the value of obtaining an 
accurate group difference estimate, and allow some leeway in precision. More 
precise estimates and more power could be achieved through future larger studies. 
 
 
To assess the vaccine-induced and total antibody (IgG and IgA) 
response and IgA/IgG plus IgA/IgM ratios in the sera at days 28 and 56. 
 
The serum antibody response described by the proportion of 4X increases or 
conversion from undetectable to detectable response in virus-specific  
antibody after two immunizations  in intervention and control groups will be 
estimated and 95% confidence interval will be described for both groups, 
overall and by stratum. The proportion difference with 95% confidence 
interval will be reported, overall and by stratum. Due to possible variation in 
virus strains and  the influenza virus vaccine formula in different flu 
seasons, analyses by season will also be described. Continuous titer values 
in both groups, overall or by stratum, will also be described and compared 
with point estimate along with 95% confidence intervals. 
 
Titer ratios, such as IgA/IgG and IgA/IgM will be summarized with 
descriptive statistics, including mean, standard deviation, median and range, 
for each intervention group, overall or by season. Two-sample tests (t-test or 
Wilcoxon rank-sum test) will be applied whenever appropriate. 
 
We will also apply generalized estimating equations (GEE) or mixed models 
to explore titer levels longitudinally. 
 

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As sensitivity analysis, similar analytic procedures will be additionally 
implemented by treating 4X increases or conversion from undetectable to 
detectable response in virus-specific antibody after two immunizations as an 
endpoint. 
 
Analysis of Secondary Objectives 
 
To assess the functional antibody response towared the influenza virus 
vaccine in the sera 
 
Secondary analyses will examine sero-conversion defined as antibody HAI 
titers of <1:20  converting to ≥1:20, or a four-fold increase in titer for 
participants with a starting titer of ≥1:20. Sero-conversion rate will be 
estimated with proportion and 95% confidence interval for both groups, 
overall and by stratum. The rate difference will be described with point 
estimate and 95% confidence interval, overall and by stratum. Due to 
possible variation in virus strains and influenza virus vaccine compositions 
in different flu seasons, analyses by season will also be described. 
Continuous titer values in both groups, overall or by stratum, will also be 
described and compared with point estimate along with 95% confidence 
intervals. 
 
We will also apply generalized estimating equations (GEE) or mixed models 
to explore titer levels longitudinally. 
 
Analysis of Exploratory Objectives 
 
To describe the socio-demographic characteristics and nutritional diet 
among children participating in the trial.  
 
Socio-demographic and dietary intake will be described with tables and 
descriptive statistics (frequency, proportion mean, standard deviation, 
median, range, etc…). The anticipated socio-demographic  heterogeneity of 
participants may preclude further analysis, but if feasible, relationships 
between socio-demographic characteristics and dietary intake with outcomes 
will be examined.  
 
To assess the feasibility of the food diary among healthy volunteers as 
measured by completion of the diary. 

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To assess the feasibility of the food diary among healthy volunteers, the 
proportion and 95% confidence interval of participants who complete and 
return the food diary will be calculated overall and at each visit time point. 
 
To monitor serum Vitamin A and D levels at various time points. 
 
Serum Vitamin A and D levels at screening, days 0, 28 and 56 will be 
monitored, plotted and described at each time point. Summary statistics, 
such as mean, standard deviation, median and range, will be provided for all 
time points.  If feasible, paired comparisons and longitudinal modeling will 
be explored.  
12.0 OBTAINING INFORMED CONSENT 
12.1. 
Informed Consent Prior to Research Interventions  
This protocol, the informed consent document, and any subsequent modifications 
will be reviewed and approved by the IRB. 
The informed consent will describe the purpose of the study, the procedures to be 
followed, and the risks and benefits of participation. The consent discussion will 
take place in a private room and an opportunity to ask questions will be given. A 
copy of the signed consent form will be given to the participant. The consent 
process will be documented in the research record. 
All research participants who meet eligibility criteria regardless of gender or 
minority status are fully eligible to participate in this study. 
12.2. 
Consent at Age of Majority 
Age of majority does not apply to this protocol since only children between the 
ages of 2 to 8 years old will be eligible for study participation. Participation 
should only last approximately 56 days so participants will not reach the age of 18 
before completing the study. 

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13.0 REFERENCES 
 
[1]  Harper SA, Bradley JS, Englund JA, File TM, Gravenstein S, Hayden FG,…Zimmerman 
RK. 2009. Seasonal influenza in adults and children – diagnosis, treatment, 
chemoprophylaxis, and institutional outbreak management. Clinical Infectious 
Diseases. 48: 1003-32.  
 
