Evaluation of the Effects of Age, Prior Exposure, and Previous Vaccination on the B Cell Response to Inactivated Influenza Vaccine in Healthy Adults and Children
Phase: PHASE4
Conditions: Influenza, Influenza Immunisation
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

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Evaluation of the effects of age, prior exposure, and previous 
vaccination on the B cell response to inactivated influenza 
vaccine in healthy adults and children
DMID Protocol Number: 
16-0101
Sponsored by:
National Institute of Allergy and Infectious Diseases (NIAID)
DMID Funding Mechanism:
HHSN266200700008C
Site Principal Investigator:
Angela Branche, MD
University of Rochester Medical Center
DMID Clinical Project Manager:
Melinda Tibbals, RAC, CCRA
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Statement of Compliance
The study will be carried out in accordance with Good Clinical Practice (GCP) as 
required by the following:

U.S. Code of Federal Regulations applicable to clinical studies (45 CFR 46)

21 CFR 312

ICH GCP E6

Completion of Human Subjects Protection Training

NIH Clinical Terms of Award

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SIGNATURE PAGE
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable US federal regulations and ICH guidelines.
Principal Investigator:
Signed:
Date:
Angela Branche, MD
Principal Investigator, University of Rochester
CEIRS
 

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Statement of Compliance ...............................................................................................................i
Signature Page ..............................................................................................................................ii
List of Abbreviations......................................................................................................................vi
Protocol Summary....................................................................................................................…vii
1
PROTOCOL SUMMARY.....................................................................................................vii
2
Key Roles .............................................................................................................................1
3
Background Information and Scientific Rationale.................................................................3
3.1
Background Information............................................................................................3
3.2
Scientific Rationale ...................................................................................................3
3.3
Potential Risks and Benefits .....................................................................................4
3.3.1
Potential Risks ..............................................................................................4
3.3.2
Known Potential Benefits ..............................................................................5
4
Objectives and Outcome Measures......................................................................................5
4.1
Study Objectives.......................................................................................................5
4.2
Outcome Measures...................................................................................................6
5
Study Design ........................................................................................................................6
6
Study Population...................................................................................................................7
6.1
Inclusion Criteria .......................................................................................................8
6.2
Exclusion Criteria......................................................................................................8
6.3
Handling of Withdrawals .........................................................................................10
6.4
Termination of Study...............................................................................................10
7
Study Intervention/Investigational Product .........................................................................10
7.1
Study Product Description ......................................................................................10
7.1.1
Acquisition...................................................................................................10
7.1.2
Formulation, Packaging, and Labeling........................................................10
7.1.3
Product Storage and Stability......................................................................11
7.2
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product....................................................................................................................11
7.3
Modification of Study Intervention/Investigational Product for a Participant ...........11
7.4
Accountability Procedures for the Study Intervention/Investigational Product(s)....11

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7.5
Assessment of Participant Compliance with Study Intervention/Investigational 
Product....................................................................................................................12
7.6
Concomitant Medications/Treatments ....................................................................12
8
Study Schedule...................................................................................................................12
8.1
Vaccination/Baseline (Defined as Day 0)................................................................12
8.2
Follow-up and Final Visits.......................................................................................13
8.2.1
Day 7 visit (+/- 1 day) ..................................................................................13
8.2.2
Day 28 visit (+/- 3 days) ..............................................................................14
8.2.3
Day 90 visit (+/- 7 days) ..............................................................................14
8.2.4
Blood Volumes in Children..........................................................................15
8.2.5
Surveillance for acute influenza ..................................................................15
8.3
Early Termination Visit............................................................................................16
8.4
Unscheduled Visit ...................................................................................................16
9
Study Procedures and evaluations.....................................................................................17
9.1
Clinical Evaluations.................................................................................................17
9.2
Laboratory Evaluations/Assays...............................................................................17
9.2.1
Immunogenicity Evaluations .......................................................................17
9.2.2
Specimen Collection, Preparation, Handling and Shipping.........................19
10
Assessment of Safety.........................................................................................................19
10.1
Specification of Safety Parameters.........................................................................19
11
Clinical monitoring ..............................................................................................................20
11.1
Site Monitoring Plan................................................................................................20
12
Statistical considerations ....................................................................................................20
12.1
Study Hypothesis....................................................................................................20
12.2
Sample Size Considerations...................................................................................20
12.3
Planned Interim Analyses (if applicable).................................................................21
12.3.1 Safety Review .............................................................................................21
12.3.2 Immunogenicity or Efficacy Review ............................................................21

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12.4
Final Analysis Plan..................................................................................................21
13
Source Documents and Access to Source Data/Documents .............................................22
14
Quality Control and Quality Assurance...............................................................................22
15
Ethics/Protection of Human Subjects .................................................................................23
15.1
Ethical Standard......................................................................................................23
15.2
Institutional Review Board ......................................................................................23
15.3
Informed Consent Process .....................................................................................23
15.3.1 Informed Consent/Assent Process (in Case of a Minor).............................24
15.4
Exclusion of Women, Minorities, and Children (Special Populations) ....................24
15.5
Participant Confidentiality .......................................................................................24
15.6
Study Discontinuation .............................................................................................25
15.7
Future Use of Stored Specimens............................................................................25
16
Data Handling and Record Keeping ...................................................................................25
16.1
Data Management Responsibilities ........................................................................26
16.2
Data Capture Methods............................................................................................26
16.3
Types of Data..........................................................................................................26
16.4
Timing/Reports........................................................................................................26
16.5
Study Records Retention........................................................................................27
16.6
Protocol Deviations.................................................................................................27
17
Publication Policy................................................................................................................27
18
Literature References .........................................................................................................28
19
Supplement/Appendices.....................................................................................................29

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AE
Adverse Event/Adverse Experience
ASC
Antibody Secreting Cell
CFR
Code of Federal Regulations
CIOMS
Council for International Organizations of Medical Sciences
CO
Contracting Officer
COR
Contracting Officer Representative
CRF
Case Report Form
DMID
Division of Microbiology and Infectious Diseases 
DSMB
Data and Safety Monitoring Board
FWA
Federalwide Assurance
GFPD
Genome Fragment Phage Display
GCP
Good Clinical Practice
HAI
Hemagglutination-Inhibition
ICF
Informed Consent Form
ICH
International Conference on Harmonisation
IEC
Independent or Institutional Ethics Committee
IRB
Institutional Review Board
JAMA
Journal of the American Medical Association
MBC
Memory B Cell
MN
Microneutralization
MOP
Manual of Procedures
N
Number (typically refers to participants)
NEJM
New England Journal of Medicine
NIAID
National Institute of Allergy and Infectious Diseases, NIH, 
DHHS
NIH
National Institutes of Health
OCRA
Office of Clinical Research Affairs, DMID, NIAID, NIH, DHHS
OHRP
Office for Human Research Protections
ORA
Office of Regulatory Affairs, DMID, NIAID, NIH, DHHS
PI
Principal Investigator
SAE
Serious Adverse Event/Serious Adverse Experience
SMC
Safety Monitoring Committee
SOP
Standard Operating Procedure
SPR
Surface Plasmon Resonance
WHO
World Health Organization

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1
PROTOCOL SUMMARY
Title:
Evaluation of the effects of age, prior exposure and previous vaccination 
on the B cell response to inactivated influenza vaccine in healthy adults 
and children.
Population:
240 Healthy adults and children ages 9 and over
Number of Sites:
Single site study
Study Duration:
5 years
Participant Duration: 6 months
Objectives:
Primary Objective
To evaluate the relationship between first influenza A virus exposure (inferred by age), vaccine 
history, and baseline serum antibody  response to seasonal influenza vaccine in healthy adults 
and children.
Primary Endpoint:

Measurement of the magnitude and specificity of the serum antibody responses by 
hemagglutination (HAI) and microneutralization (MN) at day 7 and 28, overall and 
stratified by age group, vaccine history, and baseline serum antibody level.


