Randomized Study of the Immunogenicity and Duration of Antibody Response Against Circulating SARS-CoV-2 Variant and Influenza Viruses Following Concomitant Versus Sequential Administration of mRNA COVID-19 Vaccine and Quadrivalent Cell Culture-based Influenza Vaccine Among Children and Adults
Phase: PHASE4
Conditions: Influenza, COVID-19
Sponsor: Duke University

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
1 
 
Cooperative Agreement IP-22-004: US Platform to Measure Effectiveness of Seasonal 
Influenza, COVID-19 and other Respiratory Virus Vaccines for the Prevention of Acute Illness in 
Ambulatory Settings 
Component D: Immunologic Study of Response to Influenza and COVID-19 Virus Vaccination 
 
Study Title: Randomized study of the immunogenicity and duration of antibody response 
against circulating SARS-CoV-2 variant and influenza viruses following concomitant 
versus sequential administration of mRNA COVID-19 vaccine and quadrivalent cell 
culture-based influenza vaccine among children and adults 
Short Title: Comparative immunogenicity of concomitant vs sequential mRNA COVID-19 and 
influenza vaccinations 
 
 
Study site (Arizona State University, Tempe) Principal Investigator 
Vel Murugan (U01 IP001180-02) 
Study site (University Hospitals Cleveland Medical Center) Principal Investigator 
 Elie Saade (U01 IP001181-02) 
Study site (Washington University in St Louis) Principal Investigator 
Stacey House (U01 IP001182-02) 
Study site (University of Pittsburgh) Principal Investigator 
Richard Zimmerman (U01 IP001184-02) 
Coordinating site (Duke University) Principal Investigator 
Emmanuel B. Walter, MD, MPH (U01 IP001194-02) 
 
Centers for Disease Control and Prevention (CDC) Co-Investigators 
Jessie Chung, Brendan Flannery, Kelsey Sumner, Min Levine, Suryaprakash Sambhara, 
Shivaprakash Gangappa, Melissa Coughlin 
 
Version Number: 2 
2 October 2023 
 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
2 
 
STATEMENT OF COMPLIANCE 
• 
This trial will be conducted in compliance with the protocol, the International Conference 
on Harmonization (ICH) Guideline E6—Good Clinical Practice (GCP), and the applicable 
guidelines and regulatory requirements from the United States (US) Code of Federal 
Regulations (CFR), 45 CFR Part 46. 
• 
All study personnel with participant contact have completed Human Participants 
Protection Training. 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
3 
 
TABLE OF CONTENTS 
1 
TITLE OF PROJECT .............................................................................................................................................. 11 
1.1 
STUDY SPONSOR ................................................................................................................................................................... 11 
1.2 
INVESTIGATORS ...................................................................................................................................................................... 11 
2 
ABBREVIATIONS ................................................................................................................................................... 12 
3 
BACKGROUND ....................................................................................................................................................... 12 
3.1 
PROTOCOL SUMMARY .......................................................................................................................................................... 12 
3.2 
PROTOCOL BACKGROUND AND RATIONALE ................................................................................................................. 13 
3.3 
VACCINES BACKGROUND AND RATIONALE ................................................................................................................... 14 
3.3.1 
COVID-19 Vaccines .................................................................................................................................................... 14 
3.3.2 
Influenza Vaccines ...................................................................................................................................................... 14 
4 
STUDY OBJECTIVES AND OUTCOME MEASURES ............................................................................... 15 
4.1 
PRIMARY .................................................................................................................................................................................... 15 
4.2 
EXPLORATORY ........................................................................................................................................................................ 16 
5 
METHODS ................................................................................................................................................................. 20 
5.1 
MAIN STUDY DESIGN ............................................................................................................................................................. 20 
5.2 
LABORATORY STUDIES ........................................................................................................................................................ 21 
5.2.1 
Influenza Antibody Assays ...................................................................................................................................... 21 
5.2.2 
SARS-CoV-2 Antibody Assays ............................................................................................................................. 22 
5.2.3 
Future studies ................................................................................................................................................................ 23 
5.3 
STUDY ENROLLMENT AND WITHDRAWAL ...................................................................................................................... 23 
5.3.1 
Participant Inclusion Criteria .................................................................................................................................. 23 
5.3.2 
Participant Exclusion Criteria ................................................................................................................................. 23 
5.3.3 
Temporary Delay Criteria (Visits 1 and 2) ....................................................................................................... 24 
5.3.4 
Recruitment ..................................................................................................................................................................... 25 
5.3.5 
Enrollment ........................................................................................................................................................................ 26 
5.3.6 
Participant Withdrawal ............................................................................................................................................... 27 
5.4 
STUDY SCHEDULE, PROCEDURES, & EVALUATIONS ................................................................................................ 27 
5.4.1 
Schedule of events and data collection............................................................................................................ 27 
5.4.2 
Randomization ............................................................................................................................................................... 32 
5.4.3 
Deviation from Protocol............................................................................................................................................. 33 
5.5 
DATA COLLECTION................................................................................................................................................................. 33 
5.5.1 
Participant Demographics and Vaccine History .......................................................................................... 33 
5.5.2 
Follow-up Surveillance for Vaccine Reactogenicity and Safety and Self-Reported ARI or 
Change in Health Status Resulting in Hospitalization .............................................................................. 33 
5.5.3 
Study Product Supply, Storage, and Administration ................................................................................. 34 
5.5.4 
Biospecimens Collection & Handling ................................................................................................................. 35 
5.6 
VACCINE SAFETY AND REACTOGENICITY ..................................................................................................................... 36 
5.7 
MEDICAL RECORD EXTRACTION ...................................................................................................................................... 36 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
4 
 
