Randomized Open-Label Trial to Compare the Immunogenicity of Cell Culture-Based and Recombinant Unadjuvanted Quadrivalent Influenza Vaccines to Conventional Egg-Based Unadjuvanted Quadrivalent Influenza Vaccines Among Healthcare Personnel Aged 18-64 Years
Phase: PHASE4
Conditions: Influenza
Sponsor: Centers for Disease Control and Prevention

 
 
       
1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomized Open-Label Trial to Compare the Immunogenicity of Cell Culture-Based and 
Recombinant Unadjuvanted Quadrivalent Influenza Vaccines to Conventional Egg-Based 
Unadjuvanted Quadrivalent Influenza Vaccines among Healthcare Personnel Aged 18-64 Years 
 
NCT03722589. 
 
Document Date:  October 29, 2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
       
2 
 
 
 
 
 
 
 
421. Title of Project:  Randomized Open-Label Trial to Compare the Immunogenicity of Cell 
Culture-Based and Recombinant Unadjuvanted Quadrivalent Influenza Vaccines to Conventional 
Egg-Based Unadjuvanted Quadrivalent Influenza Vaccines among Healthcare Personnel Aged 
18-64 Years 
 
Table of Contents 
Content  
 
 
 
 
 
 
 
 
 
Page Number 
2.0 List of Abbreviations……………………………………………………………………………………………………......................... 3 
3.0 Project Summary………………………………………………………………………………………………………………………………... 3 
        
Study Sponsors/Investigators…………………….…………………………………………………………………..…………… 4 
4.0 Introduction…………………..……………………………………………………………………………………………………………………. 5 
 
4.1 
Background Information……………………….………………………………………………………………………. 5 
 
4.2 
Justification……………………………………………………………………………….………………………………….. 9 
 
4.3 
Expected Benefits from Proposed Study………………………….………………………………………..…. 10 
5a. Study Objectives………………………………………………………………………………………………………………………….………. 10 
 
 
5a.1 
Primary Objectives………………………………………………………………………………............. 10 
 
 
5a.2 
Secondary Objectives………………………………………………………………………………………. 11 
5a.3 
Exploratory Objectives…………………………………………………………………………………….. 11 
5b. Study Endpoints………………………………………………………………………………………………………………………............. 12 
 
 
5b.1 
Primary Endpoints…………………………………………………………………………………........... 12 
 
 
5b.2 
Secondary Endpoints……………………………………………………………………………………….. 13 
5b.3 
Exploratory Endpoints……………………………………………………………………………………… 13 
6.0 Study Location…………………………………………………………………………………………………………………………............. 17 
7.0 Methods………………………………………………………………………………………………………………………………….............. 17 
 
7.1 
Study Population…………………………………………………………………………………………………………… 17 
 
7.2 
Inclusion Criteria……………………………………………………………………………………………….............. 17 
 
7.3 
Exclusion Criteria………………………………………………………………………………………….……............. 18 
 
7.4 
Discontinuation/Withdrawal criteria…………………………………………………………….……............. 18 
 
 
7.5 
Implementation/Monitoring Procedures……………………………………………………………............. 18 
 
 
 
 
Study Design…………………………………………………………………………………………………….. 19 
 
 
 
Consent/Screening/Enrollment………………………………………………………………………. 19 
 
 
 
Study Visit 1, Year 1…………………………………………………………………………………………. 20 
 
 
 
 
Randomization…………………………………………………………………………………… 20 
Opt-In Consent for Longitudinal PBMC Collection:……………………………… 20 
 
 
 
 
 
Blood Draw ……………….…..………………………………………………………………….. 21 
 
 
 
 
Blinding………………………………………………………………………………………………. 21 
 
 
 
 
Post-Vaccination Safety Monitoring ………………………………………………….. 21 
 
 
 
Study Visit 1b, Year 1………………………………………………………………………………………… 21 
Blood Draw ……………….…..…………………………………………………………………… 21 
 
Study Visit 2, Year 1…………………………………………………………………………….. 21 
Blood Draw ……………….…..…………………………………………………………………… 22 
Study Visit 3, Year 1…………………………………………………………………………................... 22 
Blood Draw ……………….…..…………………………………………………………………… 22 
 
 
 
Study Visit 1, Year 2………………………………………………………………………………………….. 22 
Randomization of Participants from Year 1 of the Study…………………….. 22 

 
 
       
3 
 
Randomization of Newly Enrolled Participants in Year 2…………………….. 22 
Blood Draw ……………….…..………………………………………………………………….. 22 
Vaccine Administration……………………………………………………………………….. 23 
Study Visits 1b, 2, and 3 of Year 2……………………………………………………………………… 23 
 
 
 
Monitoring of Adverse Events…………………………………………………………………………… 23 
 
 
 
Active Surveillance for Influenza-Like Illness……………………………........................... 23 
 
 
 
Electronic Medical Record Extraction/Abstraction……………………………………………. 24 
Protocol Deviations………………………………………………………………………………………….. 24 
Data Linkage with Previous Study of Influenza Vaccine Effectiveness during the 2010-
2011 Influenza Season……………………………………………………………………………………… 24 
Protocol Completion or Termination………………………………………………………………… 25 
 
Site Monitoring Plan…………………………………………………………………………… 25 
Study Design Flow Diagram ………………………………………………………………............................................ 26 
 
 
 
Statistical Analysis………………………………………………………………………………............... 28 
 
 
 
Power/Sample Size Calculations…………………………………………………………............... 28 
 
 
 
Laboratory Protocol……………………………………………………………………………............... 29 
 
 
 
 
Specimen Collection/ Storage…………………………………………………………….. 29 
 
 
 
 
Specimen Testing……………………………………………………………………………….. 31 
7.6 Vaccine Product……………………………………………………………………………………………………….……........................ 33 
8.0 Ethical Considerations…………………………………………………………………………………………………..……………………… 34 
 
8.1 
Possible Risks…………………………………………………………………………………………………………………. 34 
 
8.2 
Provisions for protecting privacy/ confidentiality …………………………………..……………………… 35 
 
8.3 
Consent forms for study volunteers ……………………………..………………………………………………. 35 
9.0 Study Oversight, Management, and Guidance for Decision Making ………………………………………….……...... 36 
10.0 References………………………………………………………………………………………………..……………............................. 36 
 
  
 
List of Appendices 
 
Appendix A:  Schedule of Events  
 
Appendix B:  Eligibility Screening Form 
 
Appendix C:  Consent Form 
 
Appendix D:  Enrollment Questionnaire 
 
Appendix E:  Pre-vaccination Questionnaire/Vaccine Administration Form 
 
Appendix F1:  Blood Specimen Collection/Tracking Form 
 
Appendix F2:  Influenza-like Illness Data Collection and Respiratory Specimen Collection Form 
 
Appendix G:  EMR Chart Extraction/Abstraction Form 
 
Appendix H:  Protocol Deviation Log 
 
Appendix I:  Study Status Change Form 
 
Appendix J:  Active Surveillance Tracking Form 
 
Appendix K:  Human Sera Collection Guidelines for Influenza Serology   
 
Appendix J:  Guidelines for Freezing Human PBMCs 
 
Protocol Version 5, Modified on May 24, 2018, (29Aug2018) 
 
 
 
 
 
 
 
 
 
 

 
 
       
4 
 
 
 
 
 
 
 
 
 
 
2. List of Abbreviations 
 
 
ADCC 
Antibody-dependent cell-mediated cytotoxicity 
GMT 
Geometric mean titers 
HCP 
Healthcare Personnel 
HA 
Hemagglutinin 
HI 
Hemagglutination Inhibition 
ILI 
Influenza-like illness 
MDCK 
Madin-Darby Canine Kidney 
MFR 
Mean fold rise  
MN 
Microneutralization  
NA 
Neuraminidase 
NAI 
Neuraminidase antibody mediated inhibition 
rHA 
Recombinant Hemagluttinins 
SCR 
Seroconversion rate 
 
3. Project Summary  
This randomized, open-label trial will assess humoral and cell-mediated immune 
responses to cell culture-based and recombinant unadjuvanted quadrivalent influenza vaccines 
compared to conventional egg-based unadjuvanted quadrivalent standard dose (15µg of HA per 
strain) influenza vaccines among persons aged 18-64 years.  The trial will be conducted at two 
sites in the United States during two influenza seasons (2018-19 and 2019-20).  Stratified 
enrollment procedures will be used to enroll a mix of participants based on age and reported 
receipt of influenza vaccine during the prior season as a proxy for frequency of influenza 
vaccination.   
Eligible participants at each site will be randomized 2:2:1:1 to receive a single dose of 
cell culture-based vaccine (Flucelvax™ Quadrivalent by Seqirus, Inc., 15µg of HA per strain) 
versus recombinant vaccine (Flublok® Quadrivalent by Sanofi Pasteur, 45µg of HA per strain) 
versus one of two standard dose egg-based vaccines (Fluzone® Quadrivalent by Sanofi Pasteur, 
15µg of HA per strain and Fluarix® Quadrivalent by GlaxoSmithKlein, 15µg of HA per strain) 
during August-September of 2018 and again during August-September of 2019.  All study 

 
 
       
5 
 
vaccines are licensed for use in adults aged >18 years in the United States.  Participants will have 
blood collected just prior to vaccination and at approximately 28 days and 6 months post-
vaccination (or at the end of influenza virus circulation as determined by available surveillance 
data) to evaluate humoral immune responses to vaccination. Additional blood will be collected 
from a subset of participants pre-vaccination and at approximately 7 days, 28 days and 6 months 
post-vaccination (or at the end of influenza virus circulation) to evaluate longitudinal cell-
mediated immune responses to vaccination.  Active surveillance with mid-turbinate nasal swab 
collection for influenza-like illness (ILI) defined as new onset of cough or worsening of chronic 
cough within the preceding 7 days will be conducted during the period of influenza circulation at 
each study site.  Additional blood will also be collected at 6 months post-vaccination (or at the 
end of influenza virus circulation) from participants with reverse-transcription polymerase chain 
reaction (RT-PCR)-confirmed ILI during the influenza season to evaluate cell-mediated immune 
responses to natural influenza virus infection.   
Sites will aim to enroll 864 participants (432 per site) at the start of the 2018-19 season, 
including up to 200 participants (up to 100 per site) who will contribute additional blood at all 
study visits to evaluate cell-mediated immune responses to vaccination.  Efforts will be made to 
retain participants enrolled in the first year of the study for both years of the study.  Sites will 
also enroll additional participants at the start of the 2019-20 season to make up for participants 
who withdraw or are lost to follow-up prior to the start of the 2019-20 season.  Both participants 
and study investigators will be aware of study arm assignments with the exception of laboratory 
investigators who will be blinded to study arm assignment until testing is completed, as 
appropriate.     
Relative efficacy of single doses of study vaccines will be assessed by comparing 
immunologic responses to vaccination among participants between study arms using Fluzone® 
Quadrivalent and Fluarix® Quadrivalent as the comparator groups for participants in the 
Flucelvax™ Quadrivalent or Flublok® Quadrivalent arms.  In addition, the effect of frequency of 
prior vaccination during the preceding five years on immunologic responses to vaccine will be 
evaluated in subgroup analysis.  Both humoral (influenza antibody) and cell-mediated (influenza- 
specific CD4 and CD8 T cell) immune responses will be evaluated.  
Study Sponsor 
Influenza Division, U.S. Centers for Disease Control and Prevention 

