The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets
Phase: PHASE1
Conditions: Influenza
Sponsor: Stanford University

 
 
 
 
The Effects of Attenuated Versus Inactivated Flu 
Vaccine in Twin Sets     
 
Study Protocol and Statistical Analysis Plan 
NCT03898986 
August 13, 2019 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
1 
 
 
 
 
 
 
 
 
 
 
 
 
 
The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets 
 
 
 
 
 
 
 
 
 
 
 
 
 
Funding Mechanism: 5U19AI05722915 
 
Other Identifying Numbers:  IRB Protocol: 50200 
 
U19 CCHI Center Principal Investigator: Mark M. Davis, PhD 
 
Clinical Core Principal Investigator/Protocol Director:  Philip M. Grant, MD 
 
 
 
 
Version Number: 1 
 
 
 
13AUG2019 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
2 
 
 
TABLE OF CONTENTS 
 
1 
Protocol Summary 
2 
Objectives ............................................................................................................................. 5 
3 
Background and Rationale ................................................................................................... 6 
3.1 
Potential Risks and Benefits ..................................................................................... 6 
4 
Study Design ........................................................................................................................ 8 
5 
Study Enrollment and Withdrawal ........................................................................................ 9 
5.1 
Subject Inclusion Criteria .......................................................................................... 9 
5.2 
Subject Exclusion Criteria ......................................................................................... 9 
5.3 
Treatment Assignment Procedures ........................................................................ 10 
5.4 
Study Product Description ...................................................................................... 10 
5.5 
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product ................................................................................................................... 11 
5.6 
Accountability Procedures for the Study Intervention/Investigational Product(s) ... 12 
5.7 
Concomitant Medications ....................................................................................... 12 
6 
Study Schedule .................................................................................................................. 13 
6.1 
Schedule of Events ................................................................................................. 13 
6.2 
Day 0 ...................................................................................................................... 14 
6.3 
Post-vaccination Visits ............................................................................................ 14 
6.4 
Early Termination Visit ............................................................................................ 14 
6.5 
Definition of Evaluations ......................................................................................... 14 
7 
ADVERSE EVENTS AND TOXICITY MANAGEMENT ...................................................... 17 
7.1 
Definitions of AEs, Adverse Reactions, and Serious Adverse Events .................... 17 
7.2 
Assessment of Adverse Events and Serious Adverse Event ................................. 18 
7.3 
Instructions for Reporting Adverse Events and Serious Adverse Events ............... 19 
7.4 
Reporting Procedures ............................................................................................. 19 
7.5 
Safety Oversight ..................................................................................................... 19 
8 
CRITERIA FOR STUDY DISCONTINUATION .................................................................. 20 
8.1 
Criteria for Permanent Study Discontinuation ........................................................ 20 
9 
Statistical Considerations ................................................................................................... 21 
9.1 
Study Hypotheses .................................................................................................. 21 
9.2 
Sample Size Considerations .................................................................................. 21 
9.3 
Final Analysis Plan ................................................................................................. 21 
10 
Source Documents and Access to Source Data/Documents ............................................. 22 
11 
Quality Control and Quality Assurance .............................................................................. 23 
12 
Ethics/Protection of Human Subjects ................................................................................. 24 
12.1 
Ethical Standard ..................................................................................................... 24 
12.2 
Institutional Review Board ...................................................................................... 24 
12.3 
Subject Confidentiality ............................................................................................ 24 
12.4 
Future Use of Stored Specimens ........................................................................... 24 
13 
Data Handling and Record Keeping ................................................................................... 26 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
3 
 
13.1 
Data Management Responsibilities ........................................................................ 26 
13.2 
Data Capture Methods ........................................................................................... 26 
13.3 
Types of Data ......................................................................................................... 26 
13.4 
Timing/Reports ....................................................................................................... 26 
13.5 
Study Records Retention ........................................................................................ 27 
13.6 
Protocol Deviations ................................................................................................. 27 
14 
Publication Policy ............................................................................................................... 28 
15 
References ......................................................................................................................... 29 
16 
APPENDIX 1: Blood draw instructions for The Effects of Attenuated Versus Inactivated Flu 
Vaccine in Twin Sets (IRB-50200) ..................................................................................... 30 
 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
4 
 
1  
PROTOCOL SUMMARY 
 
Title: The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets 
Phase:  IV 
Population: Up to 40 healthy children and adult twins (40 total twin pairs), ages 2-20 years, will  
be randomized to either Group A or Group B (1 twin pair to group A and 1 twin pair to Group B): 
 
Group A:  Up to 10 monozygotic (MZ) and 10 dizygotic (DZ) twin volunteers will be given seasonal 
quadrivalent inactivated influenza vaccine (IIV4).  
 
Group B:  Up to 10 MZ and 10 DZ twin volunteers will be given quadrivalent live, attenuated influenza 
vaccine (LAIV4).  
 
Number of Sites: 1 
Study Duration: 60 months for enrollment (48 months for enrollment), study completion, analysis 
Subject Participation Duration: 4-5 weeks 
Description of Agent or Intervention:   
Quadrivalent, Inactivated Influenza Vaccine (IIV4) 
Quadrivalent, Live Attenuated Influenza Vaccine (LAIV4)  
Objectives: 
Primary: Determine the proportion of participants experiencing Grade 2 or Higher 
Adverse Effects 
Secondary: Determine HAI Titers at Week 4 after vaccination 
Description of Study Design:  This is a Phase IV study of healthy monozygotic and dizygotic children 
and adults who will receive the current seasonal influenza vaccine. Twin pairs will be randomized to 
either Group A or Group B (one twin to Group A and one twin to Group B). There are no exclusions for 
gender, ethnicity or race. The volunteers will be enrolled into one of 2 groups over a 4-year period. 
Immunization will be at Day 0, blood samples for immunogenicity assays will be collected prior to 
immunization and at Days 7 (6-8) and 28 (21-35).  
 
Group A: Up to 10 monozygotic (MZ) and 10 dizygotic (DZ) twin volunteers, 2-20 years old, will be given 
seasonal quadrivalent inactivated influenza vaccine (IIV4). Each volunteer will complete a total of 3 
visits:  Day 0, Day 7 and Day 28.  
 
