A Comparison of CD4 T Cell Induction and Antibody Responses Between a Pure Hemagglutinin Influenza Vaccine and Licensed Subvirion Influenza Vaccine Made in Eggs or Cell Culture in Healthy Adults.
Phase: PHASE4
Conditions: Influenza
Sponsor: University of Rochester

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A comparison of CD4 T cell induction and antibody responses between a pure 
hemagglutinin influenza vaccine (rHA, Protein Sciences Corp) and licensed 
subvirion influenza vaccine made in eggs (Sanofi) or cell culture (Novartis) in 
healthy adults.
DMID Protocol Number: 15-0055
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
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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.....................................................................................................................vii
Protocol Summary ...................................................................................................................…viii
1
PROTOCOL SUMMARY....................................................................................................viii
2
Key Roles .............................................................................................................................1
3
Background Information and Scientific Rationale.................................................................3
3.1
Background Information............................................................................................3
3.2
Preliminary Data .......................................................................................................6
3.3
Rationale...................................................................................................................7
3.4
Potential Risks and Benefits .....................................................................................8
3.4.1
Potential Risks ..............................................................................................8
3.4.2
Known Potential Benefits ..............................................................................9
4
Objectives and Outcome Measures....................................................................................10
4.1
Study Objectives.....................................................................................................10
4.2
Outcome Measures.................................................................................................11
4.2.1
Primary Outcome Measures .......................................................................11
4.2.2
Secondary Outcome Measures...................................................................11
5
Study Design ......................................................................................................................12
6
Study Population.................................................................................................................13
6.1
Inclusion Criteria .....................................................................................................13
6.2
Exclusion Criteria....................................................................................................14
6.3
Handling of Withdrawals .........................................................................................16
6.4
Termination of Study...............................................................................................16
7
Study Intervention/Investigational Product .........................................................................17
7.1
Study Product Description ......................................................................................17
7.1.1
Acquisition...................................................................................................17
7.1.2
Formulation, Packaging, and Labeling........................................................17

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7.1.3
Product Storage and Stability......................................................................19
7.2
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product....................................................................................................................19
7.3
Modification of Study Intervention/Investigational Product for a Participant ...........19
7.4
Accountability Procedures for the Study Intervention/Investigational Product(s)....19
7.5
Assessment of Participant Compliance with Study Intervention/Investigational 
Product....................................................................................................................20
7.6
Concomitant Medications/Treatments ....................................................................20
8
Study Schedule...................................................................................................................21
8.1
Screening (Day -21 to Day 0) .................................................................................21
8.2
Vaccination (Defined as Day 0) ..............................................................................21
8.3
Follow-up and Final Visits.......................................................................................22
8.3.1
Day 7 visit (± 1 day) ....................................................................................22
8.3.2
Day 14 visit (± 1 day) ..................................................................................23
8.3.3
Day 28 visit (± 2 days).................................................................................23
8.3.4
Influenza surveillance..................................................................................24
8.3.5
Final study visit (Day 180 +/- 14 days)........................................................24
8.4
Early Termination Visit............................................................................................25
8.5
Unscheduled Visit ...................................................................................................25
9
Study Procedures and evaluations.....................................................................................26
9.1
Clinical Evaluations.................................................................................................26
9.2
Laboratory Evaluations/Assays...............................................................................26
9.2.1
Immunogenicity Evaluations .......................................................................26
9.2.2
Specimen Collection, Preparation, Handling and Shipping.........................27
9.3
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings .....................................................................................27
10
Assessment of Safety.........................................................................................................28
10.1
Specification of Safety Parameters.........................................................................28
10.2
Safety Reporting: VAERS and Adverse Events of Special Interest (AESI) ............28

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10.3
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings .....................................................................................29
10.4
Reporting Procedures.............................................................................................29
10.5
Other Adverse Events (if applicable) ......................................................................29
10.6
Reporting of Pregnancy ..........................................................................................30
10.7
Type and Duration of Follow-up of Participants after Adverse Events....................30
10.8
Safety Oversight – Indendent Safety Monitor (ISM) ...............................................30
11
Clinical monitoring ..............................................................................................................31
11.1
Site Monitoring Plan................................................................................................31
12
Statistical considerations ....................................................................................................32
12.1
Study Hypothesis....................................................................................................32
12.2
Sample Size Considerations...................................................................................32
12.3
Planned Interim Analyses (if applicable).................................................................32
12.3.1 Safety Review .............................................................................................32
12.3.2 Immunogenicity or Efficacy Review ............................................................32
12.4
Final Analysis Plan..................................................................................................33
13
Source Documents and Access to Source Data/Documents .............................................34
14
Quality Control and Quality Assurance...............................................................................35
15
Ethics/Protection of Human Subjects .................................................................................36
15.1
Ethical Standard......................................................................................................36
15.2
Institutional Review Board ......................................................................................36
15.3
Informed Consent Process .....................................................................................36
15.3.1 Informed Consent/Assent Process (in Case of a Minor).............................37
15.4
Exclusion of Women, Minorities, and Children (Special Populations) ....................37
15.5
Participant Confidentiality .......................................................................................38
15.6
Study Discontinuation .............................................................................................38
15.7
Future Use of Stored Specimens............................................................................38
16
Data Handling and Record Keeping ...................................................................................40

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16.1
Data Management Responsibilities ........................................................................40
16.2
Data Capture Methods............................................................................................40
16.3
Types of Data..........................................................................................................40
16.4
Timing/Reports........................................................................................................41
16.5
Study Records Retention........................................................................................41
16.6
Protocol Deviations.................................................................................................41
17
Publication Policy................................................................................................................42
18
Literature References .........................................................................................................43
19
Supplement/Appendices.....................................................................................................45

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AE
Adverse Event/Adverse Experience
CFR
Code of Federal Regulations
CIOMS
Council for International Organizations of Medical Sciences
CRF
Case Report Form
DMID
Division of Microbiology and Infectious Diseases 
DSMB
Data and Safety Monitoring Board
FWA
Federalwide Assurance
GCP
Good Clinical Practice
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
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
WHO
World Health Organization

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1
PROTOCOL SUMMARY
Title:
A comparison of CD4 T cell induction and antibody responses between a 
pure hemagglutinin influenza vaccine (rHA, Protein Sciences Corp) and 
licensed subvirion influenza vaccine made in eggs (Sanofi) or cell culture 
(Novartis) in healthy adults.
Population:
Healthy adults ages 18 to 49 years
Number of Sites:
Single site study
Study Duration:
7 years
Subject Duration:
6 months
Agents:
Flublok (baculovirus expressed HA), Flucelvax (inactivated vaccine 
generated in mammalian cell culture, Fluzone SD (inactivated vaccine 
generated in eggs, administered at 15 ug per HA), Fluzone HD 
(inactivated vaccine generated in eggs, administered at 60 ug per HA)
Primary Objective

Comparison of the serum antibody response to immunization with pure HA, cell- derived 
and egg derived subvirion vaccines in adults age 18-49 years

Comparison of the HA- specific CD4 T cell response in adults receiving  pure HA, cell-
derived or egg-derived split or subvirion vaccines
Secondary Objectives

Comparison of the epitope specificity of B cells following vaccination with HA or 
subvirion vaccines
Evaluation of the different vaccine formulations to induce HA specific antibodies that 
recognize circulating viruses
Tertiary Objective

Confirmation of the safety of the licensed vaccines in healthy adults


Evaluation of the effect of repeated vaccination on the immune response

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Schematic of Study Design:
Because it is possible that prior exposure will impact the relative impact of peptide specific help 
on the antibody response, subjects will be screened for pre-vaccination antibody. Subjects will 
be divided into relatively high (greater than 1:8) or low (equal to or less than 1:8) pre-vaccination 
antibody based on pH1N1.  The basic study design for the healthy adults age 18-49 years is 
described below. Subjects will be queried regarding prior history of influenza vaccination, and 
stratified into those who have not been vaccinated in the previous year and those who have, 
followed by randomized assignment to receive one of four licensed vaccines.  Study subjects 
who participated in a previous year can be re-enrolled and will receive the same vaccine they 
received previously.  Because the goal of the protocol is to evaluate the immune response to 
the vaccine and is not intended as a formal comparison of the adverse event profile, the vaccine 
assignment is not blinded.  Following vaccination, subjects will have samples of blood obtained 
at the time points illustrated above.  The last study visit will take place approximately 6 months 
after vaccination in order to test the durability of the response.