 
 
       
6 
 
Investigators 
Baylor Scott & White Health, Texas A&M Health Science Center, College of Medicine, 
Temple, Texas 
• Manjusha Gaglani, MBBS 
• Arundhati Rao, Ph. D (Molecular Virology Lab Director)  
• Marcelo Pando, Ph. D (PBMC Processing Lab Director)  
• Sheila Dobin, Ph. D (PBMC Storage Lab Director)  
The Center for Health Research, Kaiser Permanente Northwest, Portland, Oregon 
• Allison Naleway, PhD 
• Michelle Henninger, PhD 
• Holly Groom, MS 
 
U.S. Centers for Disease Control and Prevention 
• Fatimah S. Dawood, MD 
• Brendan Flannery, PhD 
• Mark Thompson, PhD 
• Alicia Fry, MD, MPH 
• Lauren Beacham,  
• Sarah Spencer, PhD 
• Vic Veguilla, MPH 
• Min Levine, PhD 
• Prakash Sambhara, DVM, PhD 
• Shiva Gangappa, DVM, PhD 
• Ian York, DVM, MSc, PhD 
 
Advisors 
Center for Clinical Epidemiology & Population Health at the Marshfield Clinic Research 
Foundation in Wisconsin 
• Edward A. Belongia, MD 
 

 
 
       
7 
 
4.   INTRODUCTION 
4.1 Background Information:  
 
Influenza is estimated to result in 9.2-35.6 million illnesses, 140,000-710,000 
hospitalizations and 12,000-56,000 deaths in the United States.1  Influenza vaccination is the 
most effective method of preventing influenza virus infection, and annual influenza vaccination 
is recommended for all persons aged >6 months in the United States.  However, the efficacy of 
licensed influenza vaccines varies annually depending on the match between vaccine strains and 
circulating strains and by influenza virus type and subtype.  In a meta-analysis of published  
influenza vaccine effectiveness studies conducted during 2007-2015, pooled influenza vaccine 
effectiveness was estimated as 61% (95%CI 57-65) against A/H1N1 viruses circulating after the 
2009 A/H1N1 pandemic and 54% (95%CI 46-61) against influenza B viruses, but only 33% 
(95%CI 26-39) against influenza A/H3N2 viruses.2  In addition, observed influenza vaccine 
effectiveness has been lower against A/H3N2 viruses than A/H1N1 viruses for the past several 
influenza seasons in the United States.3, 4  The relatively low effectiveness of influenza vaccines 
against influenza A/H3N2 viruses is particularly concerning given that influenza A/H3N2 
viruses have been associated with higher influenza-associated hospitalization and mortality rates 
among adults.5, 6 
 
Questions about the preventive benefit of influenza vaccines are especially relevant to 
healthcare personnel (HCP). Although HCP are believed to be at increased risk of influenza virus 
infection7, there is limited information on the effectiveness of influenza vaccines among HCP.8 
Reports of reduced influenza vaccine effectiveness among persons with a history of frequent 
influenza vaccination in some studies9, 10 and seasons11 also make it especially important to 
examine influenza vaccine effects among HCP who are among the most vaccinated target 
populations. Recent studies of influenza vaccine immunogenicity among HCP have 
demonstrated that repeated vaccination can blunt the antibody response to hemagglutinin12 and 
neuraminidase.13  
 
The conventional method of inactivated influenza vaccine (IIV) production relies on 
propagation in embryonated chicken eggs of an egg-adapted vaccine seed strain derived from a 
circulating influenza virus.   Both the adaptation process and serial passage of influenza viruses 
in chicken eggs can result in the development of mutations that may introduce important 
antigenic differences between the vaccine strain and circulating wild-type strains.14-17 It has been 

 
 
       
8 
 
hypothesized that this phenomenon may be partially responsible for the lower vaccine 
effectiveness observed against influenza A/H3N2 viruses in some studies despite a reportedly 
good match between vaccine seed strains and circulating strains.16, 18, 19  Historically, the 
immunogenicity of influenza vaccines has been assessed by measuring antibody responses to 
egg-grown influenza viruses which may be a suboptimal measure of efficacy if egg-grown 
viruses differ antigenically from circulating wild-type viruses because of mutations acquired 
during egg passage.   
During the past decade, inactivated influenza vaccines manufactured using alternative 
vaccine virus production technologies have been licensed in the United States, including a cell 
culture-based influenza vaccine (Flucelvax™ by Seqirus, Inc.) licensed for persons aged > 4 
years and a recombinant influenza vaccine (Flublok® by Sanofi Pasteur) licensed for persons age 
> 18 years.  Studies evaluating these vaccines have measured antibody responses to cell culture-
grown viruses in some cases which may be more representative of immune responses to 
circulating viruses if cell culture-grown viruses possess fewer mutations acquired during the 
adaptation and viral passage process. 
Flucelvax™ is a subunit vaccine formulated from influenza virus cultivated in Madin-
Darby Canine Kidney (MDCK) cells.   A version known as Optaflu is also licensed in Europe.  
Although cultivation of vaccine strains is done using MDCK cell lines, seed viruses for the 
vaccine may be either egg-derived or cell culture-derived.  In a phase III, randomized, placebo-
controlled, observer-blinded trial comparing either cell culture-based trivalent IIV (Optaflu) or 
egg-based trivalent IIV (Aggripal) to placebo among 11,404 adults aged 18-49 years, the cell 
culture-based vaccine was 70% (lower bound of the 95% CI=55%) efficacious against all 
culture-confirmed influenza-like illness (ILI), 84% (lower bound of the CI=61%) against ILI 
associated with vaccine-like viruses, and 59% (lower bound of the CI=34%) against ILI 
associated with drifted viruses.20  Efficacy was similar for these outcomes for the cell culture- 
and egg-based vaccines, but relative efficacy was not assessed. In other studies evaluating the 
immunogenicity of Flucelvax™ or Optaflu compared to standard dose egg-based vaccines, 
immune responses to the cell culture-based vaccines were generally similar or non-inferior to 
egg-based vaccines when evaluating responses against homologous egg-grown virus antigens.21, 
22  However, some studies demonstrated a better immune response to cell culture-based vaccine 
compared to egg-based vaccines when immune responses were evaluated against homologous 

 
 
       
9 
 
cell culture-grown virus antigens.21, 23, 24 Early trials of Flucelvax™ used vaccine produced in 
cell culture from egg-derived seed viruses (likely resulting in vaccine viruses that were more 
similar to those used in egg-based vaccines), whereas more recent formulations of Flucelvax™ 
(e.g. the 2017-2018 formulation) included an H3N2 vaccine strain grown from a cell culture-
derived seed virus.  The 2018-2019 formulation of Flucelvax™ will be made from cell-culture 
derived seed strains for both the H3N2 and H1N1 vaccine strains. 
Flublok® is a recombinant vaccine formulated with purified HA protein 
from cell-culture isolates of the vaccine reference virus.  HA is grown using baculovirus vector 
technology in an insect cell line from the fall armyworm.  Since production of Flublok® does not 
use egg-adapted seed strains, the recombinant HA (rHA) antigens contained in the vaccine are 
thought to completely match those in the vaccine reference virus.25   In contrast to most other 
vaccines licensed for use in adults aged 18-64 years in the United States, Flublok® contains 45 
μg of HA per strain.  In a phase III randomized placebo-controlled trial of trivalent Flublok® 
among healthy adults aged 18-49 years, Flublok® was 45% (95%CI 19-63%) efficacious against 
culture-confirmed influenza during the 2007-2008 influenza season when the majority of 
circulating influenza A/H3N2 and B viruses were drifted from the vaccine strains.  Flublok® was 
also found to be highly immunogenic for all three vaccine strains with 99% of participants 
having an HI titer of >1:40 against the H1N1 vaccine strain at 28 days post-vaccination, 97% 
against the H3N2 vaccine strain, and 96% against the B vaccine strain; HI was measured against 
baculovirus derived rHAs.26  In a subsequent phase III randomized trial comparing quadrivalent 
Flublok® to quadrivalent Fluarix, an egg-based vaccine, among adults aged 18-49 years, 
Flublok® and Fluarix recipients had similar post-vaccination HI titers against all four vaccine 
components using egg-grown antigens.27 Randomized trials in adults aged >50 years also 
demonstrated similar immune responses at 28 days post-vaccination as measured by HI against 
baculovirus derived rHAs between trivalent Flublok® and Fluzone®28 and showed that Flublok® 
was more efficacious than Fluarix against RT-PCR-confirmed ILI (relative vaccine efficacy, 
30%, 95% 10-47%) during the 2014-2015 influenza season when drifted A/H3N2 viruses 
predominated.29   
Both Flucelvax™ and FluBlok® were licensed in the United States based on 
demonstrations of clinical efficacy and non-inferiority using immunogenicity outcomes 
according to the criteria of the Centers for Biologic Evaluation and Research.  Studies assessing 

 
 
       
10 
 
the immunogenicity of Flucelvax largely assessed immune response against homologous egg-
based antigens, although some studies also assessed responses against homologous cell-grown 
antigens.  In contrast, studies assessing Flublok assessed immune responses against baculovirus-
derived rHAs.  To date, there are few data directly comparing the immunogenicity of cell 
culture-based and recombinant influenza vaccines to egg-based vaccines using the same 
immunogenicity outcome measures against the same antigenic targets for all three vaccine types, 
including evaluation of response to cell-grown viruses that may be more representative of 
circulating strains.  It also remains unclear whether cell culture-based or recombinant influenza 
vaccines that are free from mutations in the viral HA introduced during the egg adaptation and 
passage process of egg-based vaccine production are more effective against circulating influenza 
viruses.  This randomized, open-label trial will assess humoral and cell-mediated immune 
responses to cell culture-based and recombinant quadrivalent influenza vaccines compared to 
two conventional egg-based quadrivalent standard dose (15µg of HA per strain) influenza 
vaccines among persons aged 18-64 years using both cell-grown and egg-grown vaccine 
reference viruses as antigenic targets.  Two egg-based influenza vaccines will be included as 
comparators in this trial since other vaccine components that vary by vaccine regardless of 
method of vaccine virus growth might influence immune response to vaccination including 
neuraminidase (NA) content, preservatives, and other substances retained as part of the 
inactivation or purification processes.   The primary study hypothesis is that a single dose of cell 
culture-based or recombinant influenza vaccine may be more immunogenic than a single dose of 
split-virus/subvirion egg-based influenza vaccine to cell-grown viruses that are more 
representative of circulating strains, regardless of the vaccine product among persons aged 18-64 
years.   
4.2  
Justification: 
 
Influenza vaccines are the most effective method of influenza prevention and are 
recommended for all persons aged >6 months in the United States.  However, the efficacy of 
licensed influenza vaccines varies annually depending on the match between vaccine strains and 
circulating strains and varies by influenza virus type and subtype.  Data from the last several 
influenza seasons showing lower vaccine effectiveness against influenza A/H3N2 viruses 
combined with studies documenting challenges with growing recently circulating influenza 
A/H3N2 viruses in embryonated chicken eggs for vaccine production have raised questions 

 
 
       
11 
 
about the impact of egg adaptation and propagation of vaccine viruses on the effectiveness of 
egg-based influenza vaccines.  At the same time, influenza vaccines made by alternative methods 
of production that avoid the egg adaptation and/or egg propagation steps are now licensed in the 
United States, including cell culture-based vaccines and recombinant influenza vaccines.  At 
present, the Advisory Committee on Immunization practices does not preferentially recommend 
any specific influenza vaccine for use in adults in the United States.  Studies are needed to 
compare the immunogenicity of conventional egg-based vaccines versus vaccines manufactured 
using alternative methods of production using standard assays and endpoints.  Data from such 
studies will inform whether a preferential recommendation for vaccines based on a specific 
method of production are warranted.   
  