Group B: Up to 10 MZ and 10 DZ twin volunteers, 2-20 years old will be given seasonal quadrivalent 
live, attenuated influenza vaccine (LAIV4). Each volunteer will complete a total of 3 visits: Day 0, Day 7 
and Day 28. 
Estimated Time to Complete Enrollment: Participants will be enrolled Aug-Feb in each of 4 years  
  
 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
5 
 
2 
OBJECTIVES 
Primary: Determine the proportion of participants experiencing Grade 2 or Higher Adverse Effects 
 
Secondary: Determine HAI Titers at Week 4 after vaccination 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
6 
 
3 
BACKGROUND AND RATIONALE 
Influenza virus infection represents a major cause of morbidity and mortality in the United States, 
resulting in 9.2-35.6 million illnesses, 140,000-710,000 hospitalizations, and 12,000-56,000 deaths 
annually since 2010 (1). This results in life-years lost, quality of life reduced, and an economic and 
medical cost of between $71 billion and $167 billion per year. Along with the fact that influenza can 
trigger deadly pandemics such as the 1918 pandemic that resulted in the deaths of 50 million to 
100 million people (3-5% of the global population), these data illustrate the high level of priority 
needed to curb the public health threat of influenza. Influenza continues to be a problem due to the 
problems with the current vaccine strategy. Now our vaccines incorporate only three or four of the 
large number of circulating strains, with no adjuvant except for the elderly, and provide only 10-
60% protection from influenza infection (2). Annual modifications of the vaccine are required each 
year, based on predictions of the next season’s circulating strains. This strategy is often thwarted 
by the annual antigenic changes that occur in influenza virus composition. In addition to the issues 
with seasonal influenza, pandemic influenza typically starts with unpredicted strains, for which 
entirely new vaccines are required, significantly hindering our preparedness. These reasons and 
more highlight the necessity for a better understanding of the key components of an effective 
immune response and how this could lead to a universal vaccine. In study, we will use twins to 
investigate the role of genetics in immune response to both inactivated influenza vaccine (IIV4) and 
live attenuated inactivated vaccine (LAIV4). 
 
3.1 
Potential Risks and Benefits 
3.1.1 Potential Risks 
 
This protocol will immunize children and adult participants with either IIV4 (intramuscular) or 
LAIV4 (intranasal) vaccines that are licensed and recommended for use in the age groups 
studied. The discomforts of this study are those of receiving IM injection or intranasal 
application of the vaccine, and blood drawn from an arm vein, and possible reactions to the 
vaccine. Drawing blood causes transient discomfort and may cause fainting. Infection at the 
site where blood will be drawn or where the vaccination is given is extremely unlikely, but is 
a potential risk. Bruising at the site of blood drawing may occur, but can be prevented or 
lessened by applying pressure for several minutes immediately after the blood draw. 
Immediate allergic reactions to vaccine, including anaphylaxis, are in general extremely 
rare (approximately 1 person in 4,000,000), and might occur as a skin rash such as hives, 
difficulty breathing, fainting, drop in the blood pressure and death. Such reactions can 
usually be stopped by emergency medications administered by study personnel. Vaccine 
recipients may develop systemic reactions such as fever, headaches, body aches, and 
fatigue. These reactions are usually greatest within the first 24 to 72 hours after vaccination 
and last 1 to 2 days. Analgesics (e.g., aspirin or Tylenolâ) and rest will generally relieve or 
moderate these symptoms. Other hypersensitivity reactions, including Arthus reactions 
resulting in large local swelling reactions, are also possible. Although Guillain-Barré 
syndrome may have been associated with the 1976-77 inactivated swine influenza vaccine 
and TIV vaccines used in early 1990's, subsequent inactivated vaccines have not been 
associated with an increased risk of this condition. LAIV4 has the potential for transmission 
of influenza to close contacts with severely weakened immune systems (requiring care in a 
protected environment, such as a bone marrow transplant unit).  It is not known whether 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
7 
 
LAIV4 (FluMist Quadrivalent) is excreted in human milk. Because some viruses are 
excreted in human milk, lactating women will not be eligible for this study. 
 
3.1.2 Known Potential Benefits 
 
Participants given the seasonal influenza vaccine are likely to experience decreased 
frequency and severity of subsequent influenza infection. The beneficial role of influenza 
vaccination has been recognized increasingly over the past several years as more 
information has become available about the high rate of morbidity and mortality from this 
respiratory pathogen.  

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
8 
 
4 
STUDY DESIGN 
 
This is a phase IV study of 40 healthy 2-20 year old volunteers (all members of twin sets) who are 
given licensed seasonal influenza vaccine. Following confirmation of written informed consent (and 
assent, if applicable), baseline blood samples will be drawn from all study participants prior to 
immunization. The volunteers will be enrolled into one of 2 groups (randomized by twin pair): 
 
Group A: Up to 10 healthy MZ and 10 healthy DZ twin volunteers, 2-20 year old, will be given 
seasonal quadrivalent inactivated influenza vaccine (IIV4). Each volunteer will complete a total of 3 
visits: Day 0 (pre-immunization), and Day 7 +/- 1  and Day 28 +/-  7 (post-immunization). All visits 
will consist of drawing blood for study assays and monitoring for adverse events (SAEs) and 
adverse events (AEs). 
 
Group B: Up to 10 healthy MZ and 10 healthy DZ twin volunteers, 2-20 year old, will be given 
quadrivalent live, attenuated influenza vaccine (LAIV4). Each volunteer will complete a total of 3 
visits: Day 0 (pre-immunization), and Day 7 +/- 1 and Day 28 +/- 7 (post-immunization). All visits 
will consist of drawing blood for study assays and monitoring for serious adverse events (SAEs) 
and adverse events (AEs). 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
9 
 
5 
STUDY ENROLLMENT AND WITHDRAWAL 
 
5.1 
Subject Inclusion Criteria 
 
1. 
2-20 years old with a weight of ≥10.0 kilograms 
2. 
Member of a monozygotic (MZ) or dizygotic (DZ) twin set 
3. 
Willing to complete the informed consent process (and assent for minors 2-17 years of 
age) 
4. 
Twin pair also agreeable and eligible for study inclusion 
5. 
Availability for follow-up for the planned duration of the study (at least 28 days after 
immunization) 
 
