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

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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: 
Year 2 Protocol Amendment 
 
NCT03722589 
 
Document Date: July 11, 2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. Protocol Title  
Randomized Open-Label Trial to Compare the Immunogenicity of Cell Culture-Based and Recombinant 
Unadjuvanted Influenza Vaccines to Egg-Based Influenza Vaccines among Healthcare Personnel Aged 18-
65 Years:   
Year Two Amendment 
 
Background 
The first year of this randomized, open-label trial conducted during the 2018-2019 influenza season 
assessed humoral and cell-mediated immune responses to cell culture-based and recombinant unadjuvanted 
quadrivalent influenza vaccines compared to conventional egg-based unadjuvanted quadrivalent SD (SD) (15µg 
of HA per strain) influenza vaccines among healthcare personnel (HCP) aged 18-64 years at two sites in the 
United States.  Eligible participants were 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 egg-based SD vaccines (Fluzone® 
Quadrivalent by Sanofi Pasteur, 15µg of HA per strain and Fluarix® Quadrivalent by GlaxoSmithKlein, 15µg of 
HA per strain) during October 2018.  The first year of the trial was designed to address the question of whether 
HCP who receive cell culture-based or recombinant influenza vaccines for the first time (after receipt of egg-
based SD vaccines in previous seasons) have higher antibody titers (surrogates for immune protection1, 2) against 
circulating influenza viruses compared to HCP who receive egg-based SD vaccines.   
Since the trial began, preliminary data from another randomized trial comparing cell culture-based, 
recombinant and egg-based SD vaccines have become available and suggest that recombinant vaccine induces 
higher antibody titers against both egg- and cell-grown vaccine reference viruses compared to egg-based vaccines, 
whereas cell culture-based vaccine does not (personal communication, Min Levine, CDC).  Data from 
observational vaccine effectiveness analyses comparing cell culture-based vaccine to egg-based vaccines are 
mixed with some analyses suggesting little to no difference in effectiveness between vaccines.3, 4  However, it is 
unknown whether repeated vaccination with cell culture-based or recombinant vaccines provides additional 
benefit compared to first-time receipt of these vaccines or whether the improved humoral immune response to 
recombinant vaccine that contains three times the antigen dose of SD influenza vaccines is driven by response to 
recombinant antigen, higher antigen dose, or both.  
 
 
Year two of this trial will examine two hypotheses.  The first hypothesis is that repeated vaccination with 
cell culture-based or recombinant vaccines boosts antibody titers against cell-grown viruses more than first-time 

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vaccination with these vaccines, after receipt of egg-based SD vaccines.  To address this hypothesis, participants 
from year one of the trial who received cell culture-based or recombinant influenza vaccines will be re-
randomized to receive one of these two vaccines in year two of the trial and participants who received egg-based 
SD vaccines will be re-randomized to receive a cell-culture based, recombinant or egg-based SD vaccine 
(represented by Fluzone® Quadrivalent).  The second hypothesis is that higher antigen doses, regardless of 
whether egg- or cell-grown, will result in higher antibody titers against cell-grown viruses.  To address this 
hypothesis, the trial will enroll additional participants at the Kaiser Permanente Northwest site into a non-
randomized vaccine arm in which participants will receive Fluzone® High-Dose influenza vaccine.  Fluzone® 
High-Dose is an egg-based vaccine that contains four times the antigen content (60 μg of HA per strain) compared 
to SD influenza vaccine and is licensed for use in persons aged >65 years. In this trial, Fluzone® High-Dose will 
be used off-label among persons aged 18-64 years (see Rationale for Off-Label Use of Fluzone® High-Dose 
Among Healthcare Personnel Aged 18-64 Years).   
Year two of this trial may also afford the opportunity to evaluate whether cell culture-based, recombinant, 
or high-dose egg-based vaccines boost antibody response among HCP with low responses to SD egg-based 
vaccines, if low-responders are identified among participants who received SD egg-based vaccines in year one.  
Year two of this trial will also investigate differences in cell-mediated immune responses to repeated versus first-
time vaccination with cell-based and recombinant vaccines in a subset of participants (approximately 100) at the 
Baylor Scott & White Health site.  Effects of repeated vaccination with non-egg-based vaccines on cell-mediated 
immune responses have not been studied. 
Rationale for Off-Label Use of Fluzone High-Dose among Healthcare Personnel Aged 18-64 Years 
Off-label use of Fluzone® High-Dose has been studied in randomized controlled trials in healthcare 
personnel aged 18-64 years,5 adults with HIV infection6, and adult and pediatric oncology patients7-9 and has been 
shown to be well tolerated.  In a randomized trial comparing Fluzone® High-Dose to Fluzone® Quadrivalent in 
HCP aged 18-64 years in Canada, fatigue, weakness and injection site reactions were more frequently reported 
after Fluzone® High-Dose, but the majority of adverse events were mild and none required medical attention.5  In 
the same study, 92% of Fluzone® High-Dose recipients reported willingness to receive the same vaccine in the 
future based on their experiences with adverse events.  Other trials of off-label use of Fluzone® High-Dose have 
confirmed a similar safety profile with either no difference in adverse events compared to Fluzone® Quadrivalent 
or more reactogenicity or systemic events that were generally mild and self-limited among Fluzone® High-Dose 
recipients.6-9  These findings are consistent with a large pre-licensure trial comparing the safety profile of 
Fluzone® High-Dose to Fluzone® Quadrivalent among persons aged >65 years which found that solicited 
injection-site reactions and systemic adverse events were more common after Fluzone® High-Dose but most 
reactions occurred within three days of vaccination and were mild and self-limited.10 A formal Investigational 