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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
Email: angela_branche@urmc.rochester.edu
DMID Clinical Project Manager:
Melinda Tibbals, RAC, CCRA
Respiratory Disease Branch/DMID/NIAID 
5601 Fishers Lane, Room 8A18
Rockville, MD 20892-9825
Phone:  240-627-3387 
Email: melinda.tibbals@nih.gov
DMID Scientific Lead:
Marciela DeGrace, Ph.D.
Respiratory Diseases Branch/DMID/NIAID
5601 Fishers Lane Room 8E16
Bethesda, MD 20892-9825
Phone:  240-627-3460
Email:  marciela.degrace@nih.gov
DMID Medical Officer
Francisco Leyva, MD, PhD, ScM
Division of Microbiology and Infectious 
Diseases
NIAID, NIH
5601 Fishers Lane, Room 8E27 
Bethesda, MD 20892-9825
Phone:  240-627-3347
Email: francisco.leyva@nih.gov

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Scientific Investigators
David Topham, PhD
Department of Microbiology and Immunology
Center for Vaccine Biology and Immunology
University of Rochester Medical Center
601 Elmwood Avenue, Rochester NY 14642
Email: david_topham@urmc.rochester.edu
Andrea Sant, PhD
Department of Microbiology and Immunology
University of Rochester Medical Center
601 Elmwood Avenue, Rochester NY 14642
Email: andrea_sant@urmc.rochester.edu
Patrick Wilson, PhD
Department of Immunology
University of Chicago
924 E. 57th Street R422
Chicago, IL 60637
Email: wilsonp@uchicago.edu
Scott Hensley, PhD
Department of Microbiology
University of Pennsylvania
02A Johnson Pavilion
3610 Hamilton Walk
Philadelphia, PA 19104
Email: hensley@pennmedicine.upenn.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 prevaccination 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.
Growth of virus in embryonated hen’s eggs has been used for production of IIV since these 
vaccines were first introduced.  For the influenza A components of the vaccine, the selected 
reference antigenic variant for that season is typically reassorted with the high-yield laboratory 
influenza A/PR-8 virus to generate a seed virus that can replicate to high titer in eggs.  In some 
cases, the seed virus may require additional passage in eggs to achieve adequate growth levels 
in this substrate.  Harvested virions are subsequently concentrated, chemically inactivated, and 
disrupted with detergent, and the HA and NA proteins are partially purified by chromatography 
and other proprietary methods to yield a preparation referred to as split-product, subvirion, or 
subunit vaccine, depending on the manufacturer.  In addition to containing a standardized 
amount of HA protein as determined by single radial immunodiffusion (SRID), licensed IIV 
preparations contain variable, but substantial amounts of NA as well as matrix (M) and 

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nucleoprotein (NP).  Thus, it is possible that repeated exposure to the highly conserved NP and 
M proteins plays a role in the effect of prior vaccination on subsequent antibody responses.
Recently, two inactivated vaccines made from alternative substrates have been licensed in the 
US for seasonal use.  These include mammalian cell culture grown inactivated vaccine 
(FluCelVax) [4] licensed for individuals ages 4 and above, and a purified recombinant 
baculovirus-expressed HA vaccine (FluBlok) made in insect cells [5] and licensed for ages 18 
and above.  Cell culture grown vaccine is made using a different type of seed virus and 
purification process which could result in a different mixture of proteins in the final preparation.  
In contrast, the baculovirus-expressed HA vaccine is a completely different preparation that 
contains over 95% pure HA protein without any other components of influenza virus.  The 
availability of these additional licensed forms of influenza vaccine has provided an opportunity to 
evaluate the role that prior immunity to the various components of the vaccine plays in the 
antibody response, and to specifically address the hypothesis that CD4 recognition of epitopes 
specifically on the HA protein plays the major role in help for HA-specific B cell responses.
The basic composition of vaccines currently licensed in the US for seasonal influenza that are 
being used in this study are shown below:

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3.2
Preliminary Data 
We have previously demonstrated a wide breadth of CD4 repertoire to influenza, which includes 
up to 100 distinct peptides specificities in the primary response [6-8].  However, different 
populations of CD4 T cells, distinguished by their antigen specificity, may not contribute to the 
all functions important in development of a specific immune response.  In particular, it has been 
demonstrated that protein vaccines and some enveloped viruses [9], delivery of help for 
antibody responses requires that the B cell and CD4 T cell recognize the epitopes from the 
Vaccine
Fluzone (SD)
Fluzone (HD)
Flublok
FluCelVax
Production
Eggs (avian)
Eggs (avian)
Sf9 cells (insect)
MDCK cells (mammalian
Inactivation
Formaldehyde
Formaldehyde
NA
b prioprolactone
Disruption
Triton X-100
Triton X-100
NA
Cetyltrimethyl-
ammonium bromide
Purification
Sucrose gradient, 
additional 
concentration
Sucrose gradient, 
additional 
concentration
Column chromatography
ND
Dose
15 ug/HA
60 ug/HA
45 ug/HA
15 ug/HA
Indication
6 mo, and older
65 yrs and older
18 yrs and older
4 yrs and older
Components§
H1N1
A/Michigan/45/2015 
X-275*
A/Michigan/45/2015 
X-275*
A/Michigan/45/2015
A/Singapore/GP1908/20
15 IVR-180**
H3N2
A/Hong 
Kong/4801/2014 X-
263-B*
A/Hong 
Kong/4801/2014 X-
263-B*
A/Hong Kong/4801/2014
A/Singapore/GP2050/20
15
B Yamagata
B/Phuket/3073/2013
NA
B/Phuket/307/2013
B/Utah/9/2014
B Victoria
B/Brisbane/60/2008
B/Brisbane/60/2008
B/Brisbane/60/2008
B/Hong Kong/259/2010
Source:  Individual 2017-2018 FDA package inserts 
§ All virus components are antigenically consistent with current WHO recommendations, however egg-grown viruses 
may contain egg adaptation changes not contained in mammalian cell or baculovirus expressed vaccines
*X-275 and X-263-B represent reassortants with A/PR/8 (H1N1) for high yield growth in eggs, the HA and NA are 
derived from the vaccine viruses
**IVR-180 represents a reassortant with A/Texas/1/77 (H3N2) selected for high-yield growth in mammalian cell culture, 
the HA and NA are derived from the vaccine virus
NA – Not applicable.  ND – Not described in the package insert