[2] Sommer A, Katz J, Tarwotjo I. 1984. Increased risk of respiratory disease and diarrhea in 
children with preexisting mild vitamin A deficiency. Am.J.Clin.Nutr. 40: 1090-
1095. 
 
[3]  Chesney RW. 2010. Vitamin D and The Magic Mountain: the anti-infectious role of the 
vitamin. J.Pediatr. 156: 698-703. 
 
[4]  Manaseki-Holland S, Qader G, Isaq MM et al. 2010. Effects of vitamin D 
supplementation to children diagnosed with pneumonia in Kabul: a randomised 
controlled trial. Trop.Med.Int.Health. 15: 1148-1155. 
 
[5]  Urashima M, Segawa T, Okazaki M, Kurihara M, Wada Y, Ida H. 2010. Randomized 
trial of vitamin D supplementation to prevent seasonal influenza A in 
schoolchildren. Am.J Clin.Nutr. 91: 1255-1260. 
 
[6]  Belderbos ME, Houben ML, Wilbrink B et al. 2011. Cord blood vitamin D deficiency is 
associated with respiratory syncytial virus bronchiolitis. Pediatrics. 127: e1513-
e1520. 
 
[7]  Block SL, Falloon J, Hirschfield JA et al. 2012. Immunogenicity and safety of a 
quadrivalent live attenuated influenza vaccine in children. Pediatr.Infect.Dis.J. 31: 
745-751. 
 
[8]  Ross AC. 2012. Vitamin A and retinoic acid in T cell-related immunity. Am.J Clin.Nutr. 
96: 1166S-1172S. 
 
[9]  Pino-Lagos K, Benson MJ, Noelle RJ. 2008. Retinoic acid in the immune system. 
Ann.N.Y.Acad.Sci. 1143: 170-187. 
 [10]  Mora JR, Iwata M, Von Andrian UH. 2008. Vitamin effects on the immune system: 
vitamins A and D take centre stage. Nat.Rev.Immunol. 8: 685-698. 
 [11]  Barria MI, Garrido JL, Stein C et al. 2013. Localized mucosal response to intranasal live 
attenuated influenza vaccine in adults. J Infect.Dis. 207: 115-124. 

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 [12]  Surman SL, Rudraraju R, Sealy R, Jones B, Hurwitz JL. 2012. Vitamin A deficiency 
disrupts vaccine-induced antibody-forming cells and the balance of IgA/IgG 
isotypes in the upper and lower respiratory tract. Viral Immunology. 25: 341-344. 
 [13]  Butler JC, Havens PL, Sowell AL et al. 1993. Measles severity and serum retinol 
(vitamin A) concentration among children in the United States. Pediatrics. 91: 
1176-1181. 
 [14]  Sommer A. 1993. Vitamin A, infectious disease, and childhood mortality: a 2 solution? J 
Infect.Dis. 167: 1003-1007. 
 [15]  Sommer A, Tarwotjo I, Djunaedi E et al. 1986. Impact of vitamin A supplementation on 
childhood mortality. A randomised controlled community trial. Lancet. 1: 1169-
1173. 
 [16]  Al TZ, Piskunov A, Rochette-Egly C. 2013. Vitamin A and retinoid signaling: genomic 
and nongenomic effects. J Lipid Res. 54: 1761-1775. 
 [17]  Chesney RW, Han X. 2013. Differential regulation of TauT by calcitriol and retinoic acid 
via VDR/RXR in LLC-PK1 and MCF-7 cells. Adv.Exp.Med.Biol. 776: 291-305. 
 [18]  Ross AC. 2007. Vitamin A supplementation and retinoic acid treatment in the regulation 
of antibody responses in vivo. Vitam.Horm. 75: 197-222. 
 [19]  Bahl R, Bhandari N, Kant S, Molbak K, Ostergaard E, Bhan MK. 2002. Effect of vitamin 
A administered at Expanded Program on Immunization contacts on antibody 
response to oral polio vaccine. Eur.J Clin.Nutr. 56: 321-325. 
 [20]  Benn CS, Balde A, George E et al. 2002. Effect of vitamin A supplementation on 
measles-specific antibody levels in Guinea-Bissau. Lancet. 359: 1313-1314. 
 [21]  Li-Ng M, Aloia JF, Pollack S et al. 2009. A randomized controlled trial of vitamin D3 
supplementation for the prevention of symptomatic upper respiratory tract 
infections. Epidemiol.Infect. 137: 1396-1404. 
 [22]  Vijayaraghavan K, Radhaiah G, Prakasam BS, Sarma KV, Reddy V. 1990. Effect of 
massive dose vitamin A on morbidity and mortality in Indian children. Lancet. 
336: 1342-1345. 
 [23]  Cooper C, Thorne A. 2011. Vitamin D supplementation does not increase 
immunogenicity of seasonal influenza vaccine in HIV-infected adults. HIV 
Clin.Trials. 12: 275-276. 