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New Drug waiver was obtained for this trial from the US Food and Drug Administration Division of Vaccines 
and Related Products (Appendix M, FDA IND Exemption Letter). 
HCP are believed to be at increased risk of influenza virus infection and are one of the most vaccinated 
target populations in the United States.  The frequent influenza vaccination history of HCP may predispose them 
to a lower antibody response to SD influenza vaccines, as some studies of influenza vaccine immunogenicity 
among HCP suggest that repeated vaccination can blunt the antibody response to hemagglutinin11 and 
neuraminidase.12  In addition, some studies have found that influenza vaccines are less effective in persons with a 
history of frequent influenza vaccination,13, 14 Fluzone® High-Dose has been shown to elicit higher antibody titers 
to some or all vaccine strains compared to Fluzone® Quadrivalent among persons aged >65 years and other 
populations in which the vaccine has been used off-label.5-8  Year two of this trial will evaluate whether Fluzone® 
High-Dose elicits an improved immune response in HCP aged 18-64 years with a history of frequent vaccination 
compared to cell-culture based, recombinant, and egg-based SD influenza vaccines.  The addition of Fluzone® 
High-Dose vaccine in year two will also provide the opportunity to evaluate the role of higher antigen content 
versus vaccine type (egg-based vs. recombinant) in eliciting improved immune responses to vaccination if higher 
post-vaccination titers are observed among Flublok® Quadrivalent  recipients.   
STUDY OBJECTIVES, YEAR TWO 
• 
Primary Objectives 
1) Compare serologic responses after two consecutive years of vaccination with cell culture-based 
influenza vaccine (Flucelvax™ Quadrivalent ) or recombinant influenza vaccine (Flublok® 
Quadrivalent ) versus first time receipt of cell culture-based or recombinant vaccines (following 
vaccination with egg-based SD influenza vaccines during the prior season) at approximately 28 
days after receipt of the 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  
2) Compare serologic responses after cell culture-based influenza vaccine (Flucelvax™ 
Quadrivalent) versus recombinant influenza vaccine (Flublok® Quadrivalent) at approximately 
28 days after receipt of the 2019-20 vaccines, regardless of prior vaccination status, 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  
3) Compare serologic responses after two consecutive years of vaccination with cell culture-based 
influenza vaccine (Flucelvax™ Quadrivalent ) or recombinant influenza vaccine (Flublok® 