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same protein antigen.  In the case of influenza antibodies specific for HA and NA, CD4 T cells 
specific for epitopes in these proteins may be the only ones relevant for development of the 
antibody response.  This help for high affinity Ab production may be primarily provided by cells 
belonging to the Tfh lineage (reviewed in [10]).   However, most studies of the cellular immune 
response to influenza vaccine have not separately tracked the specific CD4 cells with these 
combined properties, leaving the contribution of these cells undetected.  In contrast, monitoring 
CD4 T cells that do not have these specificities or function will likely contribute noise to the 
assessment of CD4 help for Ab responses to influenza.
In the first full year of this study, we found that there were unexpected differences in the serum 
antibody responses to vaccination between the study groups.  In particular, serum HAI antibody 
responses to the baculovirus-expressed HA were more frequent and the mean fold rise was 
greater than those to the egg-grown vaccine, possibly consistent with the hypothesis that a 
more directed CD4 T cell response is more effective at providing help for antibody.
Interpretation of these results is complicated by the fact that the dosage level of the rHA vaccine 
was approximately 3 times that of the egg or cell culture vaccines, so that the more vigorous 
antibody responses may simply be dose related rather than reflective of CD4 specificity.  
Despite randomization, pre-vaccination titers were highest in the Fluzone group, as well as 
history of prior vaccination (18/22 in Fluzone, 16/22 in Flublok, and 13/26 in Flucelvax).  In 
addition, we were unable to find significant numbers of subjects with pre-vaccination serum 
antibody titers of <= 1:8 to the pH1N1 component of the vaccines. 

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For these reasons, we modified our protocol by :
1.  Adding the high dose influenza vaccine (Fluzone HD).  This may allow separation of the 
effects of dose from the effects of vaccine composition
2.  Eliminating screening for low titers of antibody to pH1N1 and instead stratifying 
randomization based on history of prior vaccination
3.  Continuing to encourage re-enrollment in subsequent years with assignment to the same 
group, in order to assess the effect of vaccine composition on the response to revaccination. 
Secondly, in the 2017-2018 we noted that the Flucelvax H3 component was as poorly 
immunogenic as that of Fluzone SD despite the differences in manufacturing (cell-based 
derivation versus egg-based). Post-vaccination antibody titers in Flucelvax recipients were low 
to all H3N2 viruses tested, including the egg-based, cell-based and two wild-type H3N2 strains. 
Recent findings have demonstrated that egg adaptation of vaccine viruses during vaccine 
development for products made in eggs has led to decreased protection against wild-type H3N2 
viruses for products like Fluzone SD. However, we would have expected Flucelvax which avoids 
issues of egg adaptation, since vaccine viruses are grown in cell culture, to be more 
immunogenic to wild-type H3N2 strains. The reason for our findings of poor immunogenicity of 
the Flucelvax H3 components which was similar to egg-based Fluzone SD remains unclear and 
further study is needed outside the scope of the current project.
For this reason, though we have now chosen to eliminate the Flucelvax arm of the study for 
newly enrolled subjects.  However, to preserve the ability to assess the secondary aim of the 
effect of repeated vaccination without introducing confounders, subjects who have received 2 or 
more consecutive years of Flucelvax will continue to receive the same vaccine in subsequent 
years.

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3.3
Rationale
Our observations to date lead to the following model:  Traditional egg-derived TIV have 
contaminating internal virion proteins that preferentially elicit memory CD4 T cells specific for 
these proteins.  These CD4 T cells will have limited efficacy as helpers for the neutralizing Ab 
response and will suppress the CD4 T cell response to new HA epitopes in the vaccine.  The 
current study will test this hypothesis by comparing CD4 cell responses to specific epitopes, and 
the subsequent B cell and antibody response, in subjects receiving a vaccine containing only 
HA protein to vaccines with more complex antigenic characteristics.  The CD4 T cell reactivity to 
pools of unique, conserved, and total pH1 HA peptides as well as H3, influenza B HA, NP, and 
M1 peptides will be quantified using cytokine Elispot assays and flow cytometry, and then 
compared to the subsequent antibody and B cell response.
We will also use this study as an opportunity to evaluate the effects of prior vaccination.  Recent 
studies have emphasized the potential negative effect of vaccination in prior years on both the 
immune response as well as the protective effectiveness of current vaccine.  In order to 
evaluate this phenomenon in the context of multiple vaccine formulations, we will ascertain the 
prior vaccination history of the subjects and stratify vaccination based on vaccine history.
In addition, subjects who participated in this study in a previous year are eligible to re-enroll, and 
will receive the same vaccine that they were randomized to previously.  This will allow an 
evaluation of differences between vaccine formulations in the responsiveness to multiple 
vaccinations.  
Furthermore, recent studies indicate reports that the glycosylation pattern of viral 
hemagglutinins produced in cell culture can vary depending on the host cell used [11, 12], and 
that this can affect CD4 T cell immunogenicity and antibody recognition.  As a cell culture-based 
influenza vaccine production platform offers many advantages and may eventually supplant the 
traditional egg-based approach, it is of great value to understand the CD4 T cell response 
induced by this vaccine and how this affects neutralizing Ab production.
Recent data have also suggested that the failure of seasonal influenza infection to induce 
substantial levels of stalk specific antibody may be due to the relatively inaccessible nature of 
this epitope.  As part of this study, we will also compare the specificity of the human antibody 
response between the vaccine groups, with the hypothesis that the rHA vaccine will more 
readily allow targeting of these important, broadly conserved epitopes.  We have compelling 
preliminary data demonstrating that multiple antibodies that we have isolated have a particularly 
slow on rate when they bind to the HA-stalk versus the HA-globular head epitopes on whole 
virions, but not on recombinant HA trimmers expressed in baculovirus.  We hypothesize that a 
free, recombinant HA vaccine will allow more efficient targeting of the HA-Stalk epitopes. 

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3.4
Potential Risks and Benefits
3.4.1 Potential Risks
The risks and discomforts of this study include risks associated with the vaccine, the risks 
associated with study procedures (blood drawing 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.  Although high-dose vaccine is not indicated in individuals 
under 65 years of age, clinical trials of such vaccines in healthy adults suggest that high dose 
vaccine is association with slight increases in mild local reactogenicity but are otherwise well 
tolerated [13].  Systemic symptoms like fever and malaise occur at rates equal to placebo.  
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) [14]. No cases of GBS have 
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.
Personal health information of the subjects will be collected to determine eligibility and to 
evaluate safety 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.
3.4.2 Known Potential Benefits
All four products being used in this study are licensed vaccines for seasonal influenza and 
would be expected to provide some protection against influenza illness caused by the strains 
contained within the vaccine.

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4
OBJECTIVES AND OUTCOME MEASURES
4.1 Study Objectives
Primary Objective

Comparison of the serum antibody response to immunization with pure HA, cell-derived 
and egg derived subvirion vaccines in adults age 18-49 years

Comparison of the HA- specific CD4 T cell response in adults receiving  pure HA, cell-
derived  vaccine egg-derived split or subvirion vaccines
Secondary Objectives

Comparison of the epitope specificity of B cells following vaccination with HA or 
subvirion vaccines
Evaluation of the different vaccine formulations to induce HA specific antibodies that 
recognize circulating viruses
Exploratory  Objective

Confirmation of the safety of the licensed vaccines in healthy adults


Evaluation of the effect of repeated vaccination on the immune response
4.2 Outcome Measures
4.2.1 Primary Outcome Measures
The primary outcome measure for this study is:

Mean change in the specificity, quantity and quality of serum antibody titers to the 
vaccine viruses, pH1N1, H3 and influenza B  using HAI (year 1-5) and  MN for pH1N1 
and H3 viruses  (years 1-5 from Day 0 to Day 28