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 [24]  Semba RD, Muhilal, Scott AL, Natadisastra G, West KP, Jr., Sommer A. 1994. Effect of 
vitamin A supplementation on immunoglobulin G subclass responses to tetanus 
toxoid in children. Clin.Diagn.Lab Immunol. 1: 172-175. 
 [25]  Long AN, Ray MM, Nandikanti D et al. 2010. Prevalence of 25-hydroxyvitamin D 
deficiency in an urban general internal medicine academic practice. Tenn.Med. 
103: 51-2, 57. 
 [26]  Stephens D, Jackson PL, Gutierrez Y. 1996. Subclinical vitamin A deficiency: a 
potentially unrecognized problem in the United States. Pediatr.Nurs. 22: 377-89, 
456. 
 [27]  Cunningham SA, Kramer MR, Narayan KM. 2014. Incidence of childhood obesity in the 
United States. N.Engl.J Med. 370: 403-411. 
 [28]  Almekinder J, Manda W, Soko D, Lan Y, Hoover DR, Semba RD. 2000. Evaluation of 
plasma retinol-binding protein as a surrogate measure for plasma retinol 
concentrations. Scand.J.Clin.Lab Invest. 60: 199-203. 
 [29]  Chesney RW. 2012. The five paradoxes of vitamin D and the importance of sunscreen 
protection. Clin.Pediatr.(Phila). 51: 819-827. 
 [30]  Ross AC, Manson JE, Abrams SA et al. 2011. The 2011 report on dietary reference 
intakes for calcium and vitamin D from the Institute of Medicine: what clinicians 
need to know. J Clin.Endocrinol.Metab. 96: 53-58. 
 [31]  Pramyothin P, Holick MF. 2012. Vitamin D supplementation: guidelines and evidence 
for subclinical deficiency. Curr.Opin.Gastroenterol. 28: 139-150. 
 [32]  Oshansky CM, Gartland AJ, Wong SS et al. 2014. Mucosal immune responses predict 
clinical outcomes during influenza infection independently of age and viral load. 
Am.J Respir.Crit Care Med. 189: 449-462. 
 [33]  Rudraraju R, Surman SL, Jones BG, Sealy R, Woodland DL, Hurwitz JL. 2012. Reduced 
frequencies and heightened CD103 expression among virus-induced CD8(+) T 
cells in the respiratory tract airways of vitamin A-deficient mice. Clin.Vaccine 
Immunol. 19: 757-765. 
 [34]  Rudraraju R, Surman S, Jones B, Sealy R, Woodland DL, Hurwitz JL. 2011. Phenotypes 
and functions of persistent Sendai virus-induced antibody forming cells and 

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CD8+ T cells in diffuse nasal-associated lymphoid tissue typify lymphocyte 
responses of the gut. Virology. 410: 429-436. 
 [35]  Surman SL, Jones BG, Sealy RE, Rudraraju R, Hurwitz JL. 2014. Oral retinyl palmitate 
or retinoic acid corrects mucosal IgA responses toward an intranasal influenza 
virus vaccine in vitamin A deficient mice. Vaccine. 32: 2521-2524. 
[36]  Grohskopf LA, Sokolow ZL, Olsen SJ, Bresee JS, Broder KR, Karron RA. 2015. 
Prevention and control of influenza with vaccines: [Recommendations of the 
Advisory Committee on Immunization Practices, United States, 2015-2016 
influenza season]. MMWR Morb Mortal Wkly Rep. 64: 818-825.  
[37]  Belshe RB, Mendelman PM, Treanor J, King J, Gruber WC, Piedra P,…Wolff M. 1998. 
The efficacy of live attenuated, cold-adapted, trivalent, intranasal influenzavirus 
vaccine in children. N Engl J Med. 338(20); 1405-1412. 
[38]  Belshe RB, Gruber WC, Mendelman PM, Cho I, Reisinger K, Block SL,…Wolff M. 
2000. Efficacy of vaccination with live attenuated, cold-adapted, trivalent, 
intranasal influenza virus vaccine against a variant (A/Sydney) not contained in 
the vaccine. J Pediatr. 136(2): 168-175.  
[39]  Norhayati MN, Ho JJ, Azman MY. 2015. Influenza vaccines for preventing acute otittis 
media in infants and children. Cochrane Database of Systematic Reviews, 3.  
[40]  Awasthi, S., Peto, R., Read, S., Clark, S., Pande, V., Bundy, D., and team, D. (2013) 
Vitamin A supplementation every 6 months with retinol in 1 million pre-school 
children in north India: DEVTA, a cluster-randomised trial, Lancet 381, 1469-
1477. 
[41] Havens PL, Hazra R, Stephensen CB, Kiser JJ, Flynn PM, Wilson CM,…Mulligan K. 
2014. Vitamin D3 supplementation increases fibroblast growth factor-23 in HIV-
infected youths treated with tenofovir disoproxil fumorate. Anitivrial therapy. 19: 
613-618. 
[42]  Ruiter, B., Patil, S. U., and Shreffler, W. G. (2015) Vitamins A and D have antagonistic 
effects on expression of effector cytokines and gut-homing integrin in human 
innate lymphoid cells, Clinical and experimental allergy : journal of the British 
Society for Allergy and Clinical Immunology. 
[43] Uzum, A. K., Salman, S., Telci, A., Boztepe, H., Tanakol, R., Alagol, F., and Ozbey, N. 
C. (2010) Effects of vitamin D replacement therapy on serum FGF23 