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Quadrivalent) versus egg-based SD vaccine (Fluzone Quadrivalent) at approximately 28 days 
after receipt of the 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  
4) Compare immune responses after first time vaccination with cell culture-based influenza vaccine 
(Flucelvax™ Quadrivalent ), recombinant influenza vaccine (Flublok® Quadrivalent ), or high-
dose egg-based vaccine (Fluzone® High-Dose) versus repeated vaccination with egg-based SD 
vaccine at approximately 28 days after receipt of the 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  
Secondary Objectives 
1) Compare serologic 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  
• 
Exploratory Objectives 
1) Characterize cell-mediated immune responses of B cells, innate lymphoid cells (ILC), CD4 and 
CD8 T-cells by multiparametric flowcytometry with antigen tags, B-cell receptor seq and 
antibody-seq to Flucelvax™ Quadrivalent, Flublok® Quadrivalent, Fluzone® Quadrivalent, and 
Fluzone® High-Dose at approximately 7 and 28 days after vaccination in a sub-set of vaccinees. 
2) Characterize the avidity of antibodies produced in response to Flucelvax™ Quadrivalent, 
Flublok® Quadrivalent, Fluzone® Quadrivalent and Fluzone® High-Dose at approximately 7 and 
28 days after vaccination. 
3) Compare immune responses among persons vaccinated with either a single dose of Flucelvax™ 
Quadrivalent, Flublok® Quadrivalent, or Fluzone® High-Dose versus a single dose of Fluzone® 
Quadrivalent at approximately 28 days after vaccination measured by  
▪ 
NAI and antibody-dependent cell-mediated cytotoxicity (ADCC) titers  
▪ 
Other relevant immunological markers 
 
STUDY ENDPOINTS, YEAR TWO 
Primary Endpoints 

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• 
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: 
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  
o GMT ratio defined as the ratio of GMTs between study arms at 28 days post-vaccination 
• 
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 
o GMT ratio 
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, MFR, and 
GMT ratio 
• 
HI 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 
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, GMT ratio and post-vaccination titers > seropositive thresholds at 1:40, 1:80, 
1:160 as measured by HI 
o SCR, GMT, MFR, and GMT ratio as measured by MN, as appropriate 
• 
GMT as measured by NAI and ADCC pre- and post-vaccination. 
• 
Mean percentages of strain-specific activation markers on T, B and ILC populations, frequencies of 
antibody secreting plasmablasts and memory B cells to hemagglutinin (HA) with HA-tags,  interferon-
gamma (IFN- gamma), interleukin 2 (IL-2),  and Tumor Necrosis Factor-alpha (TNF-alpha) secreting T 

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cell responses to wild-type cell-grown strains, B cell repertoire, antibody-seq, fine-specificities of 
antibody reactivities and single cell transcriptome analysis (where feasible) at approximately 7 and 28 
days post-vaccination. 
• 
Indicators of immune response to vaccination based on other immunologic assays not listed above (as 
appropriate) 
 
METHODS, YEAR TWO MODIFICATIONS 
Unless otherwise specified in the sections below, study methods will remain the same as those described in the 
original protocol.  
Inclusion Criteria  
• 
Healthcare personnel, including dentists and other dental health personnel  
• 
Have been receiving routine medical care in the Baylor Scott & White Health System for at least one 
month before enrolling in the study  
or  
enrolled in the Kaiser Permanente health network for at least one month before enrolling in the study 
• 
Aged 18-65 years for participants who were originally enrolled in year one of the study 
• 
Aged 18-64 years for participants newly enrolled in year two of the study 
• 
Available and willing to participate in study follow-up through approximately 1 month after study 
vaccination in the 2019-2020 influenza season  
• 
Received an egg-based SD influenza vaccine during the 2018-2019 influenza season (for participants 
enrolled for the first-time during year two) 
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 research staff believes may interfere with successful completion of the 
study 