5.7.1 
Prior Vaccination .......................................................................................................................................................... 36 
5.7.2 
Prior Lab-Confirmed Illness .................................................................................................................................... 37 
6 
HUMAN SUBJECTS PROTECTIONS ............................................................................................................. 37 
6.1 
INSTITUTIONAL REVIEW BOARD REVIEW ....................................................................................................................... 37 
6.2 
PARTICIPANT CONFIDENTIALITY ....................................................................................................................................... 38 
6.3 
RISKS AND BENEFITS TO PARTICIPANTS ....................................................................................................................... 39 
6.4 
RETURN OF RESULTS ........................................................................................................................................................... 41 
6.5 
COMPENSATION ...................................................................................................................................................................... 42 
7 
DATA MANAGEMENT, TRANSFER, AND SECURITY ............................................................................ 42 
7.1 
DATA MANAGEMENT AND TRANSFER ............................................................................................................................. 42 
7.2 
DATA SECURITY ...................................................................................................................................................................... 43 
8 
STATISTICAL ANALYSIS ................................................................................................................................... 44 
8.1 
STUDY POPULATIONS ........................................................................................................................................................... 44 
8.2 
ANALYSIS PLAN OF YEAR 2 COMPONENT D ................................................................................................................ 44 
REFERENCES ....................................................................................................................................................................................... 46 
LAB APPENDICES ................................................................................................................................................................ 47 
LAB APPENDIX A: RESPIRATORY SPECIMEN TESTING FOR INFLUENZA .............................................................................. 48 
LAB APPENDIX B: HUMAN SERA COLLECTION GUIDELINES FOR INFLUENZA SEROLOGY ............................................ 49 
LAB APPENDIX C: PERIPHERAL BLOOD MONONUCLEAR CELLS ........................................................................................... 50 
 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
5 
 
PROTOCOL SUMMARY 
Title:  
Randomized study of the immunogenicity and duration of antibody 
response against circulating SARS-CoV-2 variant and influenza 
viruses following concomitant versus sequential administration of 
mRNA COVID-19 vaccine and quadrivalent cell culture-based 
influenza vaccine among children and adults 
Phase: 
Phase IV 
Population: 
600 persons aged 18-64 years and 200 children aged 6-11 years for 
whom a single dose of 2023-24 influenza vaccine and a single dose 
of mRNA COVID-19 vaccine is indicated as recommended by ACIP 
Clinical Sites: 
Four study sites: Arizona State University Tempe; University 
Hospitals of Cleveland; Washington University St. Louis; University of 
Pittsburgh 
Coordinating Site: 
Duke University 
Study Duration: 
12 months total 
Enrollment: Aug — Oct 2023 
Vaccination: Aug — Nov 2023 
Blood draws: Aug 2023 — May 2024 
Active surveillance for illness: Aug 2023 — May 2024 
Laboratory testing: Nov 2023 — Jul 2024 
Participant Duration and 
Number of Participants: 
• 
Up to 10 months (Aug 2023 — May 2024)  
• 
800 total participants (600 aged 18-64 years, 200 aged 6-11 
years) at 4 sites (only two sites will enroll children aged 6-11 
years) 
Randomization: 
Adults aged 18-64 years, 2:1:1 permuted block randomization 
i. 
Concomitant ccIIV4 + COVID-19 mRNA (day 0)  
[n = 300] 
ii. 
Sequential ccIIV4 (day 0), COVID-19 mRNA (day 28) [n = 
150] 
iii. 
Sequential COVID-19 mRNA (day 0), ccIIV4 (day 28) [n = 
150] 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
6 
 
 
Children aged 6-11 years, 2:1:1 permuted block randomization 
i. 
Concomitant ccIIV4 + Moderna COVID-19 mRNA (day 0)  
[n = 100]  
ii. 
Sequential ccIIV4 (day 0), Moderna COVID-19 mRNA (day 
28) [n = 50] 
iii. 
Sequential Moderna COVID-19 mRNA (day 0), ccIIV4 (day 
28) [n = 50] 
 