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
Mean change in the CD4 T cell reactivity to pools of  total pHA peptides derived from the 
pH1N1 HA, as well as H3, influenza B HA, NP, and M1 peptides will be quantified using 
cytokine Elispot  From Day 0 to Day 14
4.2.2 Secondary Outcome Measures
Secondary and tertiary outcome measures are listed below:
Secondary Outcome Measures

Antibody specificity of cloned plasmablasts from subjects in each group


Correlations of CD4 T cell ELISPOT number (Day 0 to Day 14)  and antibody titers (Day 
0 to Day 28) in subjects from each group (year 1-3), R value per cohort will be reported
Tertiary Outcome Measure

Vaccine related adverse events

Mean change in the CD4 T cell reactivity to pools of total pHA peptides derived from the 
pH1N1 HA, as well as H3, influenza B HA, NP, and M1 peptides will be quantified using 
cytokine Elispot from Day 0 to Day 14 for subjects with successive years of vaccination

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5
STUDY DESIGN
Subjects age 18-49 years will be stratified by their prior vaccination history, followed by 
randomized assignment to receive one of three licensed vaccines: Fluzone SD, Fluzone HD and 
Flublok.  Block randomization will be performed separately for the two groups.  Study subjects 
who participated in a previous year can be re-enrolled and receive the same vaccine as 
previously (Fluzone SD, Fluzone HD, Flublok, Flucelvax). Because the goal of the protocol is to 
evaluate the immune response to the vaccine and is not intended as a formal comparison of the 
adverse event profile, the vaccine assignment is not blinded.  Following vaccination, subjects 
will have blood sample obtained at the time points illustrated above.  The last study visit will take 
place 6 months after vaccination.

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6
STUDY POPULATION
Up to 500 subjects will be enrolled from the existing population of healthy adults residing in 
Rochester, NY and within the University of Rochester community, males and non-pregnant 
females aged 18-49 years, inclusive. Pregnancy status will be verified at screening and 
enrollment prior to vaccination. Women of childbearing potential will be asked to use a barrier 
method of birth control or an FDA approved form of birth control.
Study subjects will be recruited via placement of flyers in key locations on campus.  Based on 
previous experience, we anticipate enrollment of 40 to 60 percent non-pregnant females in
the study.
6.1
Inclusion Criteria
Subjects must meet all of the following inclusion criteria to participate in this study:
1.
Aged between 18 and 49 years of age (inclusive). (Participants who are returning for a 
subsequent year may be ≥ 50 years.)
2.
Female subjects of childbearing potential must fulfill one of the following: (i) not able to bear 
children because she has been surgically sterilized (tubal ligation or hysterectomy) or (ii) 
agrees to practice effective methods of contraception that may include, but are not limited to 
abstinence, barrier methods, monogamous relationship with vasectomized partner, birth 
control pills, patches, hormonal shots or hormonal implants, NuvaRing and IUDs 
(intrauterine devices), from 30 days prior to study enrollment through 30 days following 
receipt of the last dose of vaccine. 
3.
Female subjects of childbearing potential must have a negative urine pregnancy test within 
24 hours prior to vaccination.
4.
Are in good health1.
1As determined by medical history and targeted physical examination to evaluate acute or currently ongoing 
chronic medical diagnoses or conditions, defined as those that have been present for at least 90 days, which 
would affect the assessment of the safety of subjects or the immunogenicity of study vaccinations. Chronic 
medical diagnoses or conditions should be stable for the last 60 days. This includes no change in chronic 
prescription medication, dose, or frequency as a result of deterioration of the chronic medical diagnosis or 
condition in the 60 days prior to enrollment. Any prescription change that is due to change of health care 
provider, insurance company, etc., or that is done for financial reasons, as long as in the same class of 
medication, will not be considered a deviation of this inclusion criterion. Any change in prescription 
medication due to improvement of a disease outcome, as determined by the site principal investigator or 
appropriate sub-investigator, will not be considered a deviation of this inclusion criterion. Subjects may be on 

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chronic or as needed (prn) medications if, in the opinion of the site principal investigator or appropriate sub-
investigator, they pose no additional risk to subject safety or assessment of reactogenicity and 
immunogenicity and do not indicate a worsening of medical diagnosis or condition. Similarly, medication 
changes subsequent to enrollment and study vaccination are acceptable provided there was no deterioration 
in the subject’s chronic medical condition that necessitated a medication change, and there is no additional 
risk to the subject or interference with the evaluation of responses to study vaccination. Note: Topical, nasal, 
and inhaled medications (with the exception of inhaled corticosteroids as outlined in the Subject Exclusion 
Criteria (see Section 5.1.2), herbals, vitamins, and supplements are permitted.
5.
Have normal vital signs (heart rate >55 to <100 bpm; blood pressure: systolic ≥ 90 mm Hg 
and ≤150 mm Hg; diastolic ≤ 90 mm Hg; oral temperature <100.0ºF);  
6.
The subject is able to understand and comply with the planned study procedures, including 
being available for all study visits.
7.
The subject has provided informed consent prior to any study procedures.
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 a history of severe reactions following previous immunization with licensed 
or unlicensed influenza virus vaccines. 
3.
Subject report of a history of Guillain-Barre syndrome within 6 weeks of receipt of a previous 
influenza vaccine.
4.
The subject is a woman who is pregnant or breastfeeding or intends to become pregnant 
during the study period between enrollment and 30 days following receipt of vaccine.
5.
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. 
6.
The subject received immunoglobulin or another blood product within the 3 months prior to 
enrollment in this study.  
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.
The subject has long-term (greater than 2 weeks) use of oral or parenteral steroids 

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9.
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.
10. 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 (defines 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.   
11. Subjects with an active infection or that haves 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.
12. 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.
13. 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.
14. Have been hospitalized for psychiatric illness, history of suicide attempt, or confinement for 
danger to self or others, within the past 10 years.
15. The subject has a diagnosis of schizophrenia, bi-polar disease, or other severe (disabling) 
chronic psychiatric diagnosis, or is receiving psychiatric drugs.  Subjects who are receiving 
antidepressant drugs and are stable for at least 3 months prior to enrollment without 
decompensation are allowed enrollment into the study.  
16. The subject has a history of alcohol or drug abuse in the 5 years prior to enrollment.
17. The subject has a known human immunodeficiency virus, hepatitis B, or hepatitis C 
infection.
18. The subject has any condition that the principal investigator (PI) believes may interfere with 
successful completion of the study.
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 

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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.

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7
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT
7.1
Study Product Description
Fluzone Standard Dose (Sanofi Pasteur).  Quadrivalent subvirion vaccine (IIV4) produced in 
embryonated hen’s eggs and administered intramuscularly at a dose of 15 ug of HA (as 
determined by SRID) per component.
Fluzone High Dose (Sanofi Pasteur).  Trivalent subvirion vaccine (IIV3) produced in 
embryonated hen’s eggs and administered intramuscularly at a dose of 60 ug of HA (as 
determined by SRID) per component.
FluCelVax (Novartis).  Quadrivalent subunit vaccine produced in MDCK mammalian cell culture 
(ccIV4) administered intramuscularly at a dose of 15 ug of HA (determined by SRID) per 
component
FluBlok (Protein Sciences) Quadrivalent purified recombinant hemagglutinin vaccine (rIV4) 
produced in insect cells and administered intramuscularly at a dose of 45 ug of HA (determined 
by SRID) per component.
All four products are indicated for the prevention of influenza in adults, although Fluzone HD is 
only indicated for prevention in adults ages 65 and older.  
7.1.1 Acquisition
All four vaccines will be ordered from the hospital pharmacy which will acquire the vaccine 
through distributors.
7.1.2 Formulation, Packaging, and Labeling
Fluzone HD and SD (Sanofi Pasteur)
Fluzone (Influenza Vaccine) for intramuscular injection is an inactivated influenza vaccine, 
prepared from influenza viruses propagated in embryonated chicken eggs. The virus-containing 
allantoic fluid is harvested and inactivated with formaldehyde. Influenza virus is concentrated 
and purified in a linear sucrose density gradient solution using a continuous flow centrifuge. The 
virus is then chemically disrupted using a non-ionic surfactant, octylphenol ethoxylate (Triton® 
X-100), 11 producing a “split virus”. The split virus is further purified and then suspended in 
sodium 12 phosphate-buffered isotonic sodium chloride solution. 