Serum antibody response on day 90 after vaccination by HAI and MN overall and 
stratified by acute antibody response.
Secondary Objectives
Evaluate factors related to failure of vaccine to provide protection against symptomatic influenza 
and the immune response to infection in vaccinated individuals by prospective surveillance of 
the vaccine cohort.

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Secondary Endpoints
Description of demographic and clinical characteristics and immune response to vaccination 
among subjects who develop symptomatic influenza (Vaccine Failures) and among subjects 
who do not.
Exploratory Objective
To evaluate the relationship between first influenza A virus exposure (inferred by age), vaccine 
history, and Memory B cell (MBC) specificity, and the magnitude and breadth of the subsequent 
B cell response to seasonal influenza vaccine in healthy adults and children.
Exploratory Endpoints

Frequency and specificity of peripheral blood antibody secreting cells (ASC) on day 7 
post vaccination (initial response), overall and stratified by age group, vaccine history, 
and baseline serum antibody level 

Frequency and specificity of peripheral blood memory B cells (PMBC) on day 28 post 
vaccination (memory response), overall and stratified by age group, vaccine history, and 
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Schematic of Study Design:
* Note:  For children weighing less than 100 lbs, only 15 mL of blood will be drawn at each time 
point.and plasma will be used for antibody testing.  Subjects will be queried regarding any blood 
being drawn for other purposes such as clinical testing, and that the total drawn will not exceed 
the above amounts
In addition, beginning on day 48, subjects will be contacted by phone once every two weeks 
during the influenza season and asked about influenza symptoms. Subjects who report 
symptoms will be asked to return for a clinic visit and specimen collection.  Influenza season will 
be defined as when community laboratories detect 4 or more positives in any one week for two 
weeks in a row.

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2
KEY ROLES
Principal Investigator
Angela Branche, MD
University of Rochester 
Infectious Diseases Division
University of Rochester Medical Center
601 Elmwood Avenue
Rochester, NY 14642
Phone 585-275-5871
Fax 585-442-9328
Email:angela_branche@urmc.rochester.edu
DMID Clinical Project Manager:
Melinda Tibbals, RAC, CCRA
DMID, NIAID, NIH 
5601 Fishers Lane
Rockville, MD  20892-9825
Tel: 240-627-3387 
Email: melinda.tibbals@nih.gov
DMID Scientific Lead:
Erik J. Stemmy, Ph.D.
DMID, NIAID, NIH 
5601 Fishers Lane
Rockville, MD  20892-9825 
Tel:  240-627-3380
Fax:  301-496-8030
Email:  erik.stemmy@nih.gov
DMID Medical Monitor
Venus Shahamatdar, MD
DMID, NIAID, NIH 
5601 Fishers Lane, Room 7E54
Rockville, MD 20892-9825
Phone: 240-627-3369
Email: venus.shahamatdar@nih.gov
DMID Medical Officer
Trinka Coster, MD, MSDMID, NIAID, NIH
5601 Fishers Lane
Rockville, Maryland 20892-9826
Phone: 301-761-7736
E-mail: trinka.coster@nih.gov

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Scientific Investigators
David Topham PhD
University of Rochester
601 Elmwood Avenue
Rochester NY
Phone 585-273-1403
Fax 585-273-2452
Email:  David Topham@urmc.rochester.edu
Mark Sangster PhD
University of Rochester
601 Elmwood Avenue
Rochester NY
Phone 585-273-2350
Fax 585-273-24552
Email:  Mark Sangster@urmc.rochester.edu

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3
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
3.1
Background Information 
Inactivated influenza vaccines (IIVs) have been in use in the US and worldwide for the 
prevention of illness due to seasonal and pandemic influenza since their licensure 
approximately 60 years ago.  Multiple studies over many years have evaluated the 
immunogenicity of these vaccines in various populations.  In general, serum antibody responses 
to IIV are vigorous in healthy young adults with low levels of pre-vaccination antibody.  Nasal 
antibody responses can also be detected, although they are not as vigorous as those seen after 
live vaccination.
The serum antibody response to IIV is impacted by several factors, including the age of the 
subject, the presence of chronic diseases, and the use of immune modulating drugs.  In 
addition, the prior exposure of the subject to influenza vaccine or infection appears to have a 
substantial impact on both the immune response as well as the protective efficacy of IIV.  For 
seasonal influenza vaccine, the magnitude of the serum antibody response is lower in 
individuals who begin with higher titers, and highest in those with low prevaccination titers.  
Consistent with this observation, individuals with prior vaccination, and who therefore have 
higher baseline antibody titers, tend to have less vigorous antibody responses to revaccination.  
The impact of prior vaccination on protection is controversial.  Some studies have suggested 
that vaccine efficacy is higher in those who were not vaccinated in the previous season than in 
those who were [1, 2].  Other studies have not seen this association [3].  The mechanisms by 
which prior exposure to vaccine or infection might modify the subsequent response to IIV are 
not known, but would have obvious implications for the design of new influenza vaccines and 
vaccine strategies.
3.2
Scientific Rationale 
This protocol will address the hypothesis that vaccination and infection drive adaptation of the 
antibody secreting and memory B cell populations, and differ based on the antigenicity of the 
vaccine strains and immune history of the subject.  This protocol will address whether initial 
influenza priming, most likely occurring during childhood, affects later-in-life immune responses 
to influenza vaccination. Recent data in the literature [4, 5], and our own preliminary data 
suggest that initial encounters with influenza have a long lasting effect on circulating antibodies 
and the specificity of the memory B cell repertoire.  This protocol will test whether qualitative 
and quantitative differences in circulating antibodies and memory B cells can strongly influence 
subsequent immune responses to seasonal influenza vaccines. This protocol will perform 