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• You are not willing or able to get the licensed flu vaccines being used in this study;  
• 
For enrollment into the non-randomized Fluzone® High-Dose arm at KPNW, the following exclusion 
criterion will also apply for female HCP aged 18-49 years: 
o  Pregnant by participant report or study-administered urine pregnancy test 
Study Design:  Year two of the trial will be open-label and will include both randomized and non-randomized 
arms (Figure 3). Since the proposed trial design is a modification of the original protocol, the consent process will 
be repeated for all participants from year one of the trial.  Participants from year one who received Flucelvax™ 
Quadrivalent  or Flublok® Quadrivalent  will be randomized 1:1 to receive Flucelvax™ Quadrivalent  or 
Flublok® Quadrivalent  in year two (Figure 3).  Participants who received an egg-based SD vaccine in year one 
(Fluzone® Quadrivalent or Fluarix Quadrivalent) will be randomized 1:1:1 to receive Flucelvax™ Quadrivalent , 
Flublok® Quadrivalent , or Fluzone® Quadrivalent in year two.   
Both sites may also aim to enroll additional participants to achieve a total of 150 participants per site 
(including participants who continue from year one plus additional newly enrolled participants) who received 
egg-based SD influenza vaccine during the 2018-2019 influenza season and who will be randomized 1:1:1 to 
receive Flucelvax™ Quadrivalent, Flublok® Quadrivalent, or Fluzone® Quadrivalent in year two.  The KPNW 
site will also enroll up to 80 new participants for a non-randomized study arm that will receive Fluzone® High-
Dose.    
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. Stratified enrollment 
will be used for both randomized and non-randomized arms to ensure an even mix of age groups (18-44 years and 
45-65 years) in each study arm.   
Figure 3  Stratified randomization and sample size goals for groups with new enrollees per site 
 
 
 
 
 
 
Flucelvax in year one 
AND 
Age 18-44 years 
Flucelvax in year one 
AND 
Age 45-65 years
Randomized 
1:1 
Randomized 
1:1 
Flucelvax  
 
Flublok 
Flucelvax 
  
Flublok 

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Egg-based vaccine in year 
one or newly enrolled AND  
Age 45-65 years, 
n=up to 150 
Egg-based vaccine in year 
one or newly enrolled AND  
Age 18-44 years,  
n=up to 150 
Flublok in year one AND 
Age 18-44 years 
Flublok in year one 
AND 
Age 45-65 years 
Newly enrolled AND  
Age 18-44 years, n=40 
(KPNW only) 
Newly enrolled AND  
Age 45-65 years, n=40 
(KPNW only) 
Randomized 
1:1 
Randomized 
1:1 
Randomized 
1:1:1 
Randomized 
1:1:1 
Not 
randomized 
Not 
randomized 
Flucelvax  
 
Flublok 
Flucelvax 
 
Flublok 
Flucelvax, n=50 
Flublok, n=50 
Fluzone SD, n=50 
Flucelvax, n=up to 50  
Flublok, n=up to 50 
Fluzone SD, n=up to 50 
Fluzone High-Dose, 
n=40 
Fluzone High-Dose, 
n=40 

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Figure 4  Study activity flow diagram, Year two 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study Visit 1b, 
Year two  
(days 6-9) 
 
(PBMC 
participants only) 
30ml whole blood 
for PBMC 
collection 
Study Visit 1, Year two (day 0 of year two) 
Enrollment interview (if newly enrolled in year two) 
Abbreviated enrollment interview (if participant from year one) 
Enroll new participants to longitudinal PBMC substudy as needed to 
maintain 100 participants in the substudy 
 
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 two (days 21-62 of year two)  
Blood collection 
20mL whole blood (all participants) 
30mL whole blood for PBMC collection  
(participants consented for longitudinal PBMC collection) 
 
 
Screening/Consent/Enrollment, Year Two 
All participants (including those enrolled in 
year one and newly enrolled)  
 