4.3  
Expected benefits from the proposed study 
Influenza results in substantial morbidity and mortality, and annual vaccination remains 
the most effective method to prevent influenza and its complications. Through this study, we will 
gain a better understanding of whether the cell culture-based vaccine Flucelvax™ Quadrivalent 
or the recombinant vaccine Flublok® Quadrivalent offer greater protection to HCP aged 18-64 
years than the conventional egg-based vaccines Fluzone® Quadrivalent and Fluarix® 
Quadrivalent as measured primarily by humoral immune responses to MDCK cell-grown 
vaccine strains. Because we will assess immune responses at 6 months  after influenza 
vaccination (or the end of influenza virus circulation) in all participants, this study will also 
provide information about how long protective immune responses to each influenza vaccine last.  
In the United States, all four study vaccines are licensed for use in adults aged >18 years 
based on phase III trials demonstrating efficacy in comparison to placebo26 or non-inferiority to 
conventional egg-based trivalent influenza vaccines.  Therefore, study participants may directly 
benefit from receipt of study vaccine by receiving protection against circulating influenza viruses 
during the 2018-19 and 2019-20 influenza seasons.  
 
5a. 
STUDY OBJECTIVES 
• Primary Objective  
o Compare immune responses among persons aged 18-64 years vaccinated with 
either a single dose of the cell culture-based vaccine Flucelvax™ Quadrivalent or 

 
 
       
12 
 
the recombinant vaccine Flublok® Quadrivalent versus a single dose of the 
conventional egg-based vaccines Fluzone® Quadrivalent or Fluarix® Quadrivalent 
at approximately 28 days after receipt of the 2018-19 and 2019-20 vaccines as 
measured by 
▪ hemagglutination inhibition (HI) titers for influenza A/H1N1, influenza 
B/Yamagata, and influenza B/Victoria strains, and 
▪ microneutralization (MN) titers for influenza A/H3N2 viruses  
• Secondary Objectives  
o Compare immune responses at 28 days post-vaccination as stated in the primary 
objective but with secondary indicators of immune response, including by:   
▪ HI titers for influenza A/H3N2 viruses, as appropriate  
▪ MN titers for other virus subtypes/lineages, as appropriate  
o Evaluate the effect of frequency of prior influenza vaccination during the 
preceding five years on immune responses after a single dose of Flucelvax™ 
Quadrivalent, Flublok® Quadrivalent, Fluzone® Quadrivalent or Fluarix® 
Quadrivalent at approximately 28 days after receipt of the 2018-19 and 2019-20 
vaccines as measured by  
▪ HI titers for influenza A/H1N1, influenza B/Yamagata, and influenza 
B/Victoria strains and  
▪ MN titers for influenza A/H3N2 viruses 
• Exploratory Objectives  
o Make the same comparisons as stated in the primary objective but at 
approximately 6 months after vaccine receipt to assess duration of immune 
responses as measured by 
▪ HI titers for all virus subtypes/lineages 
▪ MN for virus subtypes/lineages, as appropriate 
o Evaluate the relative efficacy of a single dose of the cell culture-based vaccine 
Flucelvax™ Quadrivalent or the recombinant vaccine Flublok® Quadrivalent 
versus a single dose of the conventional egg-based vaccines Fluzone® 

 
 
       
13 
 
Quadrivalent or Fluarix® Quadrivalent to prevent reverse-transcription 
polymerase chain reaction (RT-PCR)-confirmed influenza-like illness (ILI). 
o Characterize humoral immune responses as measured by HI, MN titers, 
neuraminidase antibody mediated inhibition (NAI) titers and other appropriate 
humoral immunity assays, and characterize cell-mediated immune responses of B, 
CD4 and CD8 T-cells among persons who experience ‘vaccine failure’ defined as 
the development of RT-PCR-confirmed ILI more than 14 days after receipt of 
vaccine. 
o Characterize cell-mediated immune responses of B, CD4 and CD8 T-cells to the 
cell culture-based vaccine Flucelvax™ Quadrivalent, the recombinant vaccine 
Flublok® Quadrivalent, and the conventional egg-based vaccines Fluzone® 
Quadrivalent and Fluarix® Quadrivalent at approximately 7 days, 28 days, and 6 
months after vaccination. 
o Characterize the avidity of antibodies produced in response to the cell culture-
based vaccine Flucelvax™ Quadrivalent, the recombinant vaccine Flublok® 
Quadrivalent, and the conventional egg-based vaccines Fluzone® Quadrivalent 
and Fluarix® Quadrivalent at approximately 7 and 28 days after vaccination. 
o Compare immune responses among persons aged 18-64 years vaccinated with 
either a single dose of the cell culture-based vaccine Flucelvax™ Quadrivalent or 
the recombinant vaccine Flublok® Quadrivalent versus a single dose of the 
conventional egg-based vaccines Fluzone® Quadrivalent or Fluarix® 
Quadrivalent at approximately 28 days after receipt of the 2018-19 and 2019-20 
vaccines as measured by  
▪ NAI and antibody-dependent cell-mediated cytotoxicity (ADCC) titers  
▪ Other relevant immunological markers 
5b. STUDY ENDPOINTS (Tables 1 and 2) 
Primary Endpoints 
• MN responses to the cell-grown influenza A/H3N2 (using MDCK-SIAT or other 
appropriate cell line) vaccine reference viruses for each study season at approximately 28 
days post-vaccination, including the following endpoints: 

 
 
       
14 
 
o Seroconversion rate (SCR) defined as the proportion of participants with paired 
samples that achieved ≥ 4 fold rises comparing post- versus pre-vaccination titers, 
and post vaccination titers ≥ 40.  
o Geometric mean titers (GMT) 
o MFR mean-fold rise (MFR) defined as the ratio of the post-vaccination titer value 
to the pre-vaccination value  
 
• HI responses to the cell-grown influenza A/H1N1, influenza B/Yamagata, and influenza 
B/Victoria vaccine reference viruses for each study season at approximately 28 days post-
vaccination, including the following endpoints: 
o SCR 
o GMT 
o MFR 
Secondary Endpoints 
• HI responses to cell-grown influenza A/H3N2 vaccine reference viruses for each study 
season at approximately 28 days, including the following endpoints 
o Post-vaccination titers > seropositive thresholds at 1:40, 1:80, 1:160, SCR, GMT, 
and MFR 
• HI (and MN, as appropriate) responses to cell-grown influenza A/H1N1, influenza 
B/Yamagata, and influenza B/Victoria vaccine reference viruses for each study season at 
approximately 28 days, including the following endpoints 
o Post-vaccination titers > seropositive thresholds at 1:40, 1:80, 1:160, as measured 
by HI response 
o SCR, GMT, and MFR as measured by MN response, as appropriate  
• HI and/or MN responses to egg-grown vaccine reference viruses for all vaccine viruses 
for each study season at approximately 28 days, including the following endpoints 
o SCR, GMT, MFR, and post-vaccination titers > seropositive thresholds at 1:40, 
1:80, 1:160 as measured by HI 
o SCR, GMT, and MFR as measured by MN, as appropriate 
• GMT as measured by HI for all vaccine virus subtypes/lineages at 6 months post-
vaccination using 

 
 
       
15 
 
o cell-grown vaccine reference viruses  
o egg-grown vaccine reference viruses     
Exploratory Endpoints 
• HI and/or MN responses to cell-grown wild-type influenza viruses at approximately 28 
days (if appropriate), including the following endpoints   
o SCR, GMT, MFR, and post-vaccination titers > seropositive thresholds at 1:40, 
1:80, 1:160 as measured by HI 
o SCR, GMT, and MFR as measured by MN, as appropriate 
• GMT as measured by NAI and ADCC pre- and post-vaccination. 
• Mean percentages of strain-specific T cell surface markers of activation, antibody 
secreting plasmablasts and memory B cells to hemagglutinin (HA), interferon-gamma 
(IFN- gamma), interleukin 2 (IL-2),  and Tumor Necrosis Factor-alpha (TNF-alpha) 
responses to wild-type cell-grown strains and antigen-specific B and T cell repertoire, and 
single cell transcriptome analysis (where feasible) at approximately 7 days, 28 days, and 
12 months post-vaccination. 
• Frequency of RT-PCR-confirmed ILI episodes  
• Indicators of immune response to vaccination based on other immunologic assays not 
listed above (as appropriate) 

 
 
       
16 
 
Table 1 Primary and secondary endpoints at approximately 28 days post-vaccination 
 
Cell-grown vaccine virus 
Egg-grown vaccine virus 
 
A/H3N2 
A/H1N1 
B/Yam 
B/Vic 
A/H3N2 
A/H1N1 
B/Yam 
B/Vic 
HI 
 
 
 
 
 
 
 
 
  SCR 
 
 
 
 
 
 
 
 
  GMT 
 
 
 
 
 
 
 
 
  MFR 
 
 
 
 
 
 
 
 
  Post-   
  vaccination  
  titers >  
  
seropositive    
  thresholds  
  at 1:40,  
 
 
 
 
 
 
 
 
MN 
 
 
 
 
 
 
 
 
  SCR 
 
 
 
 
 
 
 
 
  GMT 
 
 
 
 
 
 
 
 
  MFR 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Primary endpoint 
 
Secondary 
endpoint 
 
Secondary endpoint (as appropriate) 
HI:  hemagglutination inhibition; MN: microneutralization 
SCR: Seroconversion rate; GMT: Geometric mean titer; MFR: Mean fold rise 
 
 
 
 
 
 

 
 
       
17 
 
Table 2 Secondary immunogenicity endpoints at approximately 6 months post-vaccination* 
 
Cell-grown vaccine virus 
Egg-grown vaccine virus 
 
A/H3N2 A/H1N1 B/Yam B/Vic A/H3N2 A/H1N1 B/Yam B/Vic 
HI 
 
 
 
 
 
 
 
 
  SCR 
 
 
 
 
 
 
 
 
  GMT 
 
 
 
 
 
 
 
 
  MFR 
 
 
 
 
 
 
 
 
  Post-   
  vaccination  
  titers > 
  seropositive  
  thresholds  
  at 1:40,    
  1:80, 1:160 
 
 
 
 
 
 
 
 
MN 
 
 
 
 
 
 
 
 
  SCR 
 
 
 
 
 
 
 
 
  GMT 
 
 
 
 
 
 
 
 
  MFR 
 
 
 
 
 
 
 
 
 
 
Secondary endpoint 
 
Secondary endpoint (as appropriate) 
HI:  hemagglutination inhibition; MN: microneutralization;  NAI: neuraminidase antibody mediated inhibition;  ADCC:  Antibody-
Dependent Cell-Mediated Cytotoxicity 
SCR: Seroconversion rate; GMT: Geometric mean titer; MFR: Mean fold rise 
. 
 