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parallel assessment of antibody, ASC, and memory B cells over the course of an immune 
response to determine how vaccination modifies the B cell and antibody repertoires. As a 
measure of the cells responding to the vaccination, our assays will focus on the acutely 
activated antibody-secreting B cells (ASC, plasmablasts) and antibodies secreted (day 7). 
Because these ASC can be short lived, later in the response this protocol will also look at in 
vitro reactivated memory B cells and secreted antibodies at days 0, 28, and 90 as measures of 
the adapted responses before and after vaccination. Serum antibodies will also be tested for 
function, specificity, amount, and quality by conventional HAI and MN assays, as well as more 
sophisticated assays described below (ELISA, SPR, GFPDL).
3.3
Potential Risks and Benefits
3.3.1 Potential Risks
The risks and discomforts of this study include risks associated with the vaccine, administration 
of the vaccine, the risks associated with study procedures (blood drawing and nasal and throat 
swabs) and possible loss of confidentiality.
In placebo-controlled trials in adults, inactivated seasonal vaccines are associated with mild 
local pain at the site of administration.  Systemic symptoms like fever and malaise occur at rates 
equal to placebo.  According to the 
package insert, in adults, the most common (≥10%) 
injection site adverse reaction was pain (36%); the most common systemic adverse events were 
muscle aches (16%), headache (16%), and fatigue (16%). In children aged 3 through 17 years, 
the injection site adverse reactions were pain (44%), redness (23%), and swelling (19%). In 
children aged 3 through 5 years, the most common (≥10%) systemic adverse events were 
drowsiness (17%), irritability (17%), and loss of appetite (16%); in children aged 6 through 17 
years, the most common systemic adverse events were fatigue (20%), muscle aches 
(18%),headache (16%), arthralgia (10%), and gastrointestinal symptoms (10%).
During the swine influenza vaccine campaign of 1976, about 10 per 1,000,000 vaccine 
recipients in excess of the background rate developed the paralytic illness called Guillain-Barré 
Syndrome (GBS).  In the subsequent decade, no association between seasonal influenza 
vaccine and GBS was found. More extensive investigations of this potential association 
occurring in the 1990s revealed that there was a small but detectable risk of GBS in the 6 weeks 
following seasonal influenza immunization: an attributable risk of approximately 1 per 1,000,000, 
adjusted for potential confounders.  In the period since the Vaccine Adverse Event Reporting 
System (VAERS) was established in 1990, the rates of GBS reports following influenza 
vaccination have declined substantially. The annual reporting rate in that period was highest in 
the 1993-1994 influenza season (1.7 per 1,000,000 vaccinees) and lowest in the last season 
analyzed in the report, 2002-2003 (0.4 per 1,000,000 vaccinees) [6]. No cases of GBS have 

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been reported following receipt of novel H1N1 influenza vaccines to date, but the total number 
of recipients is only in the thousands. Most persons who develop GBS recover completely.
Drawing blood causes transient discomfort and may cause fainting. Bruising at the blood draw 
site may occur but can be prevented or mitigated by applying direct pressure to the draw site for 
several minutes. The use of alcohol swabbing and sterile equipment will make infection less 
likely at the site where blood will be drawn. Nasal and throat swabs can be uncomfortable and 
may cause gagging.
Personal health information of the subjects will be collected to determine eligibility and to 
evaluate safety and reactogenicity outcomes throughout the study. Research personnel will 
make every effort to keep this information confidential. Still, a risk of participation is that the 
confidentiality of this information could be lost.
3.3.2 Known Potential Benefits
Administration of licensed vaccines for seasonal influenza is recommended for all children and 
adults and would be expected to provide partial protection against influenza illness caused by 
the strains contained within the vaccine.
4
OBJECTIVES AND OUTCOME MEASURES
4.1 Study Objectives
Primary Objective
To evaluate the relationship between first influenza A virus exposure (inferred by age), vaccine 
history, and baseline serum antibody response to seasonal influenza vaccine in healthy adults 
and children
Secondary Objectives

Evaluate factors related to failure of vaccine to provide protection against symptomatic 
influenza and the immune response to infection in vaccinated individuals by prospective 
surveillance of the vaccine cohort.
Exploratory Objectives

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Evaluate the relationship between first influenza A virus exposure (inferred by age), vaccine 
history, and Memobry B cell (MBC) specificity, and the magnitidue and breath of the subsequent 
B cell response to seasonal influenza vaccine in healthy adults and children
4.2 Outcome Measures
Primary Outcome Measures:

Measurement of the magnitude and specificity of the serum antibody responses by 
hemagglutination (HAI) and microneutralization (MN) at day 7 and 28, overall and 
stratified by age group, vaccine history, and baseline serum antibody level 

Serum antibody response on day 90 after vaccination by HAI and MN and overall and 
stratified by acute antibody response
Secondary Outcome Measures

Description of demographic and clinical characteristics and immune response to 
vaccination among subjects who develop symptomatic influenza (Vaccine Failures) and 
among subjects who do not.
Exploratory Outcome Measures

Frequency and specificity of peripheral blood antibody secreting cells (ASC) on day 7 
post vaccination (initial response), overall and stratified by age group, vaccine history, 
and baseline serum antibody level 

Frequency and specificity of peripheral blood memory B cells (PMBC) on day 28 post 
vaccination (memory response), overall and stratified by age group, vaccine history, and 
baseline serum antibody level 
5
STUDY DESIGN
The study will be designed as a prospective surveillance of the immune response to seasonal 
vaccination in healthy adults of varying ages.  A schematic of the study design is shown below:

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* Note for children weighing less than 100 lbs, only 15 mL of blood will be drawn at each time 
point, and plasma will be used for antibody testing.  Subjects will be queried regarding any 
blood being drawn for other purposes such as clinical testing, and that the total drawn will not 
exceed the above amounts
Influenza season will be defined as when community laboratories detect 4 or more positives in 
any one week for two weeks in a row.  If influenza season has begun subjects will be contacted 
by phone once every two weeks starting on day 48 and asked about influenza symptoms.  
Subjects who report symptoms will be asked to return for a clinic visit and specimen collection.
6
STUDY POPULATION
The study population will consist of up to 240 medically stable adults and children ages 9 and 
older enrolled over 5 years.  Recruitment will be stratified in an attempt to achieve an even 
distribution of ages with a goal of 30 subjects in each of 8 age strata (9-19, 20-29…80+)

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6.1
Inclusion Criteria
Subjects must meet all of the following inclusion criteria to participate in this study:
1.
Aged equal to or greater than 9 years of age and weigh at least 50 pounds.
2.
The subject must be in good health, as determined by: medical history; and targeted 
physical examination, when necessary, based on medical history.  Stable medical or 
psychiatric condition is defined as: no recent increase in prescription medication, dose, or 
frequency of medication in the last 3 months and health outcomes of the specific disease 
are considered to be within acceptable limits in the last 6 months.
3.
The subject is able to understand and comply with the planned study procedures, including 
being available for all study visits.
4.
The subject/parent has provided informed consent/assent prior to any study procedures.
5.
Subjects who have not received seasonal flu vaccine for the current year.
6.2
Exclusion Criteria
Subjects who meet any of the following exclusion criteria at baseline cannot participate in the 
study:
1.
Subject report of known hypersensitivity to allergy to components of the study vaccine or 
other components of the study vaccine.
2.
Subject report of known latex allergy
3.
Subject report of a history of severe reactions following previous immunization with licensed 
or unlicensed influenza virus vaccines. 
4.
Subject report of a history of Guillain-Barre syndrome within 6 weeks of receipt of a previous 
influenza vaccine.
5.
The subject is a female of childbearing potential who is currently pregnant or breastfeeding 
or intends to become pregnant during the study period between enrollment and 90 days 
following receipt of vaccine.   Pregnancy will be determined by subject interview.  Pregnancy 
testing is not done in this study since there is no increased risk in pregnancy.
6.
The subject is immunosuppressed as a result of an underlying illness or treatment with 
immunosuppressive or cytotoxic drugs, or use of anticancer chemotherapy or radiation 
therapy within the preceding 36 months. 