Randomized per Figure 3 
EMR Extraction for Prior Vaccine Verification 

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Consent/Screening/Enrollment:   
Participants from year one of the study will be recruited for re-enrollment in year two of the study.  In 
addition, both sites may recruit additional participants for enrollment into the main trial for randomization to 
Flucelvax™ Quadrivalent, Fluzone® Quadrivalent or Flublok® Quadrivalent (Figure 4).  The KPNW site will 
recruit additional participants for enrollment to a non-randomized sub-study of Fluzone® High-Dose influenza 
vaccine.  All potentially eligible HCP including those who participated in year one of the trial and newly recruited 
HCP will take a self-administered survey or study staff may administer a survey to determine whether 
inclusion/exclusion criteria for year two of the trial are met (Appendix N, CDC FluVaxTrial: Eligibility Screening 
Form, Year 2).  Participants will be enrolled in the study if they meet inclusion criteria and provide consent to be 
in the second year of the study (Appendices O, Consent Form, Year 2; Appendix P, Non-Randomized Fluzone 
High-Dose Trivalent Study Consent Form).    
Additional Eligibility Screening for Fluzone High-Dose Substudy 
After providing informed consent to participate in the Fluzone® High-Dose arm of the trial, female HCP 
aged 18-49 years will be screened for pregnancy using a step-wise process.  First, women will be asked if they are 
pregnant.  Women who report that they are not pregnant will be asked to provide the start date of their last 
menstrual period.  A urine pregnancy test will be performed (with consent) for any woman who reports that she is 
uncertain about whether she is pregnant, does not know the start date of her last menstrual period, or reports a 
start date of last menstrual period that is >28 days prior to the date of screening.  Women who are pregnant based 
on self-report or a positive urine pregnancy test will be excluded from participation.  Notification of urine 
pregnancy test results will be done according to site-specific requirements.  
Study Visit 1 of Year Two [day 0] 
Participants from year one of the study will complete an enrollment questionnaire during study visit 1 of 
year two (Appendix Q, CDC HCP Flu VaxTrial:  Enrollment Questionnaire, Year 2).  All study participants will 
also complete a brief questionnaire to determine whether they are currently experiencing fever or other acute 
illness symptoms (Appendix R, Pre-vaccination Questionnaire/Vaccine Administration Form, Year 2). 

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Participants who present with moderate to 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 in Year two of the Study 
Study nurses administering vaccine will consult participants’ study files to confirm which study vaccine 
participants from year one received during the first year of the study.  For the purposes of randomization, newly 
enrolled participants into the main trial who did not participate in year one will be treated as part of the group who 
received Fluzone® Quadrivalent or Fluarix ® Quadrivalent during year one.  
Stratified block randomization will be used at each site to ensure even allocation of age groups (18-44 
years and either 45-65 years) to each study arm (Figure 3).  Within each age stratum: 
• 
participants who received Flucelvax™ Quadrivalent  in year one will be randomized 1:1 to 
receive one 0.5 mL intramuscular dose Flucelvax™ Quadrivalent or Flublok® Quadrivalent; 
• 
participants who received Flublok® Quadrivalent  in year one will be randomized 1:1 to receive 
one 0.5 mL intramuscular dose of Flucelvax™ Quadrivalent or Flublok® Quadrivalent; and 
• 
participants who received Fluzone® Quadrivalent or Fluarix ® Quadrivalent in year one or who 
were newly enrolled into the main trial will be randomized 1:1:1 to receive one 0.5 mL 
intramuscular dose of Flucelvax™ Quadrivalent, Flublok® Quadrivalent , or  Fluzone® 
Quadrivalent.   
Randomization lists will be generated using a computerized random-number generator to select randomly 
permuted group blocks of six with either three in each block assigned to Flucelvax™ Quadrivalent and Flublok® 
Quadrivalent for those who received Flucelvax™ Quadrivalent or Flublok® Quadrivalent in year one or two in 
each block assigned to Fluzone® Quadrivalent or Flucelvax™ Quadrivalent or Flublok® Quadrivalent for those 
who received Fluzone® Quadrivalent in year one or are newly enrolled to the main trial in year two. The next 
available sequential study-number will be assigned to each enrolled participant upon study-entry. 
 
Participants who are newly enrolled into the Fluzone® High-Dose substudy will not be randomized and 
will all receive Fluzone® High-Dose.   
‘Opt-In Consent for Longitudinal PBMC Collection: 
After participants are randomized, those who were in the Longitudinal PBMC Collection substudy in year 
one at BSWH will be invited to participate in longitudinal PBMC collection that requires collection of an 
additional 30mL of blood at study visits 1 and 2 in year two plus an additional study visit 1b as outlined in the 
original protocol.  Additional participants who received either Fluzone® Quadrivalent or Fluarix ® Quadrivalent in 
year one of the study may be recruited to maintain a total of 100 participants in the Longitudinal PBMC 

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Collection substudy.   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 in year two.   
KPNW will not participate in the Longitudinal PBMC Collection substudy. 
Blood Draw, Vaccine Administration, Blinding, and Post-Vaccination Safety Monitoring Procedures 
Same as described for year one of the trial.   Information related to blood collection will be recorded on blood 
collection forms (Appendix S1, S2, S3, CDC HCP Flu VaxTrial: Blood Specimen Collection/Tracking Form, 
Year 2, Study Visits 1, 1b, and 2). 
 