 
 

 
 
       
18 
 
 
6. 
LOCATIONS OF STUDY 
• Baylor Scott & White Health, Texas A&M Health Science Center, College of Medicine, 
Temple, Texas 
• The Center for Health Research, Kaiser Permanente Northwest, Portland, Oregon 
 
7.0  
METHODS 
 
7.1  
Study population: HCP who have direct contact with patients, including dentists and 
other dental health personnel, and receive medical care within two healthcare systems in 
Temple, TX and Portland, OR.  The study will prioritize enrollment of HCP who 
participated in a previous study of influenza vaccine effectiveness that was conducted at 
both study sites during the 2010-2011 influenza season. 
Study duration: Two years spanning two influenza seasons in the United States from 
August 2018 through approximately April 2020.  Participants will be enrolled beginning 
in August 2018 (or when vaccine is available) and followed for up to approximately 18 
months or through the end of influenza virus circulation during the 2019-2020 influenza 
season at each site.  Additional enrollment may be conducted beginning in August 2019 
(or when vaccine is available) to make up for participant attrition during the first year of 
the study with study participants enrolled in 2019 followed for approximately 6 months 
or through the end of influenza virus circulation during the 2019-2020 influenza season at 
each site.   
 
7.2  
  Inclusion Criteria  
• Healthcare personnel (HCP) who have direct contact with patients, including dentists and 
other dental health personnel  
• Enrolled in Scott & White Healthcare or Kaiser Permanente health network for at least 
one month 
• Aged 18-64 years 

 
 
       
19 
 
• Available and willing to participate in study follow-up through the end of the 2019-2020 
influenza season (i.e. at least approximately 18 months if enrolled during season 1 or 6 
months if enrolled during season 2)  
7.3  
Exclusion Criteria 
• Already received an influenza vaccine during the current influenza season 
• Previous hypersensitivity reaction to the study vaccines as reported by the subject 
• Received any vaccine in the 4 weeks prior to the first study visit or plans to receive a 
vaccine (other than influenza vaccine provided through the study protocol) in the 4 weeks 
following the first study visit 
• Currently participating 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 an experimental 
agent during participation in this study 
• Any condition that the principle investigator (PI) believes may interfere with successful 
completion of the study 
7.4 a Withdrawal Criteria:  
• Medical condition for which continued participation, in the opinion of the investigator, 
would pose a risk to the participant  
• As deemed necessary by the principal investigator for noncompliance or other reasons 
• Withdrawal of consent 
• Lost to follow up defined as not returning for scheduled study visits within the defined 
time periods for each visit (as described in section 7.5) for visit 2 in years 1 or 2 for all 
participants and visit 1 in year 2 for participants enrolled in year 1 of the study. 
• Receipt of influenza vaccine outside of the study and after enrollment   
7.4b 
Discontinuation Criteria: 
• Termination of the study 
 
7.5 Implementation/monitoring procedures including data collection and analysis: 

 
 
       
20 
 
Study Design: Randomized, open-label study (Overview of stratified randomization and study 
activities at each site, Figures 1 and 2) 
Consent/Screening/ Enrollment 
The study will be open to HCP meeting the eligibility criteria at the two study sites. HCP 
will be recruited using site-specific methods.  Consent, screening, and enrollment, and all study 
visits will take place at designated study site locations.  All HCP who are potentially eligible for 
the study will receive a description of the study (purposes and procedures) and will be asked to 
read, sign, and date the consent form.  The consent form will explain study details, risks, and 
benefits.  Study staff will administer the consent form to HCP.  The consent process will take 
approximately 20 minutes. In addition to receiving a copy of the full consent form, all 
participants will receive a brief summary of the consent form and an outlined schedule of study 
visits. All potential study participants will be free to decide whether or not they want to 
participate in the study.   
From the start of enrollment, sites will make efforts to recruit participants to fill each of 
the four age and prior vaccination strata shown in Figure 1.  If enrollment into the strata for 
persons with no influenza vaccination during the preceding season are not met after a certain 
period of time, sites may enroll participants with receipt of influenza vaccination during the 
preceding season into all unfilled strata if mutually agreed upon by CDC and site principle 
investigators.  During the consent process, all participants will be asked to give consent for 
collection of an additional 30mL of whole blood for PBMC collection at study visit 3 outlined 
below if they develop RT-PCR-confirmed ILI during the influenza season.   
All potential participants will take a self-administered survey or study staff may 
administer a survey to determine whether or not inclusion/exclusion criteria are met (Appendix 
B, Eligibility Screening Form). Participants will be enrolled in the study if they meet inclusion 
criteria and provide consent to be in the study (Appendix C, Consent Form).   
Stratified enrollment will be used to ensure an even mix of age groups (18-44 years and 
45-64 years) and prior vaccination status (received or did not receive influenza vaccine during 
the previous influenza season) (Figure 1).  Eight hundred and sixty-four participants will be 
enrolled in year 1 (432 at each site).  Additional participants will be enrolled at the start of year 2 
to make up for any participants who were lost to follow-up or withdrew from the study in year 1 
so that there are 864 participants (432 at each site) enrolled in the study at the start of year 2.  In 

 
 
       
21 
 
order to enroll this number of participants, site investigators will plan to screen an additional 
30% of potentially eligible HCP (i.e. screen approximately 560 participants at each site) during 
year 1. 
Study Visit 1 of Year 1 [day 0 defined as day of vaccination]:  
Study visit 1 may take place on the same day as study screening and consent or at a later 
date if screening and consent is conducted prior to study vaccine availability at the site.  
Participants will complete a brief questionnaire about demographic characteristics, health status 
(body mass index, self-perceived health status), history of chronic medical conditions during the 
preceding year, concomitant immunosuppressive medication use, household size, history of 
influenza vaccination during the preceding five years, and hours and types of patient contact 
encountered as part of work duties (Appendix D, Enrollment Questionnaire). 
All study participants will complete a brief questionnaire to determine whether they are 
currently experiencing fever or other acute illness symptoms (Appendix E, Pre-vaccination 
Questionnaire/Vaccine Administration Form). Participants who present with moderate-severe 
illness with fever and/or acute illness during the study visit will not receive study vaccine until 
their illness has resolved. In this case, a follow up appointment will be made after resolution of 
the illness, at which time the participant will receive the vaccine.  
Randomization: Stratified block randomization will be used at each site to ensure even allocation 
of age groups (18-44 years and 45-64 years) and prior vaccination status (received or did not 
receive influenza vaccine during the previous influenza season) to each study arm.  Within each 
of the four strata, participants will be randomized 2:2:1:1 to receive one 0.5 mL intramuscular 
dose of a study vaccine:  Flucelvax™ Quadrivalent, Flublok® Quadrivalent, Fluzone® 
Quadrivalent or Fluarix ® Quadrivalent.   Randomization lists will be generated using a 
computerized random-number generator to select randomly permuted group blocks of twelve 
with four in each block assigned to Flucelvax™ Quadrivalent and Flublok® Quadrivalent and 
two in each block assigned to Fluzone® Quadrivalent or Fluarix ® Quadrivalent. The next 
available sequential study-number will be assigned to each enrolled participant upon study-entry. 
‘Opt-In Consent for Longitudinal PBMC Collection: 
After participants have been randomized, they may be invited to participate in longitudinal 
PBMC collection that requires collection of an additional 30mL of blood at study visits 1, 2, and 
3 plus an additional study visit 1b as outlined below until up to 25 participants per study arm at 

 
 
       
22 
 
each site have been fully enrolled in this activity.  Participants are considered fully enrolled and 
contributing to the sample size goals for longitudinal PBMC collection once they have 
completed PBMC collection at study visits 1 and 1b.   During year 2 of the study, recruitment 
methods for PBMC collection may be modified to target selected participants based on their 
immune responses to vaccination or natural infection during year 1 of the study.  A new consent 
will be required for additional longitudinal PBMC collection activities in year 2.   
Blood Draw: All study participants will have 20 ml of venous blood drawn at the first study visit 
for serology testing. In addition, participants who consent to longitudinal PBMC collection at all 
study visits will have an additional 30 ml of venous blood drawn for collection of PBMCs to 
assess cell-mediated immune responses to vaccination. 
Vaccine administration: Prior to receiving the influenza vaccine, all study participants will 
receive an influenza vaccine fact sheet which outlines potential risks, side effects, and benefits of 
the vaccine. Participants will receive a single (0.5ml) dose of study vaccine administered in the 
deltoid muscle of the arm opposite the blood draw.  Vaccine administration will be performed by 
a qualified nurse trained in the delivery of the study vaccines and documented on Appendix E, 
Pre-vaccination Questionnaire/Vaccine Administration Form).  
Blinding: Both participants and study investigators will be aware of study arm assignments with 
the exception of laboratory investigators who will be blinded to assignment until testing is 
completed, as appropriate.     
Post vaccination safety monitoring:  Since all study vaccines are licensed for use in participants 
meeting the eligibility criteria and safety and tolerability of each vaccine have been established 
in prior studies, data on reactogenicity events and adverse events will not be solicited.  However, 
study staff will provide participants with a phone number to call in case they have any questions 
after vaccination.  
Study Visit 1b of Year 1 [days 6-9]  
Blood Draw: Participants who had blood collected at day 0 for collection of PBMCs will be 
instructed to return on day 7 (acceptable range days 6-9) to have 30 ml of venous blood drawn 
for collection of PBMCs to assess cell-mediated immune responses to vaccination. 
Study Visit 2 of Year 1 [optimal timing days 21-35 (acceptable timing days 21-62)] 

 
 