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7.
The subject has an active neoplastic disease (excluding non-melanoma skin cancer or 
prostate cancer that is stable in the absence of therapy) or a history of any hematological 
malignancy.  For this criterion, “active” is defined as having received treatment within the 
past 5 years. 
8.
Have taken high-dose inhaled corticosteroids within 30 days prior to study vaccination.
High-dose defined as per age as using inhaled high dose per reference chart 
https://www.nhlbi.nih.gov/health-pro/guidelines/current/asthma-guidelines/quick-reference-
html#estimated-comparative-daily-doses
9.
The subject received immunoglobulin or another blood product within the 3 months prior to 
enrollment in this study.
10. The subject has received an inactivated vaccine within the 2 weeks or a live vaccine within 
the 4 weeks prior to enrollment in this study or plans to receive another vaccine within the 
next 28 days after vaccination.
11. The subject has an acute or chronic medical condition that, in the opinion of the investigator 
or appropriate sub-investigator, would render vaccination unsafe or would interfere with the 
evaluation of responses. These conditions include any acute or chronic medical disease or 
conditions defined as persisting for 3 months (defined as 90 days) or longer, that would 
place the subject at an unacceptable risk of injury, render the subject unable to meet the 
requirements of the protocol, or may interfere with the evaluation of responses of the 
subject’s successful completion of the study.   
12. Subjects with an active infection or that has an acute illness or an oral temperature greater 
than 99.9F (37.7C) within 3 days prior to enrollment or vaccination.  Subjects who had an 
acute illness that was treated symptoms resolved are eligible to enroll as long as treatment 
is completed and symptoms resolved > 3 days prior to enrollment.
13. The subject is currently participating or plans to participate in a study that involves an 
experimental agent (vaccine, drug, biologic, device, blood product, or medication) or has 
received an experimental agent within 1 month prior to enrollment in this study, or expects to 
receive another experimental agent during participation in this study, or intends to donate 
blood during the study period.
14. The subject has any condition that would, in the opinion of the site investigator, place the 
subject at an unacceptable risk of injury or render the subject unable to meet the 
requirements of the protocol.
15. The subject has a history of alcohol or drug abuse in the 5 years prior to enrollment.
16. The subject has a known human immunodeficiency virus, hepatitis B, or hepatitis C 
infection.
17. Subject weighs less than 50 lbs.

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18. Subject expects to have a medical procedure during the upcoming 8 weeks that estimates 
blood loss to exceed 400 cc for adults or for children would exceed 3 ml/kg. 
6.3
Handling of Withdrawals
Participants who withdraw, or are withdrawn or terminated from the study, or are lost to follow-
up after receiving the assigned vaccine will not be replaced.  Participants who consented to the 
study but did not receive the influenza vaccine will be replaced. These subjects will be 
considered as screen failures. 
6.4
Termination of Study 
Although the study sponsor has every intention of completing the study, the sponsor reserves 
the right to terminate the study at any time for clinical or administrative reasons.  Reasons for 
termination include, but are not limited to, study closure due to internal safety review and 
recommendation, or at the discretion of DMID.
7
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT
7.1
Study Product Description
  Quadrivalent (IIV-4) split virion licensed vaccine produced in embryonated hen’s 
eggs and administered intramuscularly at a dose of not less than 15 ug of HA (as determined by 
SRID) of the following 4 influenza virus strains (2 A strains and 2 B strains): 
A/Singapore/GP1908/2015 (H1N1) IVR-180 (an A/Michigan/45/2015 [H1N1] pdm09-like virus), 
A/Singapore/INFIMH-16-0019/2016 (H3N2) NIB-104, B/Maryland/15/2016 NYMC BX-69A (a 
B/Colorado/06/2017-like virus), and B/Phuket/3073/2013.
 Quadrivalent (IIV-4) split virion licensed vaccine produced in embryonated hen’s 
eggs and administered intramuscularly at a dose of not less than 15 ug of HA (as determined by 
SRID) of the following 4 influenza virus strains (2 A strains and 2 B strains): 
A/Singapore/GP1908/2015 (H1N1) IVR-180 (an A/Michigan/45/2015 [H1N1] pdm09-like virus), 
A/Singapore/INFIMH-16-0019/2016 (H3N2) IVR-186, B/Maryland/15/2016 NYMC BX-69A, (a 
B/Colorado/06/2017-like virus), and B/Phuket/3073/2013.

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7.1.1 Acquisition
Vaccine will be ordered for each influenza season from the University of Rochester Medical 
Center hospital pharmacy which will acquire the vaccine through distributors.
7.1.2 Formulation, Packaging, and Labeling
 
 
 
 
 
 
 
 
 
 
7.1.3 Product Storage and Stability
Influenza vaccine will be stored according to manufacturer’s directions in secure, limited-access 
temperature monitored refrigerator environment at 2°C to 8°C (36°F to 46°F) until needed.  DO 
NOT FREEZE.  The temperature of the storage unit will be monitored during the duration of the 
trial, and documentation of proper dedicated storage will be maintained.  In the event of 
accidental deep-freezing or disruption of the cold chain, vaccines will not be administered; and 
the PI or the responsible person will contact the sponsor for further instructions.  
7.2
Dosage, Preparation and Administration of Study 
Intervention/Investigational Product
Vaccine will be formulated in single dose vials or syringes and will not require additional 
formulation prior to administration.  A vaccine dose of 0.5mL will be administered by a clinical 
research nurse intramuscularly (IM) in the subject’s preferred deltoid (upper arm) per the 
manufacturer’s instructions.

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7.3
Modification of Study Intervention/Investigational Product 
for a Participant
As there is only a single dose administered, there will be no dose or schedule modifications for 
any subject.
7.4
Accountability Procedures for the Study 
Intervention/Investigational Product(s)
Records of vaccine receipt and dispensation to the study subject as well as storage and 
destruction of the vaccine will be maintained according to existing standard operating 
procedures (SOPs)
7.5
Assessment of Participant Compliance with Study 
Intervention/Investigational Product 
Not applicable, single intervention within the vaccine clinic only.
7.6
Concomitant Medications/Treatments
Administration of any medication or therapies considered necessary for the subject’s welfare will 
be recorded and documented in the subject’s source documentation.  Concomitant medications 
will include all medications taken within 30 days prior to enrollment through 62 days post 
vaccination or early termination, whichever occurs first.
The following criteria will be reviewed with the subject’s during each follow up visit. If any of 
these become applicable during the study, it will be noted in the subject’s record.
1.
Use of any investigational drug or investigational vaccine other than the study article.
2.
Administration of chronic (defined as more than 14 days) immunosuppressants or other 
immune-modifying drugs (topical and nasal steroids are allowed).
3.
Receipt of a licensed vaccine.
4.
Receipt of immunoglobulins and/or any blood products.