5.2 
Subject Exclusion Criteria 
 
1.  Prior off-study vaccination with seasonal influenza vaccine within three months of study 
vaccination 
2.  Life-threatening reactions to previous influenza vaccinations 
3.  Asthma 
4.  Allergy to egg or egg products or to vaccine components including gentamicin, gelatin, 
arginine or MSG.  
5.  Active systemic or serious concurrent illness, including febrile illness on the day of 
vaccination 
6.  History of immunodeficiency (including HIV infection) 
7.  Known or suspected impairment of immunologic function; may include significant liver 
disease, diabetes mellitus treated with insulin or moderate to severe renal disease 
8.  Hospitalization in the past year for congestive heart failure or emphysema. 
9.  Chronic Hepatitis B or C. 
10.  Recent or current use of immunosuppressive medication, including systemic 
glucocorticoids (corticosteroid nasal sprays and topical steroids are permissible). Use 
of oral steroids (≤10mg prednisone-equivalent/day) may be acceptable after review by 
the investigator. 
11.  Participants in close contact with anyone who has a severely weakened immune 
system  
12.  Malignancy (other than squamous cell or basal cell skin cancer) within the past 5 years 
13.  Autoimmune disease (such as rheumatoid arthritis, lupus, etc.) treated with 
immunosuppressive medication such as plaquenil, methotrexate, prednisone, Enbrel 
14.  History of blood dyscrasias, renal disease, or hemoglobinopathies requiring regular 
medical follow up or hospitalization during the preceding year 
15. Receipt of an influenza antiviral medication within 48 hours prior to study vaccination [If 
yes, may not eligible if unable to schedule at an appropriate interval]. 
16.  Receipt of blood or blood products within the past 6 months or planned used during the 
study. 
17.  Receipt of an inactivated vaccine 14 days prior to study enrollment, or planned 
vaccinations prior to completion of last study visit (~ 28 days after study vaccination) 
18.  Receipt of a live, attenuated vaccine within 28 days prior to enrollment or planned 
vaccination during post-vaccination follow-up (~ 28 days after study vaccination) 
19.  Need for allergy immunization (that cannot be postponed) until after the last study visit. 
20.  History of Guillain–Barré syndrome 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
10 
 
21.  Pregnant or breastfeeding woman 
22.  Use of investigational agents within 30 days prior to enrollment or planned use during 
the study. 
23.  Donation of the equivalent of a unit of blood within 6 weeks prior to enrollment or 
donation of platelets within 2 weeks of enrollment or planned donation prior to 
completion of the last visit. 
24.  Any condition which, in the opinion of the investigator, might interfere with volunteer 
safety, study objectives or the ability of the participant to understand or comply with the 
study protocol. 
 
5.3 
Treatment Assignment Procedures 
 
5.3.1 Randomization Procedures 
Individuals within each pair of twins will be randomized such that one receives IIV4 (Group 
A) and the other LAIV4 (Group B). Randomization will follow a permuted block design, 
where each twin pair is a block. Random assignment within each pair will be done by 
computer. Study Intervention/Investigational Product 
 
5.4 
Study Product Description 
 
6.1.1 Acquisition 
 
The study product will be shipped from the manufacturer to the study site.  The 
quadrivalent IIV4 will be supplied by Sanofi-Pasteur as Fluzone® Quadrivalent.  The 
LAIV4 will be supplied by MedImmune as FluMist® Quadrivalent Influenza Vaccine Live, 
Nasal Spray.  
 
 
6.1.2 Formulation, Packaging, and Labeling 
 
FDA’s Vaccines and Related Biological Products Advisory Committee (VRBPAC) will make 
annual recommendations regarding the composition of the U.S. seasonal influenza 
vaccines based on global influenza virus surveillance data related to epidemiology and 
antigenic characteristics, serological responses to trivalent and quadrivalent seasonal 
vaccines, and the availability of candidate strains and reagents.   
 
The season-specific manufacturer package inserts for each study year will be attached in 
the IRB protocol submissions when available. 
 
Fluzone® Quadrivalent: Each 0.5 mL dose of Fluzone Quadrivalent (IIV4) will contain a 
total of 60 µg (15 μg of each strain) of influenza virus hemagglutinin of each of the 4 strains 
selected for the seasonal formulation. The vaccine will be supplied in a prefilled, single 
dose syringe, 0.5 mL (no preservative), single dose vial 0.5 mL (no preservative) or multi-
dose vial, 5 mL (with preservative). Each multi-dose vial contains ten 0.5 mL doses. 
 
FluMist® (Influenza Virus Vaccine) Live, Intranasal Spray. 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
11 
 
Each pre-filled refrigerated FluMist® Quadrivalent sprayer contains a single 0.2 mL dose. 
Each 0.2 mL dose contains 106.5-7.5 FFU (fluorescent focus units) of live attenuated 
influenza virus reassortants of each of the selected strains. Each 0.2 mL dose may also 
contains 0.188 mg/dose monosodium glutamate, 2.00 mg/dose hydrolyzed porcine gelatin, 
2.42 mg/dose arginine, 13.68 mg/dose sucrose, 2.26 mg/dose dibasic potassium 
phosphate, and 0.96 mg/dose monobasic potassium phosphate. Each dose contains 
residual amounts of ovalbumin (< 0.24 mcg/dose), and may also contain residual amounts 
of gentamicin sulfate (< 0.015 mcg/mL), and ethylenediaminetetraacetic acid (EDTA) (< 
0.37 mcg/dose).  FluMist® Quadrivalent contains no preservatives. It is supplied in a 
package of 10 pre-filled, single-dose (0.2 mL) intranasal sprayers. The single-use intranasal 
sprayer contains no natural rubber latex.  
 
6.1.2 Product Storage and Stability 
 
Fluzone® Quadrivalent vaccine presentations should be refrigerated at 2° to 8°C (35° to 
46°F). Vaccine that has been frozen will be discarded. Between uses, multi-dose vials will 
be returned to the recommended storage conditions at 2° to 8°C (35° to 46°F). Vaccine 
should not be used after the expiration date shown on the label.   
 
FluMist® Quadrivalent should be stored in a refrigerator between 2 to 8°C (35-46°F) upon 
receipt and until use.  The product must be used before the expiration date on the sprayer 
label and must not be frozen. The cold chain (2 to 8°C) must be maintained when 
transporting FluMist® Quadrivalent. Once FluMist® Quadrivalent has been administered; 
the sprayer should be disposed of according to the standard procedures for medical waste 
(e.g., sharps container or biohazard container). Vaccine should not be used after the 
expiration date shown on the label. 
 