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Fluzone is standardized according to United States Public Health Service requirements and is 
formulated to contain HA of each of the influenza strains recommended for the current influenza 
season.  Vaccine is formulated in sodium phosphate-buffered isotonic sodium chloride solution 
containing formaldehyde, octylphenol ethoxylate and gelatin, in single dose prefilled syringes
Flucelvax (Sequiris)
FLUCELVAX (Influenza Vaccine), a vaccine for intramuscular injection, is a “subunit” influenza 
vaccine prepared from virus propagated in Madin Darby Canine Kidney (MDCK) cells, a 
continuous cell line. These cells were adapted to grow freely in suspension in culture medium. 
The virus is inactivated with ß-propiolactone, disrupted by the detergent 
cetyltrimethylammonium bromide and purified through several process steps. Each of the 3 
virus strains is produced and purified separately then pooled to formulate the trivalent vaccine.
FLUCELVAX is a sterile, slightly opalescent suspension in phosphate buffered saline. 
FLUCELVAX is standardized according to United States Public Health Service requirements for 
the upcoming influenza season and is formulated to contain a total of 45 micrograms (mcg) 
hemagglutinin (HA) per 0.5 mL dose in the recommended ratio of 15 mcg HA of each of the 
three influenza strains recommended for that season/. Each dose of FLUCELVAX may contain 
residual amounts of MDCK cell protein (≤8.4 mcg), protein other than HA (≤ 120 mcg), MDCK 
cell DNA (≤ 10 ng), polysorbate 80 (≤ 1125 mcg), cetyltrimethlyammonium bromide (≤ 13.5 
mcg), and β-propiolactone (<0.5 mcg), which are used in the manufacturing process.
FLUCELVAX will be supplied in single dose, pre-filled syringes.  The vaccine contains no 
preservative or antibiotics, but the tip caps of the pre-filled syringes may contain natural rubber 
latex.
Flublok (Protein Sciences Corporation)
Flublok [15] is a sterile, clear, colorless solution of recombinant hemagglutinin (HA) proteins 
from three influenza viruses for intramuscular injection. It contains purified HA proteins 
produced in a continuous insect cell line (expresSF+®) that is derived from Sf9 cells of the fall 
armyworm, Spodoptera frugiperda, and grown in serum-free medium composed of chemically-
defined lipids, vitamins, amino acids, and mineral salts. Each of the three HAs is expressed in 
this cell line using a baculovirus vector (Autographa californica nuclear polyhedrosis virus), 
extracted from the cells with Triton X-100 and further purified by column chromatography. The 
purified HAs are then blended and filled into single-dose vials.
Flublok is standardized according to United States Public Health Service (USPHS) requirements 
to contain 45 mcg of each HA of the strains recommended for that season.
A single 0.5 mL dose of Flublok contains sodium chloride (4.4 mg), monobasic sodium 
phosphate (0.195 mcg), dibasic sodium phosphate (1.3 mg), and polysorbate 20 (Tween®20) 
(27.5 mcg). Each 0.5 mL dose of Flublok may also contain residual amounts of baculovirus and 

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host cell proteins (≤ 28.5 mcg), baculovirus and cellular DNA (≤ 10 ng), and Triton X-100 (≤ 100 
mcg).
Flublok contains no egg proteins, antibiotics, or preservatives.  Flublok will be supplied in single 
dose vials, and the stoppers used for the single-dose vials are not made with natural rubber 
latex.
7.1.3 Product Storage and Stability
Influenza vaccines will be stored in secure, limited-access temperature monitored refrigerator 
environment at 2°C to 8°C (.6°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
Each of the vaccines used in this study 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) n the subject’s preferred deltoid 
(upper arm) per the manufacturer’s instructions.
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)
Study vaccine will be sent to the research site at the University of Rochester prior to the start of 
the study.  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)

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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 56 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
Screening (Day -21 to Day 0)
Subjects will be screened for eligibility and for vaccine history prior to group assignment and 
vaccination.  At the screening visit, the following procedures will be performed:

The study will be explained to the subject, and the informed consent document will be 
signed.  Subjects will keep a copy of the informed consent for their information.

A medical history will be obtained, and details recorded in the case report form.

A directed 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.
8.2
Vaccination (Defined as Day 0)
Subjects will be considered enrolled if they have signed the consent form.  However, only 
subjects who met the entry criteria will be vaccinated and followed.  Note that the screening visit 
and the vaccination visit may take place on the same day.  At the vaccination visit the following 
procedures will be performed.

An interim medical history will be obtained (unless the screening and vaccination visit 
are the same) including concomitant medications, detailing any subsequent medical 
events the subject has experienced since the screening visit.

A directed physical exam will be performed, if indicated by the interval history, directed 
at the relevant portions of the exam.

A urine pregnancy test will be obtained in female subjects.  This test must be negative 
for the subject to continue in the study.

The entry criteria will be reviewed with the subject and it will be verified that the subject 
continues to meet the entry criteria.

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
A serum sample will be obtained from an arm vein for baseline antibody titers

A sample of Peripheral Blood Mononuclear Cells (PBMC) will be obtained for 
assessment of baseline influenza-specific B cells and CD4 cells

 Subjects that were not vaccinated with influenza vaccine with the prior year’s vaccine, 
will be randomized at the pharmacy into one of the three (FluBlok, Fluzone SD, Fluzone 
HD) vaccine groups, and will receive the indicated vaccine by intramuscular injection in 
open label fashion.

Repeat subjects will receive the same vaccine they received previously. (FluBlok, 
Fluzone SD, Fluzone HD, Flucelvax)

Subjects will be observed in the clinic for 30 minutes post vaccination for development of 
acute adverse events
8.3
Follow-up and Final Visits
8.3.1 Day 3 visit ((± 1 day)
Subjects will return 3 days after vaccination for the first 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 3.  On the day 3 visit, the following procedures will be 
done:

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 as described in the safety section 10.1 
and appropriate corrective actions taken if indicated

Peripheral Blood Mononuclear Cells (PBMC) will be collected from an arm vein 
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8.3.2 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.  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

If indicated by medical history, a directed physical exam will also be performed. 

Any adverse events (AE) will be recorded as described in the safety section 10.1 
and appropriate corrective actions taken if indicated

Peripheral Blood Mononuclear Cells (PBMC) will be collected from an arm vein 
for assessment of the B cell and CD4 T cell response to vaccination 
8.3.3 Day 14 visit (± 1 day)
Subjects will return for follow up approximately 14 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 history, a relevant 
physical exam will also be performed.

Any adverse events (AE) will be recorded as described in the safety section 10.1 
and appropriate corrective actions taken if indicated

Peripheral Blood Mononuclear Cells (PBMC) will be collected from an arm vein 
for assessment of the CD4 T cell response to vaccination
8.3.4 Day 28 visit (± 2 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

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
If indicated by medical history, a directed physical exam will also be performed. 