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8
STUDY SCHEDULE
8.1
Vaccination/Baseline (Defined as Day 0)
At the baseline visit, subjects will be enrolled in the study and if they meet the entry criteria will 
receive influenza vaccine.  The following procedures will be performed at the baseline visit.  

The study will be explained to the subject/parent, and the informed consent document 
will be signed.  Subjects/Parents will keep a signed copy of the informed consent for 
their information. Subjects 9 to 12 will be read an Assent Script and subjects 13-17 will 
sign an Assent Form.

A medical history will be obtained including history of prior Influenza vaccinations, and 
details recorded in the case report form.

Concomitant medications will be reviewed and recorded.

A targeted physical examination will be performed, if indicated by the medical history, 
directed at the relevant portions of the exam.

The entry criteria will be reviewed and it will be verified that the subject meets all entry 
criteria.  If the subject does not meet the entry criteria, the subject will be discontinued 
from the study, or can be rescreened in the case of entry criteria mandating temporary 
delays.
Subjects will be considered enrolled if they have signed the consent form.  However, only 
subjects who meet the entry criteria will be vaccinated and continue in the study.  The 
reasons for screen failure will be recorded for all subjects who do not meet the entry criteria.  
If the entry criteria are met:

30 mL of blood will be obtained from an arm vein (3 x 10 mL green top tubes) for 
isolation of Peripheral Blood Mononuclear Cells (PBMC).

8 mL of blood will be obtained from and arm vein (1 x 8 mL red top tube) for serum.

For children who weigh less than 100 lbs, only 15 mL of blood will be drawn

Subjects will receive vaccine by intramuscular injection in open label fashion.

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
Subjects will be observed in the clinic for 30 minutes post vaccination for development of 
acute adverse events
8.2
Follow-up and Final Visits
8.2.1 Day 7 visit (+/- 1 day)
Subjects will return one week after vaccination for the day 7 visit.  Because the timing of 
assessment of the cellular response to vaccination is critical, every effort will be made to 
ensure that subjects return on day 7.  Visits that occur out of window will still be 
evaluated but will not be included in the primary analysis.  On the day 7 visit, the 
following procedures will be done:

Interval medical history will be obtained including concomitant medications, 
reviewing any new events since the last visit.

The site of vaccination will be examined.  If indicated by medical history, a 
directed physical exam will also be performed. 

Any adverse events (AE) will be recorded and appropriate corrective actions 
taken if indicated.

30 mL of blood will be obtained from an arm vein (3 x 10 mL green top tubes) for 
isolation of Peripheral Blood Mononuclear Cells (PBMC).

8 mL of blood will be obtained from and arm vein (1 x 8 mL red top tube) for 
serum.

For children who weigh less than 100 lbs, only 15 mL of blood will be drawn
8.2.2 Day 28 visit (+/- 3 days)
Subjects will return for follow up approximately 28 days after vaccination for assessment 
of the immune response.  At that visit, the following procedures will be performed:

Interval medical history will be obtained including concomitant medications, 
reviewing any new events since the last visit.

If indicated by medical history, a directed physical exam will also be performed. 

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
Any adverse events (AE) will be recorded and appropriate corrective actions 
taken if indicated.

30 mL of blood will be obtained from an arm vein (3 x 10 mL green top tubes) for 
isolation of Peripheral Blood Mononuclear Cells (PBMC).

8 mL of blood will be obtained from and arm vein (1 x 8 mL red top tube) for 
serum.

For children who weigh less than 100 lbs, only 15 mL of blood will be drawn
8.2.3 Day 90 visit (+/- 7 days)
Subjects will return approximately 90 days after vaccination for a final follow-up visit.  At 
this visit the following procedures will take place:

Interval medical history will be obtained, including concomitant medications, 
reviewing any new events since the last visit.

If indicated by medical history, a directed physical exam will also be performed. 

Any adverse events (AE) will be recorded and appropriate corrective actions 
taken if indicated.

30 mL of blood will be obtained from an arm vein (3 x 10 mL green top tubes) for 
isolation of Peripheral Blood Mononuclear Cells (PBMC).

8 mL of blood will be obtained from and arm vein (1 x 8 mL red top tube) for 
serum. 

For children who weigh less than 100 lbs, only 15 mL of blood will be drawn
8.2.4 Blood Volumes in Children
The blood draws in this protocol will impose an upper limit of 3 mL/kg over any 8-week period 
for all pediatric patients.  This volume remains well below the allowed upper limit of 10% of total 
blood volume in an 8-week period as minimal risk. From healthy subjects who weigh at least 
100 pounds, the total amount of blood drawn in any 8 week period is 38 x 3, or 114 mL.  For 
subjects who weigh less than 100 lbs, the total blood drawn will be 45 mL.  Neither of these 
amounts will exceed the limit of 3 mL/kg

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8.2.5 Surveillance for acute influenza
Influenza season will be defined as when community laboratories detect 4 or more positives in 
any one week for two weeks in a row, and will end when influenza is no longer detected.  During 
influenza season, subjects will be contacted by phone every other week to enquire about 
influenza like symptoms.  Subjects who report symptoms meeting the case definition of 
influenza (fever or feverishness plus either cough, rhinitis, or sore throat on the same or 
consecutive days) will be asked to return for an acute illness visit.  At this visit the following 
procedures will take place

Interval medical history will be obtained, including concomitant medications, 
reviewing any new events since the last visit.

The presence and severity of respiratory symptoms will be assessed and 
recorded using a visual analog scale.

A combined nasal and throat swab will be obtained for rtRT-PCR detection of 
influenza virus.

If positive, the subject will be invited to participate in the Acute Flu study (DMID 
14-0101) to assess the immune response to infection
8.3
Early Termination Visit
If subjects discontinue from the study, they will be asked to make an early termination visit.  At 
the time of the early termination visit, the reason for early termination will be recorded, current 
health status since the last visit will be reviewed, and all concomitant medications will be 
recorded.  A targeted physical examination may be performed, as indicated, and information 
regarding AEs will be solicited. Any ongoing related AEs will be followed to resolution or until a 
stable chronic condition has been established.
Subjects will be encouraged to permit continued follow-up of AEs and to donate scheduled 
blood samples, if possible.
8.4
Unscheduled Visit
Unscheduled visits may occur at any time during the study.  Any of the following activities may 
be performed:

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
Medical history will be reviewed including concomitant medications, and updated as 
appropriate.

All concomitant medications taken since the study visit will be recorded on the 
appropriate data collection form. Previously recorded medications will be updated as 
appropriate.

Study personnel will discuss with participants and assess and record all AE/SAEs. 
Previously recorded AE/SAEs will be updated as appropriate.

Depending on the reason for the unscheduled visit, vital signs, including oral or axillary 
temperature, pulse, and blood pressure, may be obtained.

If indicated by medical history, a directed physical examination relevant to the interval 
medical history may be performed.