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concentrations in vitamin D-deficient women in short term, European journal of 
endocrinology / European Federation of Endocrine Societies 163, 825-831. 
[44] Johansson, S., and Melhus, H. (2001) Vitamin A antagonizes calcium response to vitamin 
D in man, Journal of bone and mineral research : the official journal of the 
American Society for Bone and Mineral Research 16, 1899-1905. 
[45] Surman, S. L., Jones, B. G., Rudraraju, R., Sealy, R. E., and Hurwitz, J. L. (2014) 
Intranasal administration of retinyl palmitate with a respiratory virus vaccine 
corrects impaired mucosal IgA response in the vitamin A-deficient host, Clinical 
and vaccine immunology : CVI 21, 598-601. 
[46] Penniston, K. L., and Tanumihardjo, S. A. (2006) The acute and chronic toxic effects of 
vitamin A, Am.J Clin.Nutr. 83, 191-201. 
[47] Carpenter, T. O., Pettifor, J. M., Russell, R. M., Pitha, J., Mobarhan, S., Ossip, M. S., 
Wainer, S., and Anast, C. S. (1987) Severe hypervitaminosis A in siblings: 
evidence of variable tolerance to retinol intake, The Journal of pediatrics 111, 
507-512. 
[48] Allen, L. H., and Haskell, M. (2002) Estimating the potential for vitamin A toxicity in 
women and young children, The Journal of nutrition 132, 2907S-2919S.  

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APPENDIX I: SCHEDULE OF EVALUATIONS 
Study Visit 
Screening 
1st Vaccine Visita 
 
2nd Vaccine Visit 
Follow-Up Visit 
(Off-Study) 
Early 
Discontinuation  
Study Day (Window) 
-10a 
0 
28 (+3 days) 
56 (+/- 7 days) 
 
Clinical Evaluations 
Informed Consent 
X 
 
 
 
 
Randomizationd 
 
X 
 
 
 
Medical History 
X 
X 
X 
X 
X 
Current Medication Use 
X 
X 
X 
X 
 
Socio-Demographics Questionnaire 
X 
 
  
 
 
 
 
Food Frequency Questionnaire 
 
 
 
X 
 
Compensation ($50) 
X 
X 
X 
X 
X 
Laboratory Evaluations 
Blood Sample Collectionc 
X 
X 
X 
X 
X 
Vaccine and Vitamin Administration 
Vaccine Administration 
 
Xb 
Xb 
 
 
Vitamin A and D or Placebo 
Administrationd 
 
X 
X 
 
 
Participant Diaries 
Food Diary 
 
X 
X 
 
X 
a  Day 0 (1st Vaccine Dose) must occur within 10 days of screening.  
b Participants will be observed for 30 minutes post-vaccination.  
c Serum for Vitamin and Immunologic Studies 
d  Randomization must occur prior to initiating any Day 0 visit evaluations. 

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APPENDIX II: CLINICAL AND RESEARCH TESTS 
 
No tests and/or evaluations are performed for good clinical care.  
 
All tests and/or evaluations are research tests.  
 

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APPENDIX III: TOXICITY GRADING TABLES 
 
A. Division of Microbiology and Infectious Diseases (DMID) Pediatric Toxicity Tables  
 
https://www.niaid.nih.gov/LabsAndResources/resources/DMIDClinRsrch/Documents/dmidpedto
x.pdf