Monitoring for Adverse Events 
Since the safety and tolerability of the study vaccines, including off-label use of Fluzone® High-Dose, 
have been established in prior studies and data from this study will not be used to seek a licensure change for 
Fluzone® High-Dose, 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 Two [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 Two [optimal timing days 21-35 (acceptable timing days 21-62)] 
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. 
Active Surveillance for Influenza-Like Illness 
Active surveillance for influenza-like illness will not be conducted during year two of this trial. 
Electronic Medical Record Extraction/Abstraction 
For newly enrolled participants, site investigators will extract (or abstract if necessary) data from 
participants’ electronic medical records and state immunization registries on influenza vaccination history during 
the preceding ten years to include, date of receipt, vaccine product name, vaccine lot number, and route of 
administration (Appendix T, EMR/Chart Extraction/Abstraction Form, Year 2).  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. 

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For participants who were in year one of the trial, site investigators will limit extraction (or abstraction) to 
collecting data on influenza vaccination during the 2011-2012 and 2012-2013 influenza season. 
Statistical Analysis for Year Two: 
Humoral antibody responses as measured by HI (and MN, NAI and ADCC, as appropriate) at baseline 
(day 0) and approximately 28 days post-vaccination will be compared between participants in each study arm as 
outlined in Table 5; additional comparisons may be made as appropriate.  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 post-vaccination).  Post-vaccination GMTs and MFRs at each time point post-
vaccination may be compared between study arms using repeated measures mixed models with time point and 
vaccine as well as their interaction as independent variables; ANOVA will be used to allow for adjustment for 
imbalances between vaccine groups.  Titers > seropositive thresholds at 1:40, 1:80, 1:160 and SCR may be 
compared with Chi Square tests.  Adjustment for multiple comparisons to the meet the primary objectives will be 
done using a Bonferonni correction approach. 
Cell-mediated immune responses at baseline and approximately 7 days and 28 days will be compared 
between participants in each study arm. ANOVA will be used to carry out comparisons of cell-mediated 
immunity studies, and Bonferonni correction may be used to adjust for multiple comparisons.  
All primary analyses will be conducted as intention-to-treat. All tests will be 2-tailed with a level of 
significance of .05.   
Table 5 Planned statistical comparisons for primary objectives and endpoints in year two 
Primary Objective 
Comparison Group 1 
Comparison Group 2 
Compare immune responses after 
two consecutive years of vaccination 
with cell-based influenza vaccine 
(Flucelvax™ Quadrivalent) or 
recombinant influenza vaccine 
(Flublok® Quadrivalent) versus first 
time receipt of these vaccines 
following vaccination with SD egg-
based influenza vaccines. 
Flucelvax™ 
Quadrivalent  year 
one/year two  
SD egg-based vaccine year one/Flucelvax™ 
Quadrivalent  year two 
Flublok® Quadrivalent  
year one/year two 
SD egg-based vaccine year one/Flublok® 
Quadrivalent  year two 
Compare immune responses after 
two consecutive years of vaccination 
with cell-based influenza vaccine 
(Flucelvax™ Quadrivalent), 
recombinant influenza vaccine 
(Flublok® Quadrivalent), or SD 
egg-based vaccine (Fluzone SD). 
Flucelvax™ 
Quadrivalent  year 
one/year two 
SD egg-based vaccine year one/year two 
Flublok® Quadrivalent  
year one/year two 
SD egg-based vaccine year one/year two 