5.5 
Dosage, Preparation and Administration of Study 
Intervention/Investigational Product 
 
For children between 2-3 years, IIV4 vaccine will be administered as a 0.25 mL dose, with a 
sterile, disposable syringe and needle by IM injection into the vastus lateralis muscle 
depending on the development of the deltoid. For participants 3 years or older, IIV4 vaccine 
will be administered as a 0.5 mL dose, with a sterile, disposable syringe and needle by IM 
injection into the deltoid muscle. The participant/guardian will choose whether the injection 
will be administered into the right or left deltoid. 
 
LAIV4 vaccine dosage is 0.2 mL Vaccine will be administered as an intranasal spray. Each 
sprayer contains a single dose of FluMist® Quadrivalent; approximately one-half of the 
contents should be administered into each nostril. 0.1 mL (i.e., half of the dose from a 
single FluMist sprayer) is administered into each nostril while the recipient is in an upright 
position. Insert the tip of the sprayer just inside the nose and rapidly depress the plunger 
until the dose-divider clip stops the plunger. The dose divider clip is removed from the 
sprayer to administer the second half of the dose (0.1 mL) into the other nostril. 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
12 
 
 
5.6 
Accountability Procedures for the Study 
Intervention/Investigational Product(s) 
 
The study article will be shipped by the manufacturer to the study site. Use and disposition 
of each unit of vaccine will be appropriately documented by the Clinical Core staff in 
accordance with ICH GCP.  
 
5.7 
Concomitant Medications 
 
At entry and at each study visit, the participant will be questioned about medication use and 
the information will be recorded. Medication history will include vaccinations, allergy shots 
or PPD tests received and medications taken within 28 days prior to enrollment, as well as 
vaccinations, allergy shots or PPD tests received and current prescription and over-the-
counter medications used throughout the study period. The investigator should be 
consulted regarding eligibility if the participant is taking oral steroid medications or 
medications for treatment of autoimmune disease (such as plaquenil, methotrexate, 
prednisone, Enbrel), or any other medications which might indicate a condition that 
precludes participant compliance with the protocol. 
 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
13 
 
6 
STUDY SCHEDULE 
6.1 
Schedule of Events 
 
 
Day 0 
Day 7 
Day 28 
Early Termination Visit 
Written Informed Consent/Assent 
X 
 
 
 
Eligibility Determination 
X 
 
 
 
Randomization 
X 
 
 
 
Demographics/Medical History 
X 
 
 
 
Concomitant Medications 
X 
X 
X 
X 
Signs/Symptoms/Adverse Events 
X 
X 
X 
X 
Vital Signs/Weight/Height 
X 
If clinically indicated 
Phlebotomy 
X 
X 
X 
 
IIV4 or LAIV4 Administration 
X 
 
 
 
 
 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
14 
 
 
6.2 
Day 0 
 
The following will be performed at Screening/Entry (see Section 7.6 for a detailed 
explanation of each evaluation): 
 
• 
Written Informed Consent 
• 
Eligibility Determination 
• 
Randomization 
• 
Medical History 
• 
Concomitant Medications 
• 
Signs/Symptoms/Adverse Events 
• 
Vital Signs/Weight/Height 
• 
Phlebotomy 
• 
IIV4 or LAIV Administration 
6.3 
Post-vaccination Visits 
 
Post-vaccination visits occur following the Screening/Entry Visit and occur at occur at Day 7 
(+/- 1 day) and Day 28 (+/- 7 days) after IIV or LAIV administration. 
 
The following will occur at each Post-vaccination Visit: 
 
• 
Concomitant medications 
• 
Signs/Symptoms/Adverse Events 
• 
Vital Signs, if clinically indicated 
• 
Phlebotomy 
6.4 
Early Termination Visit 
 
The Protocol Director should be notified within 48 hours if a participant terminates the study 
prior to completing all study visits.  
 
If a participant discontinues the study early for any reason then the following assessments 
for the Early Termination Visit should occur: 
 
• 
Concomitant medications 
• 
Signs/Symptoms/Adverse Effects 
• 
Vital Signs, if clinically indicated 
6.5 
Definition of Evaluations 
 
7.6.1 Written Informed Consent 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
15 
 
Participants will be given the IRB-approved informed consent form (or assent form for 
minors participating in the study) at the Day 0 Visit. A discussion of risks and possible 
benefits of participation in this study will be provided to the participants.  Participants then 
will be asked for any questions and to read/sign the consent form. The consent (and 
assent, if applicable) form will be signed prior to the performance of any study procedures. 
A copy of the informed consent (and assent, if applicable) document will be given to the 
participant for his/her records.  The rights and welfare of the participants will be protected 
by emphasizing to them that the quality of their medical care will not be adversely affected if 
they decline to participate in this study. 
 
6.6.2 Eligibility Determination 
 
At the completion of all protocol-defined screening procedures, the Eligibility Determination 
Form will be completed and signed by the Study Coordinator according to study criteria 
(see sections 4.1 and 4.2). Eligible participants giving informed consent (and assent, if 
applicable) will be enrolled in the study.  
 
6.6.3 Randomization 
After a twin-set are deemed eligible, individuals within each pair of twins will be randomized 
based on a computer code with the older twin being given the first assignment generated by 
computer and the younger twin being given the other assignment.  
 
6.6.4 Demographics/Medical History 
 
Participant demographics including date of birth, sex, and race/ethnicity will be 
documented. A medical history will be obtained and must be recorded in the source 
documents. The medication history must include: 
 
• 
All active medical diagnoses (acute or chronic); each medication on participant’s 
medication list should be assigned to a medical diagnosis 
• 
History of tonsillectomy or splenectomy, allergies, or autoimmune disease 
• 
Influenza Vaccination History over the past 5 years 
 
6.6.5 Concomitant Medications 
 
At the Entry Visit record all current and past (within the last 28 days) prescription and non-
prescription medications. At subsequent visits, record changes in medications since the last 
study visit. Additionally, record any vaccinations, allergy shots, or PPD tests received within 
the past 28 days.  
 
 
6.6.6 Signs/Symptoms/Adverse Events 
 
All Grade 2 or higher signs, symptoms, and toxicities must be documented in the 
participant’s record.  
 