Description of Study 
Procedures: 
This study is a randomized immunogenicity study in an enrolled 
cohort with active surveillance for acute respiratory illness (ARI).  
During this study, participants will be randomly assigned to receive 
an approved quadrivalent influenza vaccine (Flucelvax [ccIIV4]) and 
mRNA COVID-19 vaccine (Moderna) either concomitantly (given in 
contralateral arms) or sequentially (28 days apart).   
Blood samples from participants will be collected for measurement of 
biomarkers of immune response at baseline (visit 1; day 0), post-
vaccination 1 (visit 2; day 28), post-vaccination 2 (visit 3; day 56), 
and post-season (visit 4; day 180). Serum and peripheral blood 
mononuclear cells (PBMC) and plasma (target to collect PBMC and 
plasma samples at every visit from a subset of approximately 250 
participants; 200 adults and 50 children) samples will be isolated 
from whole blood and tested for biomarkers of vaccine 
immunogenicity, and duration of antibody responses. 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
7 
 
Participants will receive electronic surveys via email or text message 
three days after each vaccination asking about vaccine reactogenicity 
and weekly asking about change in health status and new ARI 
symptoms; those reporting illness may be asked to provide a 
respiratory swab for laboratory testing for influenza and SARS-CoV-2 
and up to 2 additional blood draws (acute [< 10 days after symptom 
onset] and convalescent [28 days after acute visit if lab-confirmed 
positive for influenza or SARS-CoV-2]). 
Specimen Collection: 
Adults aged 18-64 years, n = 600 
i. 
Sera [n = 600] 
ii. 
PBMC [target n = 200] 
Children aged 6-11 years, n = 200 
i. 
Sera [n = 200] 
ii. 
PBMC [target n = 50] 
All participants will present for 4 visits: 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
8 
 
If reporting symptoms, participants may be asked to present for two additional visits:
 
Objectives: 
Primary Objective:  
i. 
Determine baseline and post-vaccination influenza antibody titers 
by hemagglutination inhibition (HAI) (in a subset) against ccIIV4 
vaccine antigens following concomitant administration with 
mRNA vaccine or alone  
Exploratory Objective:  
ii. 
Determine baseline and post-vaccination influenza antibody titers 
by microneutralization (MN) (in a subset) against ccIIV4 vaccine 
antigens following concomitant administration with mRNA 
vaccine or alone  
iii. Determine baseline and post-vaccination SARS-CoV-2 antibody 
binding titers and ACE2 binding inhibition and viral neutralization, 
on a subset of specimens, against Moderna mRNA COVID-19 
vaccine antigens following concomitant administration with ccIIV4 
or alone 
iv. Determine duration and rate of waning of influenza HAI and MN 
antibody titers against ccIIV4 vaccine antigens and circulating 
influenza viruses 
v. Determine duration of and rate of waning of SARS-CoV-2 binding 
antibody and neutralization titers (in a subset of specimens) 
against Moderna mRNA COVID-19 vaccine antigens and 
circulating SARS-CoV-2 variants 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
9 
 
vi. Determine influenza HAI and MN antibody titers against 
circulating influenza viruses during baseline and post-vaccination  
vii. Determine SARS-CoV-2 antibody binding titers and viral 
neutralization titers (in a subset of samples) against circulating 
SARS-CoV-2 variants during baseline and post-vaccination 
viii. Compare the frequencies of antigen-specific T and B cells 
utilizing high-dimensional flowcytometric analysis of innate and 
adaptive immune cell phenotype characterization using 
fluorochrome-conjugated antigen tags, and intracellular cytokine 
staining to 2023-24 ccIIV4 and mRNA COVID-19 vaccines 
administered either concomitantly or sequentially, 28 days apart 
ix. Compare transcriptome, B cell receptor (BCR) and T cell 
receptor (TCR) diversity of antigen-specific cells and assess 
serological antibody repertoire (clonotypes) using 10X genomics 
platform, and liquid chromatography/mass spectroscopy in a 
subset of participants 
x. Investigate the waning of antigen-specific B cells and antibody 
clonotypes in a subset of participants 
xi. Describe safety of concomitant versus sequential administration 
of ccIIV4 and mRNA COVID-19 vaccination 
Outcome Measures: 
Primary Outcome Measures:  
i. 
The percent seroconversion and seroprotective titer, the 
geometric mean titer (GMT), and geometric mean fold rise 
(GMFR) in titer for each vaccination group across HAI titers for 
each 2023-24 influenza vaccine antigen 
Exploratory Outcome Measures: 
ii. 
The percent seroconversion and seroprotective titer, the 
geometric mean titer (GMT), and geometric mean fold rise 
(GMFR) in titer for each vaccination group across MN titers for 
each 2023-24 influenza vaccine antigen 
iii. GMT for SARS-CoV-2 binding antibody titer and viral 
neutralization titer (for a subset of samples) pre- and post-
vaccination with mRNA COVID-19 vaccine 
iv. GMT and geometric mean fold change in HAI or MN titers at 
baseline, post-vaccination, mid-season, and post-season against 
2023-24 circulating influenza virus and influenza vaccine 
antigens 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
10 
 