Any adverse events (AE) will be recorded as described in the safety section 10.1 
and appropriate corrective actions taken if indicated

A serum sample will be obtained from an arm vein for antibody titers 

Peripheral Blood Mononuclear Cells (PBMC) will be collected from an arm vein 
for assessment of the B cell response to vaccination
8.3.5 Influenza surveillance
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 asked to record weekly on a mobile device application 
(MyCAP) if they have developed influenza like symptoms. 
MyCAP is a mobile app that interfaces with REDCap using REDCap’s secure API. It gives 
researchers the ability to collect task oriented data on participants via the participants’ mobile 
device in real time. Enrolling the participant and subsequently communication between the 
MyCAP mobile app and REDCap server is made possible by generating a unique QR code for 
each participant from the REDCap-MyCAP web portal. The mobile app scans this QR code 
which contains the project name, site and uniquely auto-generated participant id. The participant 
id is created by REDCap and is not based upon any data entered about the participant at any 
time or linked to any personal health information or identifying information. The mobile app and 
consequently the participant, only have the capability of submitting data to the project, 
eliminating the concern of data breaches since there is no capability to view or read any data 
from the project. The app will also have the capability to be password protected. 
Once the mobile app has registered the QR code, participants will be asked to answer a weekly 
survey which they will be prompted to complete every Tuesday pertaining to the development of 
influenza like illness and associated symptoms. Data collected from participants in the mobile 
application will only be available to study staff. The following questions will be asked:
1) Have you had a fever or felt feverish in the past 5 days?
○ yes     ○ no
2) Have you had any of the following symptoms in the past 5 days: runny nose, cough, sore    
throat, body aches?
○ yes
○ no

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Subjects who do not have a mobile device compatible with the application or are unable or 
unwilling to utilize the application will be called weekly by the study staff and queried using the 
aforementioned questions. The app will send a reminder to study subjects at on Tuesday 
morning of every week to complete the surveillance questions. While there is no window built 
into the app for subjects to complete these questions it will be expected that they respond to the 
questions within 24 hours. Questions can only be answered once a week. If questions were not 
completed for the week the study team will be notified and this will be reviewed on Monday of 
the following week. If a subject misses two consecutive surveillance time points, the study team 
will call them to determine if there is an issue with the app or alternatively if they would prefer 
phone surveillances be conducted. If it is the latter case, the app will be uninstalled and weekly 
phone surveillance will be initiated. Three attempts will be made to contact subjects by phone 
and one attempt with a letter mailed to their street address. Subjects who cannot be reached by 
this method will be considered lost to follow up.   
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
8.3.6 Final study visit (Day 180 +/- 14 days)
Subjects will return approximately 180 days after vaccination for follow-up. This visit will 
be the study termination visit.  At the final 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. 

Adverse events (AE) will be recorded and corrective actions taken if indicated

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
A serum sample will be obtained from an arm vein for antibody titers

Peripheral Blood Mononuclear Cells (PBMC) will be collected from an arm vein 
for assessment of the CD4 T cell response to vaccination
8.4
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 as described in the safety section 10.1 will be recorded.
Subjects will be encouraged to permit continued follow-up of AEs and to donate scheduled 
blood samples, if possible.
8.5
Unscheduled Visit
Unscheduled visits may occur at any time during the study.  Any of the following activities may 
be performed:

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.

Information regarding AEs as described in the safety section 10.1 will be recorded. .

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.

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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.
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 through 28 days after 
vaccination or early termination, whichever occurs first.
Medications that might interfere with the evaluation of the investigational product should not be 
used unless absolutely necessary.  Medications in this category include, but are not limited to, 
glucocorticoids, i.e., oral, parenteral and high-dose inhaled steroids, and immunosuppressive or 
cytotoxic drugs.  The administration of licensed vaccines should be delayed until 21 days after 
the study vaccine.  Subjects should not receive experimental agents including vaccines for the 
duration of the study  
Targeted Physical Examination:  Licensed study clinicians (i.e., physician, physician’s assistant, 
nurse practitioner) may conduct a targeted physical examination if indicated based on review of 
history for assessment of AEs that require VAERS reporting as described in section 10.1, or if 
necessary to assist in determining eligibility.  All subjects will have vital signs (blood pressure, 
pulse, and oral temperature) and height and weight 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 A/California/07/09 virus 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.

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Microneutralization (MN) assay:  Sera will be tested by microtiter technique for neutralization of 
an A/California/07/09 x PR8 reassortant virus 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:  Peripheral Blood Mononuclear Cells (PBMC) will be obtained before and on 
day 7 and 28 after vaccine and evaluated for B-cell responses.  B cell responses will be 
evaluated by memory cell assay [16] and antibody secreting cell assay.  In addition, B cells will 
be cultured in vitro and the functional quality of secreted antibody assessed by HAI and MN 
assays
CD4 T cell responses: PBMCs collected prior to and on day 3, 7, 14 and 180 after vaccination 
will be evaluated for the presence of antigen specific CD4 cells by ELISPOT assay and flow 
cytometry using a panel of peptides spanning the H1 hemagglutinin, neuraminidase and internal 
virion proteins such as NP and M1 [17].  This assay will identify both cross reactive and H1 
specific epitope responses, as well as recall of memory cells to conserved internal virion 
proteins.  PBMC will also be evaluated for the presence of antigen specific ELISPOT and cell 
homing markers by flow cytometry.
9.2.2 Specimen Collection, Preparation, Handling and Shipping
Instructions for specimen preparation, handling, and storage are included in the Manual of 
Procedures (MOP).  Instructions for specimen shipment are included in the MOP.
9.3
Procedures to be Followed in the Event of Abnormal 
Laboratory Test Values or Abnormal Clinical Findings
Urine or serum pregnancy tests will be performed within 24 hours prior to vaccination on all 
female subjects of childbearing potential (see above).  Subjects with positive pregnancy tests 
are not eligible to participate.  No other screening or clinical laboratory tests will be routinely 
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10
ASSESSMENT OF SAFETY
10.1 Specification of Safety Parameters
Only 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.  No serious adverse events are anticipated to occur to study 
participants as a result of this protocol. 
Adverse events (AEs) related to administration of influenza vaccine include (as per Package 
Insert) injection site reactions (pain, swelling and erythema) and systemic reactions (headache 
body ache and muscle weakness). Since immunization with influenza vaccine is offered as 
standard of care to healthy adults, these anticipated reactions to the vaccine will not be 
considered reportable adverse events. Events meeting VAERS reporting requirements will be 
reported as indicated below. 
There are minimal risks associated with venipuncture for obtaining research related labs.   
Foreseeable adverse events associated with venipuncture are: mild, temporary discomfort at 
the venipuncture site, bruising, and phlebitis. Very rarely, patients may experience vaso-vagal 
syndrome during phlebotomy. Vaso-vagal reactions may include diaphoresis, nausea, syncope 
and rarely loss of consciousness. These events will not be collected. However, severe vaso-
vagal reaction, including loss of consciousness or > grade 2 hypotension will be reported as 
adverse events of special interest.
Any other adverse events that meet the reporting requirements of the Institutional Review Board 
will also be reported to DMID Medical Officer and the Clinical Project Manager.
10.2 Safety Reporting: VAERS and Adverse Events of Special 
Interest (AESI)
The investigator must report the following events to VAERS with a copy to the DMID Medical 
Officer, and the Clinical Project Manager will be notified by email, including a copy of the report 
(VAERS form). In addition, these events will be reported on the VAERS/AESI case report form: 

Any adverse event listed by the vaccine manufacturer as a contraindication to further doses 
of the vaccine (Severe allergic reaction: e.g. (Anaphylaxis or anaphylactic shock 7 days 
post-vaccination) encephalopathy or encephalitis: e.g. coma, decreased level of 
consciousness, prolonged seizures within 7days of vaccine administration, Gullian-Barre 
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
Any adverse event listed in the VAERS Table of Reportable Events Following Vaccination 
found using the following link: https://vaers.hhs.gov/docs/VAERS_Table_of_Reportable 
Events_Following_Vaccination.pdf.