Any sign or symptom that leads to discontinuation of study, regardless of grade, must be 
recorded on source documents. 
 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
16 
 
Refer to the Common Terminology for Adverse Effects, Version 5.0, November 27, 2017, 
located at 
https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/CTCAE_v5_Quic
k_Reference_8.5x11.pdf 
 
Report diagnoses for clinical events and other diseases and record whether they were 
related to study procedures.  
 
6.6.7 Vital Signs/Weight/Height 
 
Assessment of vital signs will include measurement of resting blood pressure, pulse, 
respiratory rate, and temperature. Weight should be measured in kilograms. Height should 
be measured in centimeters. 
 
6.6.8 Phlebotomy 
 
Phlebotomy will be performed at each visit using antiseptic technique. See Appendix 1: 
Blood Draw Requirements for blood draw instructions for each visit. Maximal amount for the 
blood draw will be based on weight and will not exceed 3% of total blood volume in a 24-
hour period consistent with the World Health Organization guidance. (3) 
 
6.6.9 IIV4 or LAIV Administration 
 
Participants will be administered the seasonal IIV4 vaccine in the left or right deltoid muscle 
using antiseptic technique or into the vastus lateralis (right or left) for participants younger 
than 3 or for those with limited deltoid muscle development. Participants under the age of 3 
years will receive 0.25 mL and all other participants will receive the 0.5 mL dose. 
 
LAIV4 vaccine dosage is 0.2 mL Vaccine will be administered as an intranasal spray. Each 
sprayer contains a single dose of FluMist® Quadrivalent; approximately one-half of the 
contents should be administered into each nostril. 0.1 mL (i.e., half of the dose from a 
single FluMist sprayer) is administered into each nostril while the recipient is in an upright 
position. Insert the tip of the sprayer just inside the nose and rapidly depress the plunger 
until the dose-divider clip stops the plunger. The dose divider clip is removed from the 
sprayer to administer the second half of the dose (0.1 mL) into the other nostril. 
Volunteers will be observed in clinic for 15 minutes after vaccination to monitor for any 
immediate serious reactions. 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
17 
 
7 
ADVERSE EVENTS AND TOXICITY MANAGEMENT  
7.1 
Definitions of AEs, Adverse Reactions, and Serious Adverse 
Events 
An adverse event (AE) is any untoward medical occurrence in a clinical study participant 
administered a pharmaceutical product, which does not necessarily have a causal 
relationship with the treatment. An AE can therefore be any unfavorable and/or unintended 
sign, symptom, or disease temporally associated with the use of a medicinal product, 
whether or not considered related to the medicinal product. AEs may also include pre-or 
post-treatment complications that occur as a result of protocol specified procedures, lack of 
efficacy, overdose, drug abuse/misuse reports, or occupational exposure. Preexisting 
events that increase in severity or change in nature during or as a consequence of 
participation in the clinical study will also be considered AEs. An AE does not include the 
following:  
 
• 
Medical or surgical procedures such as surgery, endoscopy, tooth extraction, and 
transfusion. The condition that led to the procedure may be an adverse event and must be 
reported   
• 
Pre-existing diseases, conditions, or laboratory abnormalities present or detected before 
the Screening Visit that do not worsen  
• 
Situations where an untoward medical occurrence has not occurred (e.g., hospitalization for 
elective surgery, social and/or convenience admissions) 
• 
Overdose without clinical sequelae 
• 
Any medical condition or clinically significant laboratory abnormality with an onset date 
before the consent form is signed and not related to a protocol-associated procedure is not 
an AE. It is considered to be pre-existing and should be documented as medical history. 
 
A serious adverse event (SAE) is defined as an event that results in the following:  
• 
Death  
• 
Life-threatening (note: The term “life-threatening” in the definition of “serious” refers to an 
event in which the participant was at risk of death at the time of the event; it does not refer 
to an event that hypothetically might have caused death if it were more severe.)  
• 
In-patient hospitalization or prolongation of existing hospitalization  
• 
Persistent or significant disability/incapacity 
• 
A congenital anomaly/birth defect  
• 
A medically important event or reaction; such events may not be immediately life-
threatening or result in death or hospitalization but may jeopardize the participant or may 
require intervention to prevent one of the other outcomes constituting SAEs. Medical and 
scientific judgment must be exercised to determine whether such an event is reportable 
under expedited reporting rules. Examples of medically important events include intensive 
treatment in an emergency room or at home for allergic bronchospasm; blood dyscrasias or 
convulsions that do not result in hospitalization; and development of drug dependency or 
drug abuse. For the avoidance of doubt, infections resulting from contaminated medicinal 
product will be considered a medically important event and be subject to expedited 
reporting requirements.  
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
18 
 
Clinical laboratory abnormalities and other abnormal assessments without clinical 
significance are not recorded as AEs or SAEs. However, laboratory abnormalities that 
require medical or surgical intervention or lead to study drug discontinuation must be 
recorded as an AE, as well as an SAE, if applicable. In addition, laboratory or other 
abnormal assessments (e.g., electrocardiogram, x-rays, vital signs) that are associated with 
signs and/or symptoms must be recorded as an AE or SAE if they meet the definition of an 
AE or SAE as described above. If the laboratory abnormality is part of a syndrome, record 
the syndrome or diagnosis (e.g., anemia), not the laboratory result (i.e., decreased 
hemoglobin).  
7.2 
Assessment of Adverse Events and Serious Adverse Event 
 
The Protocol Director is responsible for assessing AEs and SAEs for causality and severity, 
and for final review and confirmation of accuracy of event information and assessments.  
 
The Protocol Director is responsible for assessing the relationship to study drug treatment 
using clinical judgment and the following considerations:  
 
• 
No: Evidence exists that the adverse event has an etiology other than the study 
drug. For SAEs, an alternative causality must be provided (e.g., pre-existing 
condition, underlying disease, intercurrent illness, or concomitant medication).  
 
• 
Yes: There is reasonable possibility that the event may have been caused by the 
investigational medicinal product. 
 
Ineffective treatment should not be considered as causally related in the context of adverse 
event reporting. The relationship to study procedures (e.g., invasive procedures such as 
venipuncture or biopsy) should be assessed using the following considerations:  
 
• 
No: Evidence exists that the adverse event has an etiology other than the study 
procedure.  
 
• 
Yes: The adverse event occurred as a result of protocol procedures (e.g., 
venipuncture). 
 