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Compare immune responses after 
cell culture-based influenza vaccine 
(Flucelvax™ Quadrivalent) versus 
recombinant influenza vaccine 
(Flublok® Quadrivalent) at 
approximately 28 days after receipt 
of the 2019-2020 vaccines, 
regardless of prior vaccination 
status. 
Flucelvax™ 
Quadrivalent year 2 
(regardless of year one 
vaccine) 
Flublok® Quadrivalent year 2 
(regardless of year one vaccine) 
Compare immune responses after 
first time vaccination with cell-
based influenza vaccine 
(Flucelvax™ Quadrivalent), 
recombinant influenza vaccine 
(Flublok® Quadrivalent), or high-
dose egg-based vaccine (Fluzone 
High-Dose) following vaccination 
with SD egg-based influenza 
vaccines to repeated vaccination 
with SD egg-based vaccine.   
SD egg-based vaccine 
year one/Flucelvax™ 
Quadrivalent  year two 
SD egg-based vaccine year one/year two 
SD egg-based vaccine 
year one/Flublok® 
Quadrivalent  year two 
Fluzone HD year two 
SD: Standard dose; HD: High dose 
Power/Sample Size Calculations: 
Study sample size was determined based on available resources.  An overall sample size of up to 830 with 
• 
90 participants each in the Flublok® Quadrivalent year one/ Flucelvax™ Quadrivalent year two group 
and Flublok® Quadrivalent  year one/year two group 
• 
135 participants each in the Flucelvax™ Quadrivalent  year one/year two group and Flucelvax™ 
Quadrivalent  year one/ Flublok® Quadrivalent year two group 
• 
100 participants each in the SD (SD) egg-based vaccine year one/Flucelvax™ Quadrivalent  year two, SD 
egg-based vaccine year one/Flublok® Quadrivalent  year two, and SD egg-based vaccine year one/year 
two groups, and 
• 
80 participants in the Fluzone HD year two group 
would provide adequate statistical power to detect a post-vaccination GMT ratio of 1.2 between comparison 
groups as outlined in table 5 after adjusting for up to seven comparisons using a Bonferroni correction.  Table 6 
provides estimated sample sizes per comparison group required for varying post-vaccination GMT ratios after 
adjustment for up to eight comparisons.   
Table 6. Sample size calculations to detect specified post-vaccination GMT ratios between two comparison 
groups after adjustment for eight primary comparisons using a Bonferroni correction  
GMT ratio 
N per group 
1.5 
16 

16 
 
1.4 
22 
1.3 
34 
1.2 
69 
1.1 
245 
 
Vaccine Product: 
Four vaccine products will be used during the 2019-2020 season of this trial:  Fluzone® Quadrivalent, 
Flucelvax™ Quadrivalent, Flublok® Quadrivalent, and Fluzone Trivalent High Dose.  CDC or study sites will 
purchase vaccine.  Fluzone® Quadrivalent, Flucelvax™ Quadrivalent, and Flublok® Quadrivalent are inactivated 
influenza vaccines that are approved for use in adults aged >18 years in the United States.  Fluzone Trivalent 
High Dose is an inactivated vaccine that is licensed for use in adults aged >65 years in the United States and will 
be used off-label in this trial. All four vaccine products will contain vaccine strains representative of the following 
three strains in the 2019-2020 formulation:  an A/Brisbane/02/2018 (H1N1)pdm09-like virus; A/Kansas/14/2017 
(H3N2)-like virus; and a B/Colorado/06/2017-like virus (B/Victoria/2/87 lineage).  All quadrivalent vaccine 
products will also include a B/Phuket/3073/2013-like virus (B/Yamagata/16/88 lineage). 
Fluzone® High-Dose 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; the vaccine is administered intramuscularly.  Each dose is formulated to 
contain 45 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. The most 
common reactions occurring after vaccine administration in adults are pain at the injection site, myalgia, 
headache, malaise, and erythema at the injection site.  The majority of these reactions are mild to moderate.15  
References 
1. 
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antibody in protection against challenge infection with influenza A2 and B viruses. J Hyg (Lond) 1972;70:767-77. 
2. 
Potter CW, Oxford JS. Determinants of immunity to influenza infection in man. Br Med Bull 1979;35:69-
75. 
3. 
Izurieta HS, Chillarige Y, Kelman J, Wei Y, Lu Y, Xu W, Lu M, Pratt D, Chu S, Wernecke M, et al. Relative 
effectiveness of cell-cultured and egg-based influenza vaccines among the U.S. elderly, 2017-18. J Infect Dis 
2018. 
4. 
Klein N, Fireman B, Goddard K, Zerbo O, Asher J, Zhou J, King J. Vaccine Effectiveness of Flucelvax 
Relative to Inactivated Influenza Vaccine During the 2017-18 Influenza Season in Northern California. In: ID 
Week. San Francisco; 2018. 