       
23 
 
Blood Draw: All study participants will have 20 ml of venous blood drawn for serology testing. 
Participants who had blood collected at day 0 for collection of PBMCs will also have an 
additional 30 ml of venous blood drawn for collection of PBMCs. 
Study Visit 3 of Year 1 [optimal timing days 180-190 (acceptable timing days 180-210) or 
after influenza circulation has ended] 
Blood Draw: All study participants will have 20 ml of venous blood drawn for serology testing. 
Participants who had blood collected at day 0 for collection of PBMCs and participants who had 
an episode of RT-PCR-confirmed ILI will also have an additional 30 ml of venous blood drawn 
for collection of PBMCs at study visit 3.  
Study Visit 1 of Year 2 [day 0] 
All participants will complete a follow-up questionnaire during study visit 1 of Year 2 (Appendix 
D, Enrollment Questionnaire).  All study participants will also complete a brief questionnaire to 
determine whether they are currently experiencing fever or other acute illness symptoms 
(Appendix E, Pre-vaccination Questionnaire/Vaccine Administration Form). Participants who 
present with moderate-severe illness with fever and/or acute illness during the study visit will not 
receive study vaccine until their illness has resolved. In this case, a follow up appointment will 
be made after resolution of the illness, at which time the participant will receive the vaccine. 
Randomization of Participants from Year 1 of the Study:  Participants from year 1 of the study 
will remain in the same study arm to which they were assigned by randomization in year 1 (i.e. 
during study visit 1 of year 1).   Study nurses administering vaccine will consult participants’ 
study files to confirm which study vaccine participants from year 1 received during the first year 
of the study. 
Randomization of Newly Enrolled Participants in Year 2: Study staff will review participant 
allocation across age and prior vaccination strata within each study arm to determine the number 
of new participants that need to be recruited to maintain even allocation across each group.  
Stratified block randomization with randomly permuted group blocks of appropriate size will 
then be used at each site to randomize participants as needed into each of the four study arms to 
make up for attrition from year 1 enrollment. 
Blood Draw: All study participants will have 20 ml of venous blood drawn at the first study visit 
for serology testing. In addition, the participants selected and consented for longitudinal 

 
 
       
24 
 
collection of PBMCs will have an additional 30 ml of venous blood drawn for collection of 
PBMCs to assess cell-mediated immune responses to vaccination.   
Vaccine administration: Study participants will receive the study vaccine to which they are 
assigned. The same procedures for vaccine administration outlined above for study visit 1 in year 
1 will be followed for vaccine administration during this study visit.   
Study Visits 1b, 2, and 3 of Year 2 
Study visits 1b, 2, and 3 during year 2 will be conducted at the same time points (i.e. days post-
vaccination or after influenza virus circulation has ended) as in year 1 and will include the same 
study activities as in year 1.   
Monitoring of Adverse Events  
Because all four study vaccines are licensed for use in the study population and previous 
studies demonstrated a good safety profile30-33 in adults for each vaccine, active monitoring for 
adverse events will not be conducted as part of this trial.  Study staff will refer participants who 
passively report a potential adverse event to their primary care provider for further evaluation as 
needed. 
Active Surveillance for Influenza-Like Illness 
For the purposes of active surveillance, ILI will be defined as new onset of cough or 
worsening of chronic cough during the preceding seven days.  Active and passive surveillance 
for ILI will be conducted during the influenza seasons at each site defined as the time period 
when influenza viruses are detected through local (e.g. hospital) and/or state surveillance.  At 
enrollment, participants will be advised that they will receive notification from the study team 
once the influenza season begins at their site and will be instructed to report any illness meeting 
the ILI case definition to study staff as soon as they develop symptoms.  Sites may opt to give 
participants small items such as refrigerator magnets with the study phone number to remind 
participants of how to report ILI episodes.  Participants will also receive an orientation about 
self-collection of mid-turbinate nasal swabs and a self-collection swab kit.   Throughout the 
influenza season, study staff will also contact participants via telephone call, email, or text 
messaging on a weekly basis to ask whether participants have experienced onset of ILI during 
the preceding seven days. Participants who have illness meeting the ILI case definition will be 
asked to self-collect a mid-turbinate nasal swab on the same day and deliver the swab to the 
study site within 3 days of reporting the ILI episode where the day of illness report is defined as 

 
 
       
25 
 
day 0 according to standard operating procedures.  If participants prefer to have study staff 
collect the mid-turbinate nasal swab, they will be instructed to come to the study site or receive a 
home visit from study staff for swab collection within 3 days of reporting the ILI episode where 
the day of illness report is defined as day 0.  Participants will return to active surveillance 14 
days after illness onset.  At the end of the active surveillance period during each study year, 
study staff will complete an Active Surveillance Tracking Form (Appendix J) for each 
participant to capture aggregate indicators of participant response to surveillance contacts, 
including the number of weeks that the participant was under active surveillance, the number of 
surveillance contacts that the participant received, the number of contacts to which the 
participant responded, and the number of ILI episodes reported by the participant. 
In addition, electronic surveillance for participant visits for medically-attended acute 
respiratory illness (MAARI) will be conducted daily Monday through Friday during the 
influenza season.  When study staff identify MAARI episodes, they will contact participants via 
telephone call ideally within 1 day, especially for MAARI episodes identified on Monday 
through Thursday, but no later than within 3 days to confirm that the participant’s illness met the 
case definition for ILI.  Participants with ILI will be asked to self-collect a mid-turbinate nasal 
swab (or have a staff-collected swab if the participant prefers) using the same procedures as 
outlined above for active ILI surveillance. 
Electronic Medical Record Extraction/Abstraction 
When feasible, sites investigators will extract (or abstract if necessary) data from 
participants’ electronic medical records and state immunization registries on influenza 
vaccination history during at least the preceding five years to include, date of receipt, vaccine 
product name, vaccine lot number, and route of administration (Appendix G, EMR Chart 
Extraction/Abstraction Form).  Participants may be asked to sign release of medical information 
forms to allow access to vaccination records as needed to meet site-specific data access 
requirements. 
Protocol Deviations 
Study staff will report protocol deviations using a Protocol Deviation Log for each 
participant (Appendix H).  Sites may also complete additional protocol deviation forms as 
required by their local IRBs.  Sites will report deviations to their local IRBs according to site-
specific reporting requirements.   

 
 
       
26 
 
Data Linkage with Previous Study of Influenza Vaccine Effectiveness among HCP during 
the 2010-2011 Influenza Season 
 
For participants who previously participated in a CDC-sponsored study of influenza 
vaccine effectiveness that was conducted at both study sites during the 2010-2011 influenza 
season, study investigators may link data from the previous study with data collected during the 
current study.   
Protocol Completion or Termination 
 
Study staff will complete a protocol completion or termination form for each participant 
at the time the participant either completes all protocol procedures or at the time of termination if 
early termination occurs (Appendix I, Study Status Change Form). 
Site Monitoring Plan: 
CDC will conduct site monitoring through teleconference calls and site visits (as needed) 
to ensure that human subject protection, study procedures, laboratory procedures, vaccine 
administration, and data collection procedures are high quality and meet guidelines, and that the 
study is conducted in accordance with the protocol and sponsor standard operating procedures.  
CDC will summarize requests to address any areas for improvement or modification that are 
identified during teleconference calls or site visits through written communication to site PIs.  

 
 
       
27 
 
Figure 1 Stratified randomization and sample size goals per site, N=432 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Age 18-44 years AND 
Received influenza vaccine 
in preceding season 
n=144 
Age 18-44 years AND 
Did not receive influenza 
vaccine in preceding season 
n=72 
Age 45-64 years AND 
Received influenza vaccine in 
preceding season 
n=144 
Age 45-64 years AND 
Did not receive influenza 
vaccine in preceding season 
n=72 
Flucelvax, n=48 
Flublok, n=48 
Fluzone, n=24 
Fluarix, n=24 
Randomized 
2:2:1:1 
within each 
stratum to 
Flucelvax 
Flublok 
Fluzone 
Fluarix 
Flucelvax, n=24 
Flublok, n=24 
Fluzone, n=12 
Fluarix, n=12 
Flucelvax, n=48 
Flublok, n=48 
Fluzone, n=24 
Fluarix, n=24 
Flucelvax, n=24 
Flublok, n=24 
Fluzone, n=12 
Fluarix, n=12 

 
 
       
28 
 
Figure 2 Study activity flow diagram* 
*Numbers are per site 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening/Consent/Enrollment 
 
N=432 enrolled per site 
Flucelvax™ 
Quadrivalent 
n=144 per site 
 
Fluzone® 
Quadrivalent 
n=72 per site 
Flublok® 
Quadrivalent 
n=144 per site 
Study Visit 1 (day 0) 
Enrollment interview 
 
Baseline blood collection 
20mL whole blood (all participants) 
30mL whole blood for PBMC collection 
(participants consented for longitudinal PBMC collection) 
 
Vaccine administration 
Study Visit 2, Year 1 (days 21-62)  
Blood collection 
20mL whole blood (all participants) 
30mL whole blood for PBMC collection  
(participants consented for longitudinal PBMC collection) 
 
Study Visit 3, Year 1 (days 180-210 or end of influenza virus circulation)  
Blood collection 
20mL whole blood (all participants) 
30mL whole blood for PBMC collection  
(participants consented for longitudinal PBMC collection plus participants with  
RT-PCR-confirmed ILI during influenza season) 
 
Study Visit 1, Year 2 (day 0 of year 2)  
Enrollment interview (if newly enrolled in year 2) 
Baseline blood collection 
20mL whole blood (all participants) 
30mL whole blood for PBMC collection  
(participants consented for longitudinal PBMC collection ) 
 
Vaccine administration 
Active 
Surveillance 
for ILI  and 
MAARI 
during the 
influenza 
season 
Weekly 
surveillance 
contacts for 
ILI 
 
Daily 
electronic 
surveillance 
for MAARI 
 
Mid-turbinate 
nasal swab 
collection 
with illness 
meeting ILI 
case 
definition 
Randomized 2:2:1:1 
Additional screening/consent/enrollment as needed to account for 
cohort attrition during year 1  
Study Visit 1b, 
Year 1 (days 6-8) 
(PBMC 
participants 
only) 
 
30ml whole blood 
for PBMC 
collection 
Fluarix® 
Quadrivalent 
n=72 per site 

 
 