The severity grading of AEs will be assessed as Grade 1, 2, 3, or 4 using the Common 
Terminology for Adverse Effects, Version 5.0, November 27, 2017, located at 
https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/CTCAE_v5_Quic
k_Reference_8.5x11.pdf and Grade 2 or higher AEs recorded. 
 
For AEs associated with laboratory abnormalities, the event should be graded on the basis 
of the clinical severity in the context of the underlying conditions; this may or may not be in 
agreement with the grading of the laboratory abnormality. 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
19 
 
7.3 
Instructions for Reporting Adverse Events and Serious Adverse 
Events 
 
SAEs, regardless of cause or relationship, that occur after the participant first consents to 
participate in the study (i.e., signing the informed consent) and throughout the duration of 
the study, including the protocol-required post treatment follow-up period, must be reported 
on the CRF. This also includes any SAEs resulting from protocol-associated procedures 
performed from screening onwards. All AEs, regardless of cause or relationship, that occur 
from initiation of study drug until 4 weeks after last administration of study drug must be 
reported on the CRF. Any SAEs and deaths that occur after the Post-Treatment follow-up 
visit OR within 30 days of the last dose of study drug (whichever is longer), regardless of 
causality, should also be reported. All AEs should be followed up until resolution or until the 
adverse event is stable, if possible. Investigators are not obligated to actively seek SAEs 
after the 30-day period.  
 
7.4 
Reporting Procedures 
 
7.4.1 Regulatory Reporting for Studies Not Conducted Under an IND 
 
For those events meeting the previously described definition of Serious Adverse Events, 
the completion of an SAE report form is required. For SAEs related to vaccine, a VAERS 
form will be filled out and submitted to the Vaccine Adverse Event Reporting System 
(VAERS) per federal regulations. The VAERS form will simultaneously be sent to the 
sponsor.  SAEs and events that meet the Prompt Reporting guidelines (events meeting the 
criteria for Unanticipated Problems) will also be reported to the Stanford IRB as required.  
Unexpected deaths or life-threatening experiences related to the research will be reported 
to the sponsor and to the IRB within 5 working days from when the investigator learns of 
event.  SAEs not related to vaccine will be reported to the IRB on an annual basis. 
 
7.5 
Safety Oversight  
 
The Clinical Core Principal Investigator will oversee compliance with the protocol, the 
participant’s safety and any unanticipated problems involving risks to participants and will 
report these events as described above.  Unanticipated problems and serious adverse 
events will be reported to the Stanford IRB as required.  
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
20 
 
8 
CRITERIA FOR STUDY DISCONTINUATION 
8.1 
Criteria for Permanent Study Discontinuation 
 
• 
Requirement for prohibited concomitant medications (such as immunosuppressant 
medication) 
• 
Clinical reasons such that continued participation in the study may be detrimental to the 
participant’s health as judged by the Protocol Director 
• 
Failure by the participant without reasonable cause to attend study visits 
• 
Request by the participant to withdraw. 
• 
Request of the primary care provider if s/he thinks the study is no longer in the best 
interest of the participant. 
• 
Participant judged by the investigator to be at significant risk of failing to comply with the 
provisions of the protocol as to cause harm to self or seriously interfere with the validity 
of the study results. 
• 
Participant died.   
• 
At the discretion of the IRB or Study Investigators. 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
21 
 
9 
STATISTICAL CONSIDERATIONS 
 
9.1 
Study Hypotheses 
 
We hypothesize that exposure to influenza vaccination will result in immunity to influenza in 
the majority of study participants and will be well-tolerated, forming the basis of novel 
immunologic insights. 
 
9.2 
Sample Size Considerations 
 
The sample size was selected based on feasibility. 
 
9.3 
Final Analysis Plan 
 
We will report the percentage of participants having a 4-fold increase in HAI titers at Week 
4 and the percentage of participants experiencing Grade 2 or higher AEs. 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
22 
 
10 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
 
All participant information will be obtained by the investigators and their support staff and 
will remain confidential.  Specimens for research laboratory testing will be coded by a 
unique participant ID number and data for individual participants will be coded for data 
analysis.  A database containing a code key will be kept on a computer that is password 
secured or a locked cabinets and only available to clinical research staff.  Participants will 
not be identified in any reports or publications that may result from the study.  Personal 
identifierswill be removed for analyses and publications.   
 
Participant confidentiality is held strictly in trust by the participating investigators, their staff, 
and their agents. This confidentiality extends to genetic and biological sample tests, in 
addition to the clinical information relating to participating volunteers.  The study protocol, 
documentation, data, and all other information generated will be held in strict confidence.   
 
The clinical study site will permit access to all documents and records that may require 
inspection by the sponsor or its authorized representatives, including but not limited to, 
medical records (office, clinic or hospital) and pharmacy records for the participants in this 
study.   

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
23 
 
11 
QUALITY CONTROL AND QUALITY ASSURANCE 
 
Quality Control and Quality Assurance activities will generally be completed as outlined in 
the current version of the Stanford Quality Management Plan.  Chart audits for the trial will 
be conducted for research participant records utilizing the SLVP Chart Audit Tool.  Audits 
will be conducted on a random sampling of participant charts in accordance with the low 
risk to volunteers participating in the trial. Charts will be randomly selected from among 
those not previously audited.  Results of these audits will be summarized using the SLVP 
QM_QA Summary Report tool, and shared with research staff at staff meetings, as 
necessary. 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
24 
 
12 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
12.1 Ethical Standard 
 
The Protocol Director will ensure that this study is conducted in full conformity with the 
principles set forth in The Belmont Report: Ethical Principles and Guidelines for the 
Protection of Human Subjects of Research of the US National Commission for the 
Protection of Human Subjects of Biomedical and Behavioral Research (April 18, 1979) and 
codified in 45 CFR Part 46 and/or the ICH E6; 62 Federal Regulations 25691 (1997). 
   
12.2 Institutional Review Board 
 
Prior to enrollment of participants into this trial, the protocol and the informed consent form 
will be reviewed and approved by the appropriate IRB. The responsible official for the IRB 
will sign the IRB letter of approval of the protocol prior to the start of this trial and a copy will 
be provided to DAIT.  Notification of the IRB's composition and the institutions Federal Wide 
Assurance number will be provided to DAIT as needed. Should amendments to the protocol 
be required, the amendments will be reviewed by the sponsor and/or the investigators and 
submitted to the IRB. Volunteers will be compensated for their participation in this study. 
Compensation will be in accordance with the local IRB’s policies and procedures, and 
requires IRB approval. 
 