Brachial neuritis (28 days) 

Any acute complications or sequelae (including death) of above events that occurs within 
the specified time period after vaccination, 

In the judgment of the PI, any unexpected reaction to the vaccine will be reported to VAERS 
as well as the DMID Medical Officer and the Clinical Project Manager. 
The investigator must also report the following adverse events of special interest (AESI) on the 
VAERS/AESI case report form, and to DMID CPM and DMID Medical Officer. However, these 
events do not meet criteria for VAERS reporting: 

Severe vaso-vagal reaction following venipuncture, including loss of consciousness or > 
grade 2 hypotension. 

Infection or inability to breast feed following the use of the breast pump. 
All adverse events that meet the reporting requirements of the Institutional Review Board will 
also be reported to DMID Medical Officer and the Clinical Project Manager. 
10.3 Procedures to be Followed in the Event of Abnormal 
Laboratory Test Values or Abnormal Clinical Findings
Urine pregnancy tests will be performed within 24 hours prior to vaccination on all female 
subjects of childbearing potential (see above).  No other screening or clinical laboratory tests 
will be routinely obtained.
10.4 Reporting Procedures
ICH GCP 6, Section 4.11 require that an investigator notify the sponsor, regulatory authority(ies) 
and the local IRB immediately of any serious adverse event, deaths, or life-threatening 
problems that occur in the study.  Adverse events and/or laboratory abnormalities identified in 
the protocol as critical to safety evaluations should be reported to the IRB in accordance with 
reporting requirements specified by the IRB. Line lists of all adverse events and/or laboratory 
abnormalities will be reported via email to the DMID Clinical Project Manager and Medical 
Officer on a monthly basis.
10.5  Other Adverse Events (if applicable) 
Non serious adverse events will be reported with the annual report for the study

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10.6 Reporting of Pregnancy
Pregnancy events will be reported with the annual report of the study.  All pregnancies will be 
followed to termination and the result of the pregnancy will be recorded in the study record.
10.7 Type and Duration of Follow-up of Participants after 
Adverse Events
All adverse events and serious adverse events will be followed until resolved, return to baseline 
or stable as determined by the study team.  

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11
CLINICAL MONITORING 
Purpose: 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. 
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12
STATISTICAL CONSIDERATIONS
12.1 Study Hypothesis
The primary hypothesis being tested in this study is that there will be differences in the serum 
antibody response, specificity, quantity and quality of memory B cell derived polyclonal 
antibodies (MpAb) and specificity and magnitude of the HA-specific CD4 T cell response 
between recipients of egg-derived and recombinant vaccines.
12.2 Sample Size Considerations
The power analysis is based on the primary aim of comparison of serum antibody, specificity, 
memory B cell derived polyclonal antibodies (MpAb) and HA-specific CD4 T cell counts (in log 
scale to stabilize variance) between the group of subjects vaccinated with egg derived TIV IM 
and the group of subjects vaccinated with Baculovirus derived TIV IM. In this explorative study, 
the HA-specific CD4 T cell response to the Baculovirus derived vaccine is unknown and thus no 
information is available about the group difference. But our past experience indicated the 
coefficient of variation (CV) of HA-specific CD4 T cell counts varied between 0.25 and 0.4 [18]. 
If we assume CV and use percent-change differences in HA-specific CD4 T cell counts relative 
to the group with egg derived vaccine, with 45 subjects per group, we will have 80% power to 
detect at least 10% ~ 20% difference in HA-specific CD4 T cell counts between the two groups.  
This power analysis is based on two-sample t test with a two-sided significance level of 0.05 
and equal allocation of subjects to any two groups.  
12.3 Planned Interim Analyses (if applicable)
No formal interim analysis is planned.
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 efficacy is planned. Immunogenicity analysis will be done 
yearly as part of the overall analysis plan. 

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12.4 Final Analysis Plan 
We will define the groups as: G1 (egg derived vaccine) = G1a (seronegative to pH1N1 virus and 
receiving egg derived vaccine) + G1b (seropositive and receiving egg derived vaccine); G2 
(baculovirus derived vaccine) = G2a (seronegative to to pH1N1 virus and receiving baculovirus 
derived vaccine) + G2b (seropositive and receiving baculovirus derived vaccine).  The log 
transformation will be applied to stabilize variance, as suggested by our past experience [18]. 
The between-group and among-group comparison in each of the outcomes will be based on 
both standard statistical analysis approaches using summary statistics and the complicated 
random variance model approach. Specifically, each of CD4 specific cells for different Influenza 
proteins (eg HA, NP etc), memory B cell derived polyclonal antibodies,  HAI and MN titers 
among the four groups and between G1 and G2, between G1a and G2a, and between G1b and 
G2b, we will use maximum responses or maximum boost responses (maximum value after day 
0 – value at day 0) or maximum fold increase as endpoint and do group comparison by ANOVA 
for among-group comparison and two-sample t-test for between-group comparison, or their 
nonparametric analogy of Kruskal-Wallis test and Wilcoxon Rank sum test, respectively, may 
also be applied for robustness. Multiple comparisons may subject to Bonferroni correction, if 
appropriate. Correlation test such as Pearson correlation analysis, or its nonparametric analogy 
of Spearman correlation analysis, will be used to study how baseline CD4 responses and/or 
memory B cell derived polyclonal antibodies, HAI, MN titers affect the boost responses of CD4, 
memory B cell derived polyclonal antibodies, HAI and MN titers after vaccination. 
Various parametric or nonparametric mixed-effects regression approaches 99 will be used to 
study the dynamic curve of each outcome, to take account of the correlation among 
observations from the same subjects over time. Group indicator will enter the model as a 
predictor and thus can be compared simultaneous at each time point and also by difference 
from difference approach to control the effect of baseline values. Demographic information may 
be controlled by entering the model as predictors. To further investigate the effect of CD4 cell 
specificity in helping neutralizing antibody responses, regression analysis will be applied, with 
boost or fold increase of HAI or MN titers as responses, all the CD4 cell specificities as 
predictor, possibly adjusting demographic variables. Type I and III sum of squares and standard 
coefficient estimates can be used to compare the effect of CD4 specificities in helping 
neutralizing antibody responses. The parametric or nonparametric mixed-effects regression 
approaches may also be used to compare the effects to take account of within-subject 
correlation. Among-group plasmablast comparisons will be proceeded by: 1). One-way ANOVA, 
or its nonparametric analog of Kruskal-Wallis test; 2). Repeated ANOVA or its nonparametric 
analog of Friedman test for simultaneous pairwise comparison, with within-subject correlation 
taken into account.

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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 serve as source 
documents.  All study documents will be secured by key and/or password protection.