Additional laboratory tests may be obtained depending on the nature of the unscheduled 
visit.
9
STUDY PROCEDURES AND EVALUATIONS
9.1
Clinical Evaluations
Medical History:  Study personnel will take the medical history of all subjects. This history will 
include significant medical disorders of the head, eyes, ears, nose, throat, mouth, 
cardiovascular system, lungs, gastrointestinal tract, liver, pancreas, kidney, nervous system, 
blood, lymph glands, endocrine system, musculoskeletal system, skin, and genital/reproductive 
tract.  It will also include a history of allergies, cancer, immunodeficiency, psychiatric illness, 
substance abuse, and autoimmune disease.  Patients will also be queried regarding any 
previous influenza vaccinations, the date and source of vaccine, and the type of vaccine 
administered.  We will use personal history as the primary indicator but will attempt to verify 
vaccination status where possible.
Medication History:  Study personnel will record all medications, including prescription and over-
the-counter drugs (such as vitamins, minerals, supplements, homeopathic preparations and/or 
therapies), taken by the subject in the 30 days prior to enrollment.
Targeted Physical Examination:  Licensed study clinicians (i.e., physician, physician’s assistant, 
nurse practitioner) may conduct a targeted physical examination, if necessary to assist in 

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determining eligibility and height and weight will be collected.  All subjects will have vital signs 
(blood pressure, pulse, and oral temperature) measured prior to vaccination.
.
9.2
Laboratory Evaluations/Assays
9.2.1 Immunogenicity Evaluations
Serum hemagglutination-inhibition (HAI).  HAI will be performed in microtiter format using turkey 
RBCs and egg-grown, betapropriolactone-inactivated vaccine viruses as antigen.  The titer of 
antibody will be defined as the highest dilution resulting in complete inhibition of 
hemagglutination.  Sera will be treated with receptor-destroying enzyme and heat inactivated 
prior to testing at an initial starting dilution of 1:4. Sera with no detectable HAI titer will be 
assigned a titer of 1:2 for calculation purposes.
Microneutralization (MN) assay:  Sera will be tested by microtiter technique for neutralization of 
the vaccine viruses in MDCK cells.  Viral growth will be determined by ELISA of the cells 
following fixation with methanol using a combination of M- and NP-specific monoclonal 
antibodies.  The titer of antibody will be defined as the highest titer resulting in 50% inhibition of 
antigen signal compared to un-neutralized control wells.  Sera will be treated with RDE and heat 
inactivated prior to testing at an initial starting dilution of 1:10. Sera with no neutralizing titer will 
be assigned a value of 1:5 for calculation purposes. 
B cell responses:  Our previous studies have shown that the antibodies secreted by activated B 
cells (plasmablasts) and re-activated memory B cells have different specificities and affinities 
that those that accumulate in the serum.  In addition, there is evidence from analyzing secreted 
antibodies that vaccination affects the specificities and improves affinities. Therefore, the 
studies of antibody-secreting (ASC) and memory B cells require the collection of Peripheral 
Blood Mononuclear Cells (PBMC) at different times before and after vaccination for analysis. 
PBMC will be obtained before and on days 0, 7, 28, and 90 after vaccine and evaluated for B-
cell responses. First, antibody secreting cells stimulated by the vaccination, which peak 7 days 
after vaccination in the blood, will be analyzed by performing B cell ELISPOT assays on plates 
coated with recombinant HA proteins that match the influenza strains in that year’s vaccine.  In 
addition, supernatants   will be collected from cultured plasmablasts for analysis by HAI, MN, 
and ELISA as described above. Memory B cell responses will be assessed similarly at all the 
time points by reactivating the B cells in culture [7], performing B cell ELISPOT assays and 
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memory B cells provides a measure of how the vaccine modifies the B cell repertoire, as well as 
how well someone is responding to the vaccine. 
9.2.2 Specimen Collection, Preparation, Handling and Shipping
Instructions for specimen preparation, handling, and storage are included in the Manual of 
Procedures (MOP).  
10
ASSESSMENT OF SAFETY
10.1 Specification of Safety Parameters
An FDA approved, licensed seasonal inactivated influenza vaccine will be administered in this 
protocol as standard of care during influenza vaccine season, following the manufacturers’ 
instructions and safety precautions.
 is a licensed vaccine, NIAID does not expect that any new vaccine related safety signal 
will be detected in this trial.
The National Childhood Vaccine Injury Act (NCVIA) requires healthcare providers to 
report to VAERS:

Any adverse event listed by the vaccine manufacturer as a contraindication to further 
doses of the vaccine; and/or

Any adverse event listed in the VAERS Table of Reportable Events Following 
Vaccination that occurs within the specified time period after vaccination (see below).
o
Anaphylaxis or anaphylactic shock (7 days) (see halting criteria) 
o
Shoulder Injury Related to Vaccine Administration (7 days) 
o
Vasovagal syncope (7 days)
o
Guillain-Barré Syndrome (42 days) 
o
Any acute complication or sequelae (including death) of above events (interval - 
not applicable) 
In addition, CDC encourages reporting any serious, unexpected (not listed in product 
label or Investigator’s Brochure), suspected adverse reaction to VAERS and provide a 
copy of the VAERS report to DMID and local IRB as required.

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11
CLINICAL MONITORING 
The purpose of clinical monitoring is to protect the rights and well-being of human subjects in this 
study; to ensure that data are accurate, complete and verifiable from source documents; to ensure 
that conduct is in compliance with the currently approved protocol/amendments, with Good 
Clinical Practice, and with regulatory requirements.
11.1
Site Monitoring Plan
Site monitoring will be conducted using the DMID tools provided to ensure that human subject 
protection, study procedures, laboratory procedures, and data collection processes are of high 
quality and meet sponsor, GCP/ICH, and regulatory guidelines, and that the study is conducted 
in accordance with the protocol and sponsor standard operating procedures. DMID, the 
sponsoring agency, or its designee will conduct site-monitoring visits as defined in the CQMP.
A protocol-specific Clinical Quality Management Plan (CQMP) has been approved for this study 
by DMID. The Quality Assurance (QA) plan will be implemented by a weekly review of source 
documents by the CRC to determine adherence to protocol requirements. The Quality Control 
(QC) plan will be implemented by daily observation and documentation of the site’s work 
processes by study staff, to ensure that accepted procedures are followed.
Site visits may be made at standard intervals as defined by DMID and may be made more 
frequently as directed by DMID. Monitoring visits will include, but are not limited to, review of 
regulatory files, sample tracking log, CRFs, informed consent forms, medical and laboratory 
reports, and protocol compliance. Study monitors will meet with investigators to discuss any 
problems and actions to be taken and document visit findings and discussions.  The University of 
Rochester’s IRB and other regulatory agencies may conduct study monitoring visits. 
12
STATISTICAL CONSIDERATIONS
12.1 Study Hypothesis
The primary hypothesis being tested in this study is that there will be differences in the 
specificity and magnitude of the HA-specific B cell response depending on the age, previous 
vaccine history and baseline B cell reactivity.
12.2 Sample Size Considerations
Because the degree of variability in many of the measurements in this study are unknown, 
estimating the precision of the correlation of patient characteristics (age and prior vaccination 
history) with outcomes (antibody quality and quantity and B cell response) is not possible.  The 

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study will evaluate these outcomes and if trends towards a relationship are discovered 
subsequent studies will evaluate these in more detail.
However, the precision of the estimate of the proportion of subjects who achieve a pre-specified 
number of memory B cells specific for any antigen on day 28 would be determined by the 
number of subjects.  If there were 30 subjects in any particular age strata, then the half-width of 
the 95% confidence interval for the estimate of the the proportion achieving any level of mBC in 
that strata is shown below
No. of subjects
Proportion achieving mBC 
frequency
Half-width of the 95% CI for 
estimate of proportion
30
10%
10.7%
30
20
14.3
30
30
16.4
30
40
17.5
30
50
17.9
12.3 Planned Interim Analyses (if applicable)
No formal interim analysis is planned.  However, assay results will be reviewed as they become 
available
12.3.1 Safety Review 
NA, no interim analysis of safety is planned.
12.3.2 Immunogenicity or Efficacy Review
NA, no interim analysis of safety is planned.
12.4 Final Analysis Plan 
Data will be analyzed at the end of the year of enrollment.