12.3 Subject Confidentiality 
 
All participant information will be obtained by the investigators and their support staff and 
will remain confidential.  Specimens for laboratory testing will be coded by participant 
number and data for individual participants will be coded for data analysis.  A database 
containing a code key will be kept on a computer that is password secured and available 
only to study staff.  Participants will not be identified (except for age) in any reports or 
publications that may result from the study.  Personal identifiers, except for age, will be 
removed for publications. Upon completion of the study, data containing PHI will be 
retained for 50 years from the time of informed consent.  
 
Participant confidentiality is held strictly in trust by the participating investigators, their staff, 
and their agents. This confidentiality extends to genetic and biological sample tests, in 
addition to the clinical information relating to participating volunteers. 
The study protocol, documentation, data, and all other information generated will be held in 
strict confidence.   
 
The clinical study site will permit access to all documents and records that may require 
inspection by the sponsor or its authorized representatives, including but not limited to, 
medical records (office, clinic or hospital) and pharmacy records for the participants in this 
study.   
 
 
12.4 Future Use of Stored Specimens 
 
After the study is complete, residual specimens will be stored for future research.  As new 
scientific discoveries identify technologies or mediators that might be useful in studying the 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
25 
 
immune response, stored samples may be used to explore new information that could be 
made available with these advanced methods.  Volunteer specimens will be stored under a 
unique identifier. The volunteer’s name or other personal identifiers will not be available in 
any data shared with outside investigators.  New studies using stored samples will be 
reviewed by the IRB as required. 
 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
26 
 
13 
DATA HANDLING AND RECORD KEEPING 
 
The investigator is responsible to ensure the accuracy, completeness, legibility, and 
timeliness of the data reported. All source documents and data collection forms should be 
completed in a neat, legible manner to ensure accurate interpretation of data. Black or dark 
blue ink is required to ensure clarity of reproduced copies. When making changes or 
corrections, the original entry will be crossed out with a single line, and initialed and dated 
to indicate the change. DO NOT ERASE, OVERWRITE, OR USE CORRECTION FLUID 
OR TAPE ON THE ORIGINAL. 
 
Data entered into the data entry system that is derived from source documents or data 
collection forms should be consistent with the source documents and data collection forms 
or the discrepancies should be explained. 
 
13.1 Data Management Responsibilities 
 
All source documents will be reviewed by the clinical team and data entry staff, who will 
ensure that they are accurate and complete. Serious adverse events will be assessed for 
causality, and reviewed by the site PI or designee. 
 
Data collection is the responsibility of the clinical trial staff at the site under the supervision 
of the site PI. During the study, the investigator must maintain complete and accurate 
documentation for the study. 
 
The PI (or designee) will be responsible for data management, quality review, analysis, and 
reporting of the study data. 
 
13.2 Data Capture Methods 
 
Clinical data (including SAEs) will be entered into a 21 CFR Part 11-compliant data entry 
system. The data system includes password protection and internal quality checks, such as 
automatic range checks, to identify data that appear inconsistent, incomplete, or inaccurate. 
Clinical data will be entered directly from the source documents or data collection forms.  
 
13.3 Types of Data 
 
Data for this study will include clinical, safety and outcome measures. 
 
13.4 Timing/Reports 
 
 
Data will be reviewed on an ongoing basis according to the site Quality Management Plan.  
Data analysis will begin once all clinical data have been collected and verified for accuracy. 
Preliminary analyses of outcome measures will begin as soon as laboratory data are 
available. Participants will receive a unique study ID at enrollment.  All data for study 
analysis will be coded by study ID number and will be password-protected.  Coded 
protected health information will be provided only as needed for data analysis of study 
outcome measures.  Personal identifierswill be removed for publications. 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
27 
 
 
Protected health information may be disclosed as requested by The Office for Human 
Research Protections in the U.S. Department of Health and Human Services, the sponsor, 
or Stanford University Administrative Panel on Human Subjects in Medical Research and 
any other unit of Stanford University as necessary. 
 
13.5 Study Records Retention 
 
Records and documents pertaining to the conduct of this study, including CRFs, source 
documents, data collection forms, consent forms, and medication inventory records, must 
be retained by the investigator for at least 3 years and in accordance with Stanford 
University and Stanford IRB requirements and until the sponsor authorizes transfer or 
destruction of study records.   
 
13.6 Protocol Deviations 
 
A protocol deviation is any noncompliance with the clinical trial protocol or Good Clinical 
Practice (GCP).  The noncompliance may be either on the part of the participant, the 
investigator, or the study site staff.  As a result of deviations, corrective actions are to be 
developed by the site and implemented promptly.  
 
It is the responsibility of the site to use continuous vigilance to identify and report 
deviations. All deviations from the Protocol will be addressed in a study subject data 
collection form. Protocol deviations will be sent to the local IRB/IEC per their guidelines.  
The site PI/study staff is responsible for knowing and adhering to their IRB requirements.  

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
28 
 
14 
PUBLICATION POLICY 
 
Following completion of the study, the investigator may publish the results of this research 
in a scientific journal. The International Committee of Medical Journal Editors (ICMJE) 
member journals have adopted a trials-registration policy as a condition for publication. This 
policy requires that all clinical trials be registered in a public trials registry such as 
ClinicalTrials.gov, which is sponsored by the National Library of Medicine (NLM). Other 
biomedical journals are considering adopting similar policies. Any clinical trial starting 
enrollment after 27SEP2007 must be registered either on or before the onset of participant 
enrollment.  
 
The ICMJE defines a clinical trial as any research project that prospectively assigns human 
subjects to intervention or comparison groups to study the cause-and-effect relationship 
between a medical intervention and a health outcome. Studies designed for other purposes, 
such as to study pharmacokinetics or major toxicity (e.g., Phase 1 trials), would be exempt 
from this policy.  
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
29 
 
15 
REFERENCES 
 
 
1. Osterholm MT, Kelley NS, Sommer A, Belongia EA: Efficacy and effectiveness of 
influenza vaccines: a systematic review and meta-analysis, Lancet Infect Dis 2012, 12:36-
44 
 
2. Goodwin K, Viboud C, Simonsen L: Antibody response to influenza vaccination in the 
elderly: a quantitative review. Vaccine. 2006 Feb 20;24(8):1159-69. 
 