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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 sites, 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 applicable 
documentations are maintained on site.
DMID reserves the right to review site CQMP findings.  
The site 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 field 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), if applicable. The investigator’s institution will 
hold a current Federal Wide Assurance (FWA) issued by the Office for Human Protections 
(OHRP) for federally funded research.  
15.2 Institutional Review Board
The University of Rochester will provide for the review and approval of this protocol and the 
associated informed consent documents 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 tp DMID.
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 
worker 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 worker will request that a witness be present while the study 
worker reads and explains the study and what participation will entail.  If the eligible participant 

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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 
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 surrogates 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.
Subjects who participated in year 1 of the study will be allowed to participate again the following 
year. Subjects who agreed to future contact will be contacted by phone and/or letter to invite 
them to participate.
15.3.1 Informed Consent/Assent Process (in Case of a Minor)
No minors will be enrolled in this study
15.4 Exclusion of Women, Minorities, and Children (Special 
Populations)
Because not all of the vaccines being studied are licensed for use in individuals under 18, 
children will not be included in this study.  Because pregnancy could alter the immune response 
to vaccination, pregnancy is an exclusion to participation.  Otherwise there are no exclusions of 
special populations.

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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 and genetic tests in addition to the clinical information relating to participants.
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. 
To further protect subject confidentially in the United States, research participants in NYICE 
protocols are protected by a Certificate of Confidentiality from Department of Health and Human 
Services (DHHS), which can prevent disclosure of study participation even when that 
information is requested by subpoena. Participants are told of the use and limits of the 
certificate in the study consent form. 
15.6 Study Discontinuation
If the study is discontinued, enrolled subjects will continue to be followed for safety 
assessments.  No further doses of vaccine will be administered.
15.7 Future Use of Stored Specimens
Subjects will be asked for permission to keep any remaining specimen for possible use in future 
research studies, such as testing for antibodies against other viruses or bacteria. Samples will 
be stored 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. Subjects can decide if they want their samples to be used for future research or have 

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their samples destroyed at the end of the study. A subject’s decision can be changed at any 
time prior to the end of the study by notifying the study doctors or nurses in writing. However, if 
a subject consents to future use and some of their blood has already been used for research 
purposes, the information from that research may still be used.

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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.
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.
Acute surveillance clinical data will be captured using the MyCAP personal device application 
which is password protected. This aplication will be uploaded onto the subjects’ personal device 
using a unique QR code which is not linked to personal health information. The application 
interfaces directly with RedCap and data will be transmitted in real-time as it is entered. No data 
will be stored permanently on the subjects’ personal devices. 

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16.3 Types of Data
Data for this study will include subject demographics, safety assessments including 
reactogenicity events after vaccination, and research laboratory results including antibody 
responses and measurements of B cell, CD4 cell, and transcriptional responses.
16.4 Timing/Reports
The final report will include a comprehensive analysis of the data.
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.

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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 D and Monto AS. 
Influenza vaccine effectiveness in the community and in the household Clin Infect Dis  2013
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. Frey S, Vesikari T, Szymczakiewicz-Multanowska A, Lattanzi M, Izu A, Groth N and Holmes 
S. Clinical efficacy of cell culture-derived and egg-derived inactivated subunit influenza vaccines 
in healthy adults Clin Infect Dis  2010;51:997-1004
5. Treanor JJ, El Sahly HM, King JC, Jr., Graham I, Izikson R, Kohberger R, Patriarca P and 
Cox MM. Protective efficacy of a trivalent recombinant hemagglutinin protein vaccine (FluBlok) 
against influenza in healthy adults:  a randomized, placebo-controlled trial Vaccine  
2011;29:7733-7739
6. Richards KA, Chaves FA, Krafcik FR, Topham DJ, Lazarski CA and Sant AJ. Direct ex vivo 
analysis of HLA-DR1 transgenic mice reveal an exceptionally broad pattern of 
immunodominance in the rpmary HLA-DR1-restricted CD4 T-cell response to influenza virus 
hemagglutinin J Virol  2007;81:7608-7619
7. Nayak JL, Richards KA, Chaves FA and Sant AJ. Analyses of the specificity of  CD4 T cells 
during the primary immune response to influenza virus reveals dramatic MHC-linked 
assymmetries in reactivity to individual viral proteins Viral Immunology  2009;23:169-180
8. Richards KA, Chaves FA and Sant AJ. Infection of HLA-DR1 Transgenic Mice with a Human 
Isolate of Influenza A Virus (H1N1) Primes a Diverse CD4 T-Cell Repertoire That Includes CD4 
T Cells with Heterosubtypic Cross-Reactivity to Avian (H5N1) Influenza Virus J Virol  
2009;83:6566-6577
9. Sette A, Moutaftsi M, Moyron-Quiroz J, McCausland MM, Davies DH, Johnston RJ, Peters B, 
Rafii El Idrissi Benhania M, Hoffmann J, Su HP, Singh K, Garboczi N, Head S, Grey H, Felgener 
PL and Crotty S. Selective CD4+ T cell help for antibody responses to a large viral pathogen:  
deterministic linkage of specificities Immunity  2008;28:847-858
10. Crotty S. Follicular Helper CD4 T Cells (TFH) Annual Review of Immunology  2011;29:621-
663
11. Hütter J, Rödig JV, Höper D, Seeberger PH, Reichl U, Rapp E and Lepenies B. Toward 
Animal Cell Culture–Based Influenza Vaccine Design: Viral Hemagglutinin N-Glycosylation 
Markedly Impacts Immunogenicity The Journal of Immunology  2013;190:220-230

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08 February 2021
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12. Schwarzer J, Rapp E, Hennig R, Genzel Y, Jordan I, Sandig V and Reichl U. Glycan 
analysis in cell culture-based influenza vaccine production: Influence of host cell line and virus 
strain on the glycosylation pattern of viral hemagglutinin Vaccine  2009;27:4325-4336
13. Keitel WA, Couch RB, Cate TR, Hess KR, Baxter B, Quarles JM, Atmar RL and Six HR. 
High doses of purified influenza A virus hemagglutinin significantly augment serum and nasal 
secretion antibody responses in healthy young adults J Clin Microbiol  1994;32:2468-2473
14. 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
15. Ochiai H, Fujieda M, Ohfuji S, Fukushima W, Kondo K, Maeda A, Nakano T, Kamiya H, 
Hirota Y and Influenza Vaccine Epidemiology Study G. Inactivated influenza vaccine 
effectiveness against influenza-like illness among young children in Japan--with special 
reference to minimizing outcome misclassification Vaccine  2009;27:7031-5
16. 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
17. Sant AJ, Chaves FA, Krafcik FR, Lazarski CA, Menges P, Richards K and Weaver JM. 
Immunodominance in CD4 T-cell responses:  implications for immune responses to influenza 
virus and for vaccine design Expert Review of Vaccines  2007;6:357-368
18. Nayak JL, Fitzgerald T, Richards KA, Yang H, Treanor J and Sant AJ. CD4 T-cell expansion 
predicts neutralizing antibody responses to monovalent inactivated pandemic H1N1 influenza 
vaccine J Infect Dis  2013;207:297-305

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19
SUPPLEMENT/APPENDICES
Table of study procedures
Study Day (numbers denote blood volume in mL)
Procedure
d-21 
to 0
0
3
7
14
28
180
Day 0-56 
Blood 
Volume
Informed Consent
X
 
 
 
 
 
 
Medical History
X
 
 
 
 
 
 
Interval History
 
X
X
X
 
X
X
0
Directed Physical Exam*
 
X
X
X
X
X
X
0
Urine Pregnancy Test**
 
X
 
 
 
 
0
Review Entry Criteria
X
X
 
 
 
 
0
Serum for antibody
 
10
 
 
20
10
30
PBMC for B cells
 
30 
60
 
60
 
150
PBMC for CD4 T cells
 
100
50
80
50
50
280
Randomization
 
X
 
 
 
 
 
Vaccination
 
X
 
 
 
 
 
AE review
 
 
X
X
 
X
X
 
Total
140
50
140
50
80
60
460
*Examination if indicated by history
**Female subjects only