       
29 
 
Statistical Analysis:  
Humoral antibody responses as measured by HI (and MN, NAI and ADCC, as 
appropriate) at baseline (day 0), approximately 28 days and 6 months post-vaccination will be 
compared between participants in each study arm using Fluzone® Quadrivalent and Fluarix® 
Quadrivalent as the comparator groups. Participants in the Fluzone® Quadrivalent and Fluarix® 
Quadrivalent groups initially will be evaluated separately for the primary endpoints of SCR and 
GMT at 28 days post-vaccination.  If the absolute difference in SCR is <15% and there is a <2 
fold difference in post-vaccination GMT between participants in the Fluzone® Quadrivalent and 
Fluarix® Quadrivalent arms, then participants in these two study arms will be combined for the 
final analysis of the primary endpoints at 28 days post-vaccination and analyses of secondary 
endpoints.  A difference in post-vaccination GMT of >2 fold between study arms will be 
considered particularly noteworth for evaluation of the primary endpoint of GMT at 28 days 
post-vaccination.  Ninety-five percent confidence intervals will be calculated for relevant 
antibody response measures as outlined in the study endpoints at each time point (day 0 and 
approximately 28 days and 6 months post-vaccination).  Post-vaccination titers > seropositive 
thresholds at 1:40, 1:80, 1:160, SCR, GMTs, and MFRs at each time point post-vaccination will 
be compared between study arms using ANOVA.  In addition, a sensitivity analysis will be run 
using ANCOVA with baseline titers as a covariate.   
A sub-group analysis to assess prior vaccination status (number of influenza vaccinations 
received during the preceding five years) as an effect modifier of differences in GMTs between 
study arms at 28 days will also be conducted using ANCOVA. 
Cell-mediated immune responses at baseline and approximately 7 days, 28 days, and 6 
months (or at the end of influenza virus circulation) will be compared between participants in 
each study arm. Non-parametric tests will be used to carry out comparisons of cell-mediated 
immunity studies given the small sample size.  
All primary analyses will be conducted as intention-to-treat. All tests will be 2-tailed with 
a level of significance of .05.   
Power/Sample size calculations:  
 
Study sample size was determined based on available resources.  Assuming a Type 1 
error of 5%, a Type 2 error of 20%, an overall sample size of 864 with 288 participants in the 
Flucelvax® Quadrivalent and Flublok® Quadrivalent arms and 144 participants in each of the 
Fluarix® Quadrivalent and Fluzone® Quadrivalent arms would provide adequate statistical power 
to detect a 30% relative difference in SCR between study arms if the SCR among Fluarix® 

 
 
       
30 
 
Quadrivalent and Fluzone® Quadrivalent is >50%. Using the same assumptions and sample size 
would also provide adequate statistical power to detect a difference in post-vaccination GMT of 
>2 fold between study arms if post-vaccination GMT is >20.  Table 3 provides estimated sample 
sizes required for varying SCR among recipients of Fluzone Quadrivalent or Fluarix 
Quadrivalent and differences in SCR between Flucelvax™ Quadrivalent and Flublok® 
Quadrivalent recipients compared to Fluarix Quadrivalent or Fluzone Quadrivalent recipients.   
 
Based on prior experience enrolling participants at the two study sites, we will assume 
that 10% of participants from year 1 of the study will drop out by study visit 1 of year 2 of the 
study. To account for participant loss due to drop out, we will plan to enroll up to approximately 
90 additional participants in year 2.   
Table 3. Sample size calculations to detect specified differences in SCR between either 
Flucelvax™ Quadrivalent or Flublok® Quadrivalent compared to Fluzone Quadrivalent or 
Fluarix Quadrivalent at approximately 28 days post-influenza vaccination 
SCR at 28 days post-vaccination to 
either Fluzone or Fluarix 
Relative difference in SCR at 28 
days post-vaccination comparing 
either Flucelvax or Flublok to 
Fluzone or Fluarix 
n per 
Fluzone or 
Fluarix arm 
n per Flucelvax 
or Flublok arm 
40% 
10% 
 2050 
 2871 
40% 
20% 
 530 
 742 
40% 
30% 
 232 
 325 
40% 
40% 
 132 
 185 
50% 
10% 
 1350 
 1890 
50% 
20% 
 335 
 470 
50% 
30% 
 145 
203  
50% 
40% 
 80 
 112 
60% 
10% 
 866 
 1215 
60% 
20% 
 208 
 291 
60% 
30% 
 88 
 123 
60% 
40% 
 44 
 64 
 SCR: Seroconversion rate.  Assumes Fluzone and Fluarix vaccine groups are not collapsed. 
 
 
Laboratory Protocol: 
Specimen Collection/Processing/Storage:    
• Serologic studies:  20 mL of blood will be collected from all participants via venipuncture in 
10 ml serum separator tubes with clot activator and gel according to CDC guidelines 
(Appendix K, Human Sera Collection Guidelines for Influenza Serology). Each tube will be 
labeled or barcode linked with the participant ID number, study visit number, and date of 
collection.  See Table 4 for specimen collection schedule. Collected blood will be stored at 

 
 
       
31 
 
4°C immediately either by placing the sample on ice, in a 4°C refrigerator, or in a cooler with 
cold packs. Blood may be stored at 4°C for up to 18 hours. Sometime between 1 hour and 18 
hours after collection, the blood collection tube will be centrifuged to separate the clotted 
blood from the serum and the serum removed to a clean tube labeled or barcode linked with 
the study participant ID number, study visit number, and date of collection. The clotted blood 
will be discarded. The serum will then be divided into approximately 8-10 aliquots of 100 
microliters/aliquot into labeled tubes. After aliquoting, the serum samples will be 
immediately stored at -20°C or colder.  
• PBMC studies: 30 ml of whole blood will be collected at each study visit from participants 
who consent to longitudinal PBMC collection and at study visit 3 from participants who have 
RT-PCR-confirmed ILI  (see table 2 for specimen collection schedule) and placed in 
vacutainer tubes containing ethylenediaminetraacetic acid (EDTA) or heparin. PBMCs will 
be isolated from whole blood and divided into approximately six aliquots of 5x10 million 
cells/aliquot according to CDC Guidelines for Freezing Human PBMCs (Appendix L).  
Specimens will be processed within 24 hours of collection. PBMCs will be shipped to the 
Influenza Laboratory at CDC in Atlanta, Georgia, United States.  
• Mid-turbinate nasal swabs will be collected either by participant self-collection or staff 
collection from participants meeting the study case definition for ILI using flocked dacron 
swabs.  Swabs will be placed immediately into viral transport media.  Participants who self-
collect swabs will be asked to store the swab in viral transport media in their refrigerators and 
deliver the swab to the study site within 72 hours of specimen collection.  Once participant 
self-collected swabs are received at the study site, they will be processed, aliquoted and 
tested or frozen at -70 Celsius within 72 hours according to study standard operating 
procedures.  Swabs that are collected by study staff will be stored immediately at 4°C for no 
more than 72 hours prior to processing, aliquoting and testing or freezing at -70 Celsius 
according to study standard operating procedures.   
• Participant specimens (blood and respiratory samples) will be stored indefinitely for future 
use at a CDC-designated facility for future testing to evaluate aspects of respiratory infection 
and/or immune responses to influenza vaccine or influenza virus infection, as needed.  
• Participants will also be asked if they would like to be contacted about future research studies 
related to this study. 
 

 
 
       
32 
 
 
Table 4. Specimen Collection Timing and Specifications 
Study Visit 
Collection 
Type 
Collection 
Amount 
Testing Purpose 
Testing Location 
Day 0  
Serum 
20 ml 
HI and MN studies 
 
 
NAI and ADCC studies (as appropriate) 
CDC Atlanta or a 
CDC-designated lab 
 
CDC Atlanta 
 
*PBMCs 
 
30 ml total 
Cellular immune responses (B, CD4 and CD8) to 
influenza vaccine and antibody avidity 
CDC Atlanta 
6-9 days 
*PBMCs 
30 ml total 
Cellular immune responses (B, CD4 and CD8) to 
influenza vaccine and antibody avidity 
CDC Atlanta 
21-35 days 
(acceptable 
range 21-
62 days) 
Serum 
20 ml  
HI and MN studies 
 
 
NAI and ADCC studies (as appropriate) 
CDC Atlanta or a 
CDC-designated lab 
 
CDC Atlanta 
 
*PBMCs 
 
30 ml total 
Cellular immune responses (B, CD4 and CD8) to 
influenza vaccine and antibody avidity 
CDC Atlanta 
180-190 
(acceptable 
range 180-
210 days) 
Serum 
20 ml 
 
HI 
 
 
MN, NAI, and ADCC studies (as appropriate) 
CDC Atlanta or a 
CDC-designated lab 
 
CDC Atlanta 
 
*†PBMCs 
 
30 ml total 
Cellular immune responses (B, CD4 and CD8) to 
influenza vaccine 
CDC Atlanta 
Illness visit 
during 
influenza 
season** 
Mid-turbinate 
nasal swab  
N/A 
Reverse-transcription polymerase chain reaction 
for influenza A/H1N1, A/H3N2, B/Yamagata, 
B/Victoria 
Baylor Scott and 
White Health 
and 
Oregon State Public 
Health Laboratory 
*From participants who consent to longitudinal PBMC collection, estimated up to 100 per site. 
†Participants with reverse-transcription polymerase chain reaction-confirmed influenza-like 
illness during the study will have 30mL of whole blood drawn at study visit 3 for PBMC 
collection. 
**For influenza-like illness defined as illness with new onset of cough or worsening of chronic 
cough during the preceding 7 days. 
Specimen Testing: 
Sera will be tested by HI and MN to measure antibody titers against cell-grown and egg-
grown vaccine reference viruses at approximately 28 days post-vaccination compared to baseline 
samples drawn at day 0 prior to vaccination during each study season. Sera collected at 
approximately 6 months post-vaccination or at the end of influenza virus circulation at each site 
will be tested by HI (and MN, if appropriate) to evaluate duration of immune response.  Sera 
may also be tested by HI and MN against cell-grown wild-type influenza strains, as appropriate.  
MDCK-SIAT cells or another appropriate cell line will be used to cultivate cell-grown virus 
strains.  A subset of sera from each study arm may also be tested by NAI, ADCC, and other 
immunologic assays as appropriate to measure activity against antigens represented in the study 
vaccines at day 0 and at approximately 28 days and 6 months post-vaccination or at the end of 

 
 