3. Howie SR. Blood sample volumes in child health research: review of safe limits. Bull 
World Health Organ. 2011 Jan 1;89(1):46-53.  
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
30 
 
16 
APPENDIX 1: BLOOD DRAW INSTRUCTIONS FOR THE 
EFFECTS OF ATTENUATED VERSUS INACTIVATED FLU 
VACCINE IN TWIN SETS (IRB-50200) 
IRB approved 60 mL at all visits for individuals >30 kg; all blood draws will occur at CTRU and all 
samples will be sent to the HIMC for processing 
 
Blood Draw Instructions for individuals 10.0- <15.0 kilograms 
 (Max allowable volume to be collected = 23 mL) 
Visit 
Samples 
CTRU Processing Instructions 
Volume 
Collected 
Visit 1  
1 x 5 mL Serum Red Top 
1 x 2.5 mL PAXgene Tube  
1 x 10 mL Heparin Green Top  
1 x 4 mL Heparin Green Top      
Sera Aliquot:  approximately 5 
aliquots of 0.5mL/vial of the serum 
from 4 mL of blood. PAXgene saved 
for future testing; Plasma should be 
saved, and the PBMC processed 
and cryopreserved at 5-10 million 
cells/vial 
21.5 mL 
Visits 2 and 3 1 x 5 mL Serum Red Top 
1 x 10 mL Heparin Green Top  
2 X 4 mL Heparin Green Top 
 
Sera Aliquot: approximately 5 
aliquots of 0.5mL/vial of the serum 
from 4 mL of blood. Plasma should 
be saved, and the PBMC processed 
and cryopreserved at 5-10 million 
cells/vial 
23 mL 
 
 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
31 
 
 
Blood Draw Instructions for individuals 15.0- <20.0 kilograms 
(Max allowable volume to be collected = 33 mL) 
 
Visit 
Samples 
CTRU Processing Instructions 
Volume 
Collected 
Visit 1  
1 x 5 mL Serum Red Top 
1 x 2.5 mL PAXgene Tube  
2 x 10 mL Heparin Green Top  
1 x 4 mL Heparin Green Top      
Sera Aliquot:  approximately 5 
aliquots of 0.5mL/vial of the serum 
from 4 mL of blood. PAXgene saved 
for future testing; Plasma should be 
saved, and the PBMC processed 
and cryopreserved at 5-10 million 
cells/vial 
31.5 mL 
Visits 2 and 3 1 x 5 mL Serum Red Top 
2 x 10 mL Heparin Green Top  
2 X 4 mL Heparin Green Top 
 
Sera Aliquot: approximately 5 
aliquots of 0.5mL/vial of the serum 
from 4 mL of blood. Plasma should 
be saved, and the PBMC processed 
and cryopreserved at 5-10 million 
cells/vial 
33 mL 
 
 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
32 
 
 
Blood Draw Instructions for individuals 20.0- <25.0 kilograms 
(Max allowable volume to be collected = 44 mL) 
 
Visit 
Samples 
CTRU Processing Instructions 
Volume 
Collected 
Visit 1  
1 x 10 mL Serum Red Top 
1 x 2.5 mL PAXgene Tube  
3 x 10 mL Heparin Green Top  
 
Sera Aliquot:  approximately 10 
aliquots of 0.5mL/vial of the serum 
from 10mL of blood. PAXgene saved 
for future testing; Plasma should be 
saved, and the PBMC processed 
and cryopreserved at 5-10 million 
cells/vial 
42.5 mL 
Visits 2 and 3 1 x 10 mL Serum Red Top 
3 x 10 mL Heparin Green Top  
1 X 4 mL Heparin Green Top 
 
Sera Aliquot: approximately 4 
aliquots of 0.5mL/vial of the serum 
from 4 mL of blood. Plasma should 
be saved, and the PBMC processed 
and cryopreserved at 5-10 million 
cells/vial 
44 mL 
 
 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
33 
 
 
Blood Draw Instructions for individuals 25.0- <30.0 kilograms 
(Max allowable volume to be collected = 54 mL) 
 
Visit 
Samples 
CTRU Processing Instructions 
Volume 
Collected 
Visit 1  
1 x 10 mL Serum Red Top 
1 x 2.5 mL PAXgene Tube  
4 x 10 mL Heparin Green Top  
 
Sera Aliquot:  approximately 10 
aliquots of 0.5mL/vial of the serum 
from 10mL of blood. PAXgene saved 
for future testing; Plasma should be 
saved, and the PBMC processed 
and cryopreserved at 5-10 million 
cells/vial 
52.5 mL 
Visits 2 and 3 1 x 10 mL Serum Red Top 
4 x 10 mL Heparin Green Top  
1 X 4 mL Heparin Green Top 
 
Sera Aliquot: approximately 4 
aliquots of 0.5mL/vial of the serum 
from 4 mL of blood. Plasma should 
be saved, and the PBMC processed 
and cryopreserved at 5-10 million 
cells/vial 
54 mL 
 
 
 

The Effects of Attenuated Versus Inactivated Flu Vaccine in Twin Sets. V1_13AUG2019      
                                        
34 
 
Blood Draw Instructions for individuals ≥30.0 kilograms 
(Max allowable volume to be collected per IRB = 60 mL) 
 
Visit 
Samples 
CTRU Procession Instructions 
Volume 
Collected 
Visit 1  
1 x 10 mL Serum Red Top 
1 x 2.5mL PAXgene Tube  
4 x 10mL Heparin Green Top  
1 x 4mL Heparin Green Top      
Sera Aliquot:  approximately 10 
aliquots of 0.5mL/vial of the serum 
from 10mL of blood. PAXgene saved 
for future testing; Plasma should be 
saved, and the PBMC processed 
and cryopreserved at 5-10 million 
cells/vial  
56.5 mL 
Visits 2 and 3 
1 x 10 mL Serum Red Top 
5 x 10mL Heparin Green Top  
 
Sera Aliquot:  approximately 10 
aliquots of 0.5mL/vial of the serum 
from 10mL of blood. Plasma should 
be saved, and the PBMC processed 
and cryopreserved at 5-10 million 
cells/vial 
60 mL