17 
 
5. 
Volling C, Coleman BL, Katz K, Simor AE, Muller M, Powis J, McElhaney J, McGeer A. Immunogenicity and 
reactogenicity of high- vs. standard-dose trivalent inactivated influenza vaccine in healthcare workers: a pilot 
randomized controlled trial. Clin Microbiol Infect 2019;25:217-24. 
6. 
McKittrick N, Frank I, Jacobson JM, White CJ, Kim D, Kappes R, DiGiorgio C, Kenney T, Boyer J, Tebas P. 
Improved immunogenicity with high-dose seasonal influenza vaccine in HIV-infected persons: a single-center, 
parallel, randomized trial. Ann Intern Med 2013;158:19-26. 
7. 
Halasa NB, Savani BN, Asokan I, Kassim A, Simons R, Summers C, Bourgeois J, Clifton C, Vaughan LA, 
Lucid C, et al. Randomized Double-Blind Study of the Safety and Immunogenicity of Standard-Dose Trivalent 
Inactivated Influenza Vaccine versus High-Dose Trivalent Inactivated Influenza Vaccine in Adult Hematopoietic 
Stem Cell Transplantation Patients. Biol Blood Marrow Transplant 2016;22:528-35. 
8. 
Jamshed S, Walsh EE, Dimitroff LJ, Santelli JS, Falsey AR. Improved immunogenicity of high-dose 
influenza vaccine compared to standard-dose influenza vaccine in adult oncology patients younger than 65 years 
receiving chemotherapy: A pilot randomized clinical trial. Vaccine 2016;34:630-5. 
9. 
McManus M, Frangoul H, McCullers JA, Wang L, O'Shea A, Halasa N. Safety of high dose trivalent 
inactivated influenza vaccine in pediatric patients with acute lymphoblastic leukemia. Pediatr Blood Cancer 
2014;61:815-20. 
10. 
Falsey AR, Treanor JJ, Tornieporth N, Capellan J, Gorse GJ. Randomized, double-blind controlled phase 3 
trial comparing the immunogenicity of high-dose and standard-dose influenza vaccine in adults 65 years of age 
and older. J Infect Dis 2009;200:172-80. 
11. 
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. 
12. 
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. 
13. 
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. 
14. 
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. 
15. 
Fluzone High-Dose Package Insert. (Accessed June 11, 2019, at 
https://www.fda.gov/media/119870/download.) 
 
 
 
 
 
 
 
 

18 
 
 
 
 
Amendment Appendices 
Appendix Letter 
Title 
M 
FDA IND Exemption Letter 
N 
CDC Flu VaxTrial: Consent Form, Year 2 
O 
CDC Flu VaxTrial: Non-Randomized Fluzone® High-Dose Trivalent 
Study Consent Form 
P 
CDC Flu VaxTrial: Eligibility Screening Form, Year 2 
Q 
CDC HCP Flu VaxTrial: Enrollment Questionnaire, Year 2 
R 
CDC HCP Flu VaxTrial: Pre-vaccination Questionnaire/Vaccine 
Administration Form, Year 2 
T1 
CDC Flu VaxTrial: Blood Specimen Collection/Tracking Form 
Year 2 Study Visit 1 [day 0 defined as day of vaccination] 
T2 
CDC HCP Flu VaxTrial: Blood Specimen Collection/Tracking Form 
Year 2 Study Visit 1b [days 6-9 with day 7 as target] 
T3 
CDC Flu VaxTrial: Blood Specimen Collection/Tracking Form 
Year 2 Study Visit 2 [days 21-42 with days 21-35 as target] 
U 
EMR/Chart Extraction/Abstraction Form, Year 2