       
33 
 
influenza virus circulation at each site.  Sera collected at days 0 and approximately 6 months 
post-vaccination or at the end of influenza virus circulation at each site from participants who 
have RT-PCR-confirmed ILI during the study period may also be tested by a broad array of 
assays that may include HI, MN, NAI, ADCC and other assays as appropriate. 
Four parameters will be analyzed for HI: SCR, GMT, MFR, and post-vaccination titers > 
seropositive thresholds at 1:40, 1:80, and 1:160. Three parameters will be analyzed for MN:  
SCR, GMT and MFR.  SCR will be defined as the proportion of participants with paired samples 
that achieved ≥ 4 fold rise comparing post vs pre-vaccination titers and post vaccination titers ≥ 
1:40. To allow for calculation of HI GMTs, a titer of 1:5 will be assigned to vaccine non-
responders.  MFR will be defined as the mean of the ratio of post-vaccination HI/MN titer and 
pre-vaccination HI/MN titer for each subject. 
PBMCs will be tested with B and T-cell assays to understand vaccine-induced B, CD4 and 
CD8 T-cell responses. Immune responses will be quantified and characterized using 
flurochrome-conjugated HA-probes for H1 and H3 HA components of A viruses and HA-probes 
for B (Victoria and Yamagata) viruses. If reduced binding to sialic acid (RBS) mutants for B 
viruses is observed, an intracellular cytokine staining protocol will be used on influenza-
stimulated PBMCs. IFN-gamma and TNF-alpha, IL-2 and other T-cell-based cytokines will be 
examined. The mean percentages of influenza strain-specific plasmablasts and memory B cells 
and cytokine secreting CD4 and CD8 T-cells will be compared between participants in each 
study arm. Where feasible, HLA-typing and tetramer based antigen-specific T cell analysis, 
vaccine component (HA, NA, M2e and stem)-specific B and T cell repertoire and single cell 
transcriptome will be assessed. Responses will be examined at baseline and at approximately 7 
days, 28 days, and 6 months post-vaccination.  Influenza HA or NA-specific B cells may also be 
isolated from a subset of PBMC samples using flow cytometry and single-cell sorting. RNA 
libraries will then be prepared from these B cells, and the sequence of the heavy and light chain 
variable regions will be analyzed using next-generation sequencing (NGS). 
Mid-turbinate nasal swabs will be divided into 3 aliquots for testing for influenza viruses by 
RT-PCR using primers for influenza B, A/H1N1, and A/H3N2 viruses.  Specimens may also be 
cultured for influenza viruses or undergo additional influenza virus testing such as for influenza 
viral load using quantitative RT-PCR methods and for antiviral resistance using neuraminidase 
inhibition assays and sequencing. Remaining samples will be stored at -70 Celsius at a CDC-
designated facility. 
 

 
 
       
34 
 
7.6  
Vaccine Product: Four vaccine products will be used in this trial:  Fluzone® Quadrivalent, 
Fluarix® Quadrivalent, Flucelvax™ Quadrivalent, and Flublok® Quadrivalent.  CDC or study 
sites will purchase vaccine.  All four vaccines are inactivated influenza vaccines that are 
approved for use in adults aged >18 years in the United States.  All four vaccine products will 
contain vaccine strains representative of the following four strains in the 2018-2019 formulation:  
an A/Michigan/45/2015 (H1N1)pdm09-like virus; an A/Singapore/INFIMH-16-0019/2016 
(H3N2)-like virus; a B/Colorado/06/2017-like virus (B/Victoria/2/87 lineage); and a 
B/Phuket/3073/2013-like virus (B/Yamagata/16/88 lineage). 
Fluarix® Quadrivalent is a split-virus/subvirion vaccine manufactured by 
GlaxoSmithKline.  It is formulated from influenza virus grown on embryonated chicken eggs.  
Vaccine virus is harvested, concentrated and purified using zonal centrifugation, and then 
inactivated with sodium deoxycholate and formaldehyde.  The resulting vaccine suspension is 
clear and is available for use in adults as prefilled single-dose 0.5mL syringes.  Each dose is 
formulated to contain 15 μg of HA per strain.  Each 0.5mL dose of vaccine also contains 
octoxynol-10 (TRITON X-100) up to 0.115 mg, α-353 tocopheryl hydrogen succinate up to 
0.135 mg, and polysorbate 80 (Tween 80) up to 0.550 mg. Each dose may also contain residual 
amounts of hydrocortisone up to 0.0016 mcg, gentamicin sulfate up to 0.15 mcg, ovalbumin 355 
up to 0.050 mcg, formaldehyde up to 5 mcg, and sodium deoxycholate up to 65 mcg.33   
Fluzone® Quadrivalent is a split-virus/subvirion vaccine manufactured by Sanofi Pasteur.  
It is formulated from influenza virus grown on embryonated chicken eggs.  Vaccine virus is 
harvested, inactivated with formaldehyde, purified by zonal centrifugation using a sucrose 
gradient, split by Triton® X-100, and then further purified to a split-virus/subvirion form.  The 
resulting vaccine suspension is clear and is available for use in adults as a prefilled single-dose 
0.5mL syringe or a 5mL multi-dose vial; the vaccine is administered intramuscularly.  Each dose 
is formulated to contain 15 μg of HA per strain.  Each 0.5mL dose of vaccine also contains 
sodium phosphate-buffered isotonic sodium chloride solution, up to 100mcg of formaldehyde, 
and up to 250mcg of octylphenol ethoxylate. Multi-dose vials also contain the preservatives, 
thimerosal and mercury. The most common reactions occurring after vaccine administration in 
adults are pain at the injection site, myalgia, headache, and malaise.  The majority of these 
reactions are mild to moderate.32 
Flucelvax™ Quadrivalent is a subunit vaccine manufactured by Seqirus Inc.  The vaccine 
is formulated from influenza virus cultivated in MDCK cells.  Vaccine virus is harvested, 

 
 
       
35 
 
inactivated with ß-propiolactone, split by cetyltrimethylammonium bromide, and then further 
purified to a subunit form.  The resulting vaccine suspension is clear and is available for use in 
adults as a prefilled single-dose 0.5mL syringe for intramuscular administration.  Each dose is 
formulated to contain 15 μg of HA per strain.  Each 0.5mL dose of vaccine also contains 
phosphate buffered saline and up to 8.4mcg of MDCK cell protein, up to 120mcg of protein 
other than HA, up to 10ng of MDCK cell DNA, up to 1125 mcg of polysorbate, up to 13.5mcg 
of cetyltrimethlyammonium bromide, and up to 0.5mcg of β-propiolactone.  The vaccine does 
not contain preservatives.  The most common reactions occurring after vaccine administration in 
adults are pain or erythema at the injection site, headache, fatigue, myalgia, and malaise.  The 
majority of these reactions are mild to moderate.31 
Flublok® Quadrivalent is a recombinant vaccine manufactured by Sanofi Pasteur.  The 
vaccine is formulated from purified HA protein from cell-culture isolates of the vaccine 
reference virus grown using baculovirus vector technology in an insect cell line (expresSF+
®) 
from cells of the fall armyworm.  HA is removed from cells using Triton® X-100 and then 
purified by chromatography.  The resulting vaccine suspension is clear and is available for use in 
adults as a prefilled single-dose 0.5mL syringe for intramuscular administration.  Each dose is 
formulated to contain 45 μg of HA per strain.  Each 0.5mL dose of vaccine also contains 4.4mg 
of sodium chloride, 0.195mcg of monobasic sodium phosphate, 1.3mg of dibasic sodium 
phosphate, and 27.5mcg of polysorbate 20. The vaccine may also contain up to 19mcg of 
baculovirus and Spodoptera frugiperda cell proteins, up to 10ng of baculovirus and cellular 
DNA, and up to 100mcg of Triton X-100.30 
Vaccine products will not be modified for this study.  The vaccines must be stored at 2° 
C to 8° C [35.6° F to 46.4° F] until needed. Vaccine products should not be frozen and should be 
protected from natural light. Documentation of proper vaccine storage will be monitored daily 
and maintained during the duration of the trial. In the event of accidental deep-freezing or 
disruption of the cold chain, study vaccine will not be administered. 
8.0 Ethical Considerations 
8.1 Possible Risks 
The potential discomforts from study participation include having blood drawn, 
intramuscular injection of vaccine, and possible reactions to vaccine. Blood samples will be 
taken from all participants at each study visit. Participants may experience some minimal pain 
and/or bruising at the site of the blood draw. To prevent or lessen bruising, study staff will apply 
pressure or ask participants to apply pressure to the draw site for several minutes after the blood 

 
 
       
36 
 
draw. There is also a slight risk of infection associated with blood draws. Study staff will swab 
the site with alcohol and use sterile equipment to make infection at the site where blood is drawn 
or where vaccination is given extremely unlikely.   
All study vaccines are licensed for participants who meet the eligibility criteria for this 
study.  Occasionally, adult recipients of influenza vaccines may develop influenza-like reactions 
such as fever, body aches, headache, malaise, myalgia, and/or nausea. If present, these symptoms 
usually occur soon after vaccination and may last up to 1-2 days post vaccination. Some 
participants may develop reactions at the site of vaccination (redness, swelling, pain, or 
tenderness). Analgesics such as ibuprofen or acetaminophen and rest will generally relieve or 
moderate these symptoms. Acute and potentially life-threatening allergic reactions are also 
possible. Severe reactions from influenza vaccine are estimated to occur in < 1 per 4 million 
persons vaccinated. Signs of a severe allergic reaction may include shortness of breath, 
wheezing, hives, hoarseness, difficulty swallowing, swollen face/ tongue/ pharynx, tachycardia, 
and dizziness. During the swine influenza campaign in the United States in 1976, about 1 per 
100,000 vaccine recipients developed a paralytic illness called Guillain-Barré Syndrome. This 
level of risk has not been seen with other influenza vaccines. Most persons who develop 
Guillain-Barré syndrome recover completely. There may be other unknown side effects.  
8.2 Provisions for protecting privacy/ confidentiality:  
All data collected in this study will be kept confidential. All written information will be stored in 
locked cabinets with limited access, and electronic information will be stored on secured servers. 
Blood samples that will be sent to collaborating institutions for processing and analysis will not 
contain any personal identification information. Collaborating institutions will destroy any stored 
data after completion of the study and data analysis. During the study, only a few members of the 
research team will have access to personal identifying information about the study participants. 
All members of the research team, including the study investigators, research nurses, research 
assistants, and research coordinators, will have access to de-identified data during the study.  
Individuals who are not a part of the research team will not have access to the study information 
or data while the study is taking place. With an official letter and a clearly stated objective, 
scientists who are not a part of the research team may receive access to de-identified data. 
8.3 Consent forms for study volunteers: Written informed consent will be obtained for all 
potential participants prior to enrollment. In understandable language, trained project staff will 
explain study procedures to potential participants and discuss the advantages and disadvantages 
of participating. Participants will be given a copy of the full consent form. Participants will be 

 
 
       
37 
 
asked to read the full consent before agreeing to be in the study and providing their signature.  
Study coordinators will emphasize the voluntary nature of the study, the possible benefits and 
outcomes, alternatives to participation, confidentiality of participation, and the participant’s right 
to refuse and/or withdraw from the study at any time. It will be explained to participants that 
discontinuation of participation or choosing not to participate will not affect their professional 
standing.  
9.0 Study Oversight, Management, and Guidance for Decision Making 
 
A steering committee consisting of representatives from US CDC and each study site will 
provide high level input into this project.  The steering committee will review and approve a data 
usage agreement as guidance for decision making by the steering committee prior to the start of 
the study. The day-to-day project management will occur through CDC with the assistance of a 
contract research organization (to be determined); the steering committee will be consulted on 
over-arching project issues including final protocol decisions, adjudicating any protocol 
deviations that might occur, reviewing and confirming analysis plans, and making final decisions 
on analyses, manuscripts, and authorship as needed.  Upon the completion of all study 
deliverables and after a suitable moratorium, external parties may request de-identified study 
data from the steering committee as specified in U.S. Government Data Sharing guidelines.  
 