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13
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS
The site will maintain appropriate medical and research records for this study in compliance with 
ICH E6, Section 4.9 and regulatory and institutional requirements for the protection of 
confidentiality of subjects.  The site will permit authorized representatives of the DMID, its 
designees, and appropriate regulatory agencies to examine (and when required by applicable 
law, to copy) clinical records for the purposes of quality assurance reviews, audits and evaluation 
of the study safety and progress.  These representatives will be permitted access to all source 
data which include, but are not limited to, hospital records, clinical and office charts, laboratory 
notes, memoranda, pharmacy dispensing records, recorded data from automated instruments, x-
rays, and subject files kept at the laboratories involved in the study.  CRFs will consist of paper 
document collection forms which will then be entered into the database, and will serve as source 
documents.  All electronic study documents will be secured by key and/or password protection.
14
QUALITY CONTROL AND QUALITY ASSURANCE
Following a written DMID-accepted site quality management plan, the site is responsible for 
conducting routine quality assurance (QA) and quality control (QC) activities to internally 
monitor study progress and protocol compliance. 
The Principal Investigators will provide direct access to all study-related field site, source 
documents, and reports for the purpose of monitoring and auditing by the sponsor, and 
inspection by local and regulatory authorities. 
The Principal Investigators will ensure all study staff are appropriately trained and current 
documentations are maintained on site.
DMID-designated clinical monitors will verify that the clinical study is conducted and data 
generated, recorded, and reported in compliance with the protocol, GCP, and the applicable 
regulatory requirements. Clinical monitoring reports will be submitted to DMID.
The site staff will implement QC procedures with the data entry system and generate data QC 
checks that will be run on the database. Any missing data or data anomalies will be clarified and 
resolved by going back to the source documents and checking with the clinical team that 
collected the data. 

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15
ETHICS/PROTECTION OF HUMAN SUBJECTS
15.1 Ethical Standard
The investigator 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).  
15.2 Institutional Review Board
The University of Rochester will provide this protocol and the associated informed consent 
documents for the review and approval of by an appropriate ethics review committee or IRB.  
Any amendments to the protocol or consent materials must also be approved before they are 
placed into use unless it is in the best interest of the subjects’ safety to implement changes prior 
to approval.  The UR IRB operates under U.S. Federal-Wide Assurance (FWA) 
Prior to enrollment of subjects into this trial, the approved protocol and the informed consent 
form will be reviewed and approved by the appropriate IRB.  Any amendments to the protocol or 
consent materials will also be reviewed and approved by the appropriate IRB and submitted to 
the sponsor.  Notification of the IRB's composition, or the IRB’s Federal-wide Assurance 
number, will be provided.
Should amendments to the protocol be required, the amendments will be written by the PI for 
submission to the IRB and also submitted to the sponsor.  The site will submit to the sponsor a 
copy of the IRB letter of approval of the amendment.
15.3 Informed Consent Process
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in 
the study and continuing throughout the individual’s study participation. At the time the study 
staff will seek informed consent, the study worker will ask the eligible participant (or the 
participant’s representative) if the participant is literate.  If the eligible participant reports he or 
she is not literate, then the study staff will request that a witness be present while the study 
worker reads and explains the study and what participation will entail.  If the eligible subject 
accepts to take part in the study, he or she will make a mark on the signature line of the consent 
form.  The witness will also sign and date the form, if the witness is confident that the participant 

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has understood the explanation and is participating willingly.  In addition, the witness will 
complete the date line for the participant.
Extensive discussion of risks and possible benefits of this therapy will be provided to the 
participants and their families.  Consent forms describing in detail the study 
interventions/products, study procedures, and risks are given to the participant and written 
documentation of informed consent is required prior to starting intervention/administering study 
product.  Consent forms will be IRB-approved and the participant will be asked to read and 
review the document. Upon reviewing the document, the investigator will explain the research 
study to the participant and answer any questions that may arise.  The participants will sign the 
informed consent document prior to any procedures being done specifically for the study.  The 
participants should have the opportunity to discuss the study with their families or think about it 
prior to agreeing to participate.  The participants may withdraw consent at any time throughout 
the course of the study.  A copy of the informed consent document will be given to the 
participants for their 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.
15.3.1 Informed Consent/Assent Process (in Case of a Minor)
Appropriate consent will be obtained from all subjects prior to any study related procedures.  
Adults will sign informed consent, while adolescents ages 13 to 17 will sign an informed assent 
document, and consent will be obtained from a parent or legal guardian.  In rare cases where a 
subject turns 18 during the course of the study, informed consent will be obtained at the next 
study visit.  Consent from a parent or legal guardian will be obtained for children 9 to 12 years of 
age.  Children 9-12 years of age will give verbal assent.
15.4 Exclusion of Women, Minorities, and Children (Special 
Populations)
Because pregnancy could alter the immune response to vaccination, known pregnancy is an 
exclusion to participation.  Otherwise there are no exclusions of special populations.
15.5 Participant Confidentiality
Participant confidentiality is strictly held in trust by the participating investigators, their staff, and 
the sponsor(s) and their agents.  This confidentiality is extended to cover testing of biological 
specimens in addition to the clinical information relating to participants.

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The study protocol, documentation, data, and all other information generated will be held in 
strict confidence.  No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of the sponsor.
The study monitor or other authorized representatives of the sponsor may inspect all documents 
and records required to be maintained by the investigator, including but not limited to, medical 
records (office, clinic, or hospital) and pharmacy records for the participants in this study.  The 
clinical study site will permit access to such records.  A representative from the University of 
Rochester IRB may also have access to the subject’s record. 
15.6 Study Discontinuation
If the study is discontinued, enrolled subjects will continue to be followed for safety 
assessments.  
15.7 Future Use of Stored Specimens
Subjects must give permission to keep any remaining specimen for possible use in future 
research studies, such as testing for antibodies against other viruses or bacteria, in order to 
participate. Samples will be stored under this protocol (16-0101) at the local site and will not be 
sold or used directly for production of any commercial product. No human genetic tests will be 
performed on samples.  Each sample will be encoded (labeled) only with a barcode and a 
unique tracking number to protect subject’s confidentiality.  Such testing may be performed by 
collaborating laboratories located at other sites.
There are no benefits to subjects in the collection, storage and subsequent research use of 
specimens. Reports about future research done with subject’s samples will NOT be kept in their 
health records, but subject’s samples may be kept with the study records or in other secure 
areas. 
16
DATA HANDLING AND RECORD KEEPING
The site principal investigator is responsible to ensure the accuracy, completeness, legibility, and 
timeliness of the data reported. All source documents will be completed in a neat, legible manner 
to ensure accurate interpretation of data using black ink to ensure clarity of reproduced copies.  
When making changes or corrections, the original entry will be crossed out with a single line, 
initialed and dated. The original text will not be erased, overwritten, or altered with correction fluid 
or tape on the original.

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16.1 Data Management Responsibilities
All source documents and laboratory reports will be reviewed by the clinical team and data entry 
staff, who will ensure that they are accurate and complete.  
Data collection is the responsibility of the clinical study staff at the site under the supervision of 
the clinical PIs.  During the study, the investigators will maintain complete and accurate 
documentation for the study.
16.2 Data Capture Methods
Clinical data will be initially recorded on paper source documents, and then transferred to 
electronic case report forms (eCRF) within BLISS.  Source documents will be retained for 
monitoring purposes in a secure location.  Laboratory data will be directly entered into BLISS 
from laboratory notebooks  
As detailed in the CEIRS contract, overall CEIRS data sharing will adhere to the following 
schedule: 

Sequence data: provided to Data Processing Coordinating Center within 45 days

Surveillance data: provided to Data Processing Coordinating Center within 12 months

Virus phenotypic data: provided to Data Processing Coordinating Center within 12 months

Basic research data: provided to Data Processing Coordinating Center within 2 months 
post publication.
16.3 Types of Data
Data for this study will include subject demographics, clinical data, safety assessments, and 
research laboratory results including antibody responses and measurements of B cell 
responses.
16.4 Timing/Reports
The final report will include a comprehensive analysis of the data.

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16.5 Study Records Retention
Records and documents pertaining to the conduct of this clinical study, including CRFs, source 
documents, and consent forms must be retained by the investigator for at least 2 years following 
the date of completion of the study. No study records will be destroyed without prior 
authorization by DMID. These documents should be retained for a longer period, however, if 
required by local regulations.
16.6 Protocol Deviations
A protocol deviation is any noncompliance with the study protocol, GCP, or protocol-specific 
MOP requirements or institution SOPs.  The noncompliance may be either on the part of the 
subject, the site principal investigator, or other study personnel.  As a result of deviations, 
corrective actions are to be developed by the site and implemented promptly.
These practices are consistent with ICH E6:
4.5
Compliance with Protocol, Sections 4.5.1, 4.5.2, and 4.5.3
5.1
Quality Assurance and Quality Control, Section 5.1.1
5.20
Noncompliance, Sections 5.20.1, and 5.20.2
It is the responsibility of the site principal investigator and other study personnel to use 
continuous vigilance to identify and report deviations within five working days of identification of 
the protocol deviation, or within five working days of the scheduled protocol-required activity.  A 
line listing of deviations will be reported to DMID on a monthly basis.
All protocol deviations, as defined above, must be addressed in study subject data collection 
forms.  A completed copy of the DMID Protocol Deviation Form must be maintained in the 
Regulatory File, as well as a copy kept in the subject’s source document file.  Protocol 
deviations must be sent to the local IRB/IEC per its guidelines.  The site principal investigator 
and other study personnel are responsible for knowing and adhering to their IRB requirements.
17
PUBLICATION POLICY
Following completion of the study, the research investigators will share data as defined in the 
contract and as directed by the CO and COR. 

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LITERATURE REFERENCES
1. Hoskins TW, Davies JR, Smith AJ, Miller CL and Allchin A. Assessment of inactivated 
influenza A vaccine after three outbreaks of influenza A at Christ's Hospital Lancet  1979;1:33-
35
2. Ohmit SE, Petrie JG, Malosh RE, Cowling BJ, Thompson MG, Shay DK and Monto AS. 
Influenza vaccine effectiveness in the community and the household Clin Infect Dis  
2013;56:1363-9
3. Keitel WA, Cate TR, Couch RB, Huggins LL and Hess KR. Efficacy of repeated annual 
immunization with inactivated influenza virus vaccines over a five year period Vaccine  
1997;15:1114-22
4. Li Y, Myers JL, Bostick DL, Sullivan CB, Madara J, Linderman SL, Liu Q, Carter DM, 
Wrammert J, Esposito S, Principi N, Plotkin JB, Ross TM, Ahmed R, Wilson PC and Hensley 
SE. Immune history shapes specificity of pandemic H1N1 influenza antibody responses The 
Journal of Experimental Medicine  2013;210:1493-1500
5. Fonville JM, Wilks SH, James SL, Fox A, Ventresca M, Aban M, Xue L, Jones TC, Le N. M. 
H., Pham Q. T., Tran N. D., Wong Y, Mosterin A, Katzelnick LC, Labonte D, Le T. T., van der 
Net G, Skepner E, Russell CA, Kaplan TD, Rimmelzwaan GF, Masurel N, de Jong JC, Palache 
A, Beyer WEP, Le Q. M., Nguyen T. H., Wertheim HFL, Hurt AC, Osterhaus ADME, Barr IG, 
Fouchier RAM, Horby PW and Smith DJ. Antibody landscapes after influenza virus infection or 
vaccination Science  2014;346:996-1000
6. Haber P, DeStefano F, Angulo FJ, Iskander J, Shadomy SV, Weintraub E and Chen RT. 
Guilllain-Barre syndrome following influenza vaccination JAMA  2004;292:2478-2481
7. Crotty S, Aubert RD, Glidewell J and Ahmed R. Tracking human antigen-specific memory B 
cells:  a sensitive and generalized ELISPOT system Journal of Immunological Methods  
2004;286:111-122

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SUPPLEMENT/APPENDICES
Table of procedures by visit and study day
 
V1
V2
V3
 
V4
 
 
Procedure
D0
D7
D28
D48, 
D62, 
D76
D90
D104
D118, 
D132, 
D146, 
D160, 
D174, 
D188
Flu**
Illness
Informed Consent
X
 
 
 
 
 
 
 
Medical History
X
 
 
 
 
 
 
 
Interim History
 
X
X
 
X
 
 
X
Concomitant Meds
X
X
X
 
X
 
 
X
Targeted physical exam*
X
X
X
 
X
 
 
X
Review Enrollment Criteria
X
 
 
 
 
 
 
 
Peripheral blood (mL)***
38 
 38
38 
 
 38
 
 
 
Administer Vaccine
X
 
 
 
 
 
 
 
30 minute observation
X
 
 
 
 
 
 
 
Review Adverse Events
X
X
X
 
 
 
 
 
Flu surveillance phone call
 
 
 
X
 
X
X
 
NP swab for flu PCR
 
 
 
 
 
 
 
X
*As indicated by symptoms or history
Visits in italics occur only during influenza season (see protocol for definition)
** Subjects reporting flu-like symptoms will be asked to make an influenza visit
*** Blood drawing in children will not exceed 3 mL/kg in any 8 week period