10.0 References 
 
1. 
Influenza Disease Burden. (Accessed April 11, 2018, at 
https://www.cdc.gov/flu/about/disease/burden.htm.) 
2. 
Belongia EA, Simpson MD, King JP, Sundaram ME, Kelley NS, Osterholm MT, McLean HQ. Variable 
influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of test-negative design studies. 
Lancet Infect Dis 2016;16:942-51. 
3. 
Flannery B, Chung JR, Belongia EA, McLean HQ, Gaglani M, Murthy K, Zimmerman RK, Nowalk MP, 
Jackson ML, Jackson LA, et al. Interim Estimates of 2017-18 Seasonal Influenza Vaccine Effectiveness - United 
States, February 2018. MMWR Morb Mortal Wkly Rep 2018;67:180-5. 
4. 
Influenza Vaccine Effectiveness, 2016-17 US Flu VE Network & US Hospitalized Adult Influenza Vaccine 
Effectiveness Network (HAIVEN). (Accessed April 11,, 2018, at 
https://www.cdc.gov/vaccines/acip/meetings/downloads/slides-2017-06/flu-03-ferdinands.pdf.) 
5. 
Centers for Disease C, Prevention. Estimates of deaths associated with seasonal influenza --- United States, 
1976-2007. MMWR Morb Mortal Wkly Rep 2010;59:1057-62. 
6. 
Thompson WW, Shay DK, Weintraub E, Brammer L, Bridges CB, Cox NJ, Fukuda K. Influenza-
associated hospitalizations in the United States. JAMA 2004;292:1333-40. 
7. 
Kuster SP, Shah PS, Coleman BL, Lam PP, Tong A, Wormsbecker A, McGeer A. Incidence of influenza in 
healthy adults and healthcare workers: a systematic review and meta-analysis. PLoS One 2011;6:e26239. 
8. 
Wilde JA, McMillan JA, Serwint J, Butta J, O'Riordan MA, Steinhoff MC. Effectiveness of influenza 
vaccine in health care professionals: a randomized trial. JAMA 1999;281:908-13. 
9. 
McLean HQ, Thompson MG, Sundaram ME, Meece JK, McClure DL, Friedrich TC, Belongia EA. Impact 
of repeated vaccination on vaccine effectiveness against influenza A(H3N2) and B during 8 seasons. Clin Infect Dis 
2014;59:1375-85. 
10. 
Belongia EA, Skowronski DM, McLean HQ, Chambers C, Sundaram ME, De Serres G. Repeated annual 
influenza vaccination and vaccine effectiveness: review of evidence. Expert Rev Vaccines 2017;16:1-14. 

 
 
       
38 
 
11. 
Skowronski DM, Chambers C, De Serres G, Sabaiduc S, Winter AL, Dickinson JA, Gubbay JB, Fonseca 
K, Drews SJ, Charest H, et al. Serial Vaccination and the Antigenic Distance Hypothesis: Effects on Influenza 
Vaccine Effectiveness During A(H3N2) Epidemics in Canada, 2010-2011 to 2014-2015. J Infect Dis 
2017;215:1059-99. 
12. 
Thompson MG, Naleway A, Fry AM, Ball S, Spencer SM, Reynolds S, Bozeman S, Levine M, Katz JM, 
Gaglani M. Effects of Repeated Annual Inactivated Influenza Vaccination among Healthcare Personnel on Serum 
Hemagglutinin Inhibition Antibody Response to A/Perth/16/2009 (H3N2)-like virus during 2010-11. Vaccine 
2016;34:981-8. 
13. 
Laguio-Vila MR, Thompson MG, Reynolds S, Spencer SM, Gaglani M, Naleway A, Ball S, Bozeman S, 
Baker S, Martinez-Sobrido L, et al. Comparison of serum hemagglutinin and neuraminidase inhibition antibodies 
after 2010-2011 trivalent inactivated influenza vaccination in healthcare personnel. Open Forum Infect Dis 
2015;2:ofu115. 
14. 
Robertson JS. An overview of host cell selection. Dev Biol Stand 1999;98:7-11; discussion 73-4. 
15. 
Xu X, Kilbourne ED, Hall HE, Cox NJ. Nonimmunoselected intrastrain genetic variation detected in pairs 
of high-yielding influenza A (H3N2) vaccine and parental viruses. J Infect Dis 1994;170:1432-8. 
16. 
Zost SJ, Parkhouse K, Gumina ME, Kim K, Diaz Perez S, Wilson PC, Treanor JJ, Sant AJ, Cobey S, 
Hensley SE. Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies 
elicited by egg-adapted vaccine strains. Proc Natl Acad Sci U S A 2017;114:12578-83. 
17. 
Raymond DD, Stewart SM, Lee J, Ferdman J, Bajic G, Do KT, Ernandes MJ, Suphaphiphat P, Settembre 
EC, Dormitzer PR, et al. Influenza immunization elicits antibodies specific for an egg-adapted vaccine strain. Nat 
Med 2016;22:1465-9. 
18. 
Skowronski DM, Chambers C, Sabaiduc S, De Serres G, Dickinson JA, Winter AL, Drews SJ, Fonseca K, 
Charest H, Gubbay JB, et al. Interim estimates of 2014/15 vaccine effectiveness against influenza A(H3N2) from 
Canada's Sentinel Physician Surveillance Network, January 2015. Euro Surveill 2015;20. 
19. 
Skowronski DM, Janjua NZ, De Serres G, Sabaiduc S, Eshaghi A, Dickinson JA, Fonseca K, Winter AL, 
Gubbay JB, Krajden M, et al. Low 2012-13 influenza vaccine effectiveness associated with mutation in the egg-
adapted H3N2 vaccine strain not antigenic drift in circulating viruses. PLoS One 2014;9:e92153. 
20. 
Frey S, Vesikari T, Szymczakiewicz-Multanowska A, Lattanzi M, Izu A, Groth N, 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. 
21. 
Reisinger KS, Block SL, Izu A, Groth N, Holmes SJ. Subunit influenza vaccines produced from cell culture 
or in embryonated chicken eggs: comparison of safety, reactogenicity, and immunogenicity. J Infect Dis 
2009;200:849-57. 
22. 
Szymczakiewicz-Multanowska A, Groth N, Bugarini R, Lattanzi M, Casula D, Hilbert A, Tsai T, Podda A. 
Safety and immunogenicity of a novel influenza subunit vaccine produced in mammalian cell culture. J Infect Dis 
2009;200:841-8. 
23. 
Groth N, Montomoli E, Gentile C, Manini I, Bugarini R, Podda A. Safety, tolerability and immunogenicity 
of a mammalian cell-culture-derived influenza vaccine: a sequential Phase I and Phase II clinical trial. Vaccine 
2009;27:786-91. 
24. 
Vesikari T, Block SL, Guerra F, Lattanzi M, Holmes S, Izu A, Gaitatzis N, Hilbert AK, Groth N. 
Immunogenicity, safety and reactogenicity of a mammalian cell-culture-derived influenza vaccine in healthy 
children and adolescents three to seventeen years of age. Pediatr Infect Dis J 2012;31:494-500. 
25. 
Dunkle LM, Izikson R. Recombinant hemagglutinin influenza vaccine provides broader spectrum 
protection. Expert Rev Vaccines 2016;15:957-66. 
26. 
Treanor JJ, El Sahly H, King J, Graham I, Izikson R, Kohberger R, Patriarca P, Cox M. Protective efficacy 
of a trivalent recombinant hemagglutinin protein vaccine (FluBlok(R)) against influenza in healthy adults: a 
randomized, placebo-controlled trial. Vaccine 2011;29:7733-9. 
27. 
Dunkle LM, Izikson R, Patriarca PA, Goldenthal KL, Cox M, Treanor JJ. Safety and Immunogenicity of a 
Recombinant Influenza Vaccine: A Randomized Trial. Pediatrics 2018. 
28. 
Baxter R, Patriarca PA, Ensor K, Izikson R, Goldenthal KL, Cox MM. Evaluation of the safety, 
reactogenicity and immunogenicity of FluBlok(R) trivalent recombinant baculovirus-expressed hemagglutinin 
influenza vaccine administered intramuscularly to healthy adults 50-64 years of age. Vaccine 2011;29:2272-8. 
29. 
Dunkle LM, Izikson R, Patriarca P, Goldenthal KL, Muse D, Callahan J, Cox MMJ, Team PSCS. Efficacy 
of Recombinant Influenza Vaccine in Adults 50 Years of Age or Older. N Engl J Med 2017;376:2427-36. 
30. 
Protein Sciences Package Insert, Quadrivalent Influenza Vaccine. (Accessed April 13, 2018, at 
https://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM336020.pdf.) 
31. 
Flucelvax Quadrivalent Package Insert. (Accessed April 13, 2018, at 
https://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM502899.pdf.) 

 
 
       
39 
 
32. 
Sanofi Pasteur, 450/477 Fluzone® Quadrivalent Southern Hemisphere. (Accessed April 13, 2018, at 
https://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM305089.pdf.) 
33. 
FLUARIX QUADRIVALENT (Influenza Vaccine) Suspension for Intramuscular Injection. (Accessed 
May 2, 2018, at 
https://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM220624.pdf.) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
       
40 
 
 
Appendix A: Schedule of Events* 
 
 
 
Study Visit 
 
Screening/ 
Enrollment 
 
Visit 1, Year 
1 
 
Visit 1b**, 
Year 1 
 
Visit 2, Year 
1 
 
Visit 3,  
Year 1 
 
Visit 1,  
Year 2 
Study Day 
 
0 
6-9 
21-62 
180-210 
0 
Appendix B: 
Eligibility 
Screening Form 
X 
 
 
 
 
 
Appendix C: 
Consent Form 
X 
 
 
 
 
X if needed 
Appendix D: 
Enrollment 
Questionnaire  
 
X 
 
 
 
X 
Appendix E: 
Pre-vaccination 
Questionnaire/Va
ccine 
Administration 
Form 
 
X 
 
 
 
X 
Appendix F1: 
Blood Specimen 
Collection/Tracki
ng Form 
 
X 
X 
X 
X 
X 
Appendix F2: 
Influenza-like 
Illness Data 
Collection and 
Respiratory 
Specimen 
Collection Form 
 
(X) 
Appendix G: 
EMR Chart 
Extraction/Abstra
ction Form 
  
X 
 
 
 
X 
Appendix H: 
Protocol 
Deviation Log 
  
(X) 
Appendix I: 
Study Status 
Change Form 
 
X 
Appendix J: 
Active 
Surveillance 
Tracking Form 
 
X 
Parentheses indicate that forms will be completed as needed. 
*Study visits 1b, 2, and 3 are not shown for year 2.  Activities are the same as for the analogous visit in 
year 1.