v. GMT and geometric mean fold change for SARS-CoV-2 binding 
antibody and the neutralization titer (for a subset of samples) at 
baseline, post-vaccination, mid-season, and post-season against 
2023-24 circulating SARS-CoV-2 virus and mRNA COVID-19 
vaccine antigens 
vi. Percent seroconversion and seroprotective titer, GMT, GMFR in 
HAI or MN titers at baseline and post-vaccination against 2023-
24 circulating influenza virus antigens 
vii. GMT and GMFR for SARS-CoV-2 binding antibody and the 
neutralization titer (for a subset of samples) at baseline and post-
vaccination against 2023-24 circulating SARS-CoV-2 virus 
antigens 
viii. The frequency of antigen-specific T and B cells to influenza and 
SARS-CoV-2 at baseline, post-vaccination, mid-season and 
post-season, high-dimensional flowcytometric analyses of innate 
and adaptive immune cells, and ELISPOT evaluation for SARS-
CoV-2 memory B cells 
ix. BCR and TCR diversity and clonal dominance (antibody 
clonotypes) of serological repertoire in a subset of participants 
x. The frequency of antigen-specific B cells and clonal dominance 
(antibody clonotypes) to influenza and SARS-CoV-2 at baseline, 
post-vaccination, mid-season and post-season in a subset of 
participants 
xi. Number and percent of participants reporting fever or injection 
site and systemic reactions during the first 3 days following each 
vaccination and a change in health requiring hospital admission 
during the study period 
Estimated Time to Complete 
Enrollment: 
3 months: Aug — Oct 2023 
 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
11 
 
1 
TITLE OF PROJECT 
Randomized study of the immunogenicity and duration of antibody response against circulating 
SARS-CoV-2 variant and influenza viruses following concomitant versus sequential 
administration of mRNA COVID-19 vaccine and quadrivalent cell culture-based influenza 
vaccine among children and adults 
Short Title: Comparative immunogenicity of concomitant vs sequential mRNA and influenza 
vaccination 
1.1 
Study Sponsor 
Influenza Division, U.S. Centers for Disease Control and Prevention  
1.2 
Investigators 
U.S. Centers for Disease Control and Prevention  
Min Levine, PhD  
Suryaprakash Sambhara, DVM PhD 
Shivaprakash Gangappa, DVM PhD 
Melissa Coughlin, PhD 
Jessie Chung, MPH 
Brendan Flannery, PhD 
Kelsey Sumner, PhD 
 
Study Partners 
University of Pittsburgh 
Washington University in St. Louis 
University Hospitals Cleveland Medical Center 
Arizona State University, Tempe 
Duke University 
 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
12 
 
2 
ABBREVIATIONS 
ACE2  
Angiotensin Converting Enzyme 2 
ARI  
 
Acute Respiratory Illness  
BCR 
 
B cell receptor 
ccIIV4  
Flucelvax Quadrivalent 
GMT  
Geometric Mean Titers  
GMFR  
Geometric Mean Fold Rise 
HAI 
 
Hemagglutination Inhibition 
IRB  
 
Internal Review Board  
LAR 
 
Legally Authorized Representative 
MN 
 
Microneutralization 
PBMC  
Peripheral Blood Mononuclear Cells 
RT-PCR  
Reverse-Transcription Polymerase Chain Reaction 
SAE 
 
Serious Adverse Event  
TCR 
 
T cell receptor 
3 
BACKGROUND 
 
3.1 
Protocol Summary 
This protocol describes an immunologic study of response to influenza and SARS-CoV-2 
vaccination across four of the US Influenza Vaccine Effectiveness (Flu VE) Network study sites.  
This protocol meets the objectives of component D in the Flu VE Network Cooperative 
Agreement (IP16-004), including enrollment of eligible persons into a cohort, administration of a 
licensed or authorized under emergency use authorization (EUA) mRNA COVID-19 vaccine 
(Moderna COVID-19 Vaccine) and licensed quadrivalent cell culture-based influenza vaccine 
(Flucelvax; ccIIV4), collection and testing of blood specimens, administration of enrollment 
surveys, protection of personally identifiable information, transfer of a HIPAA-limited dataset to 
the Network Coordinating Center (NCC) and CDC, and creation of a coded individual-level 
dataset according to a data management agreement.   

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
13 
 
3.2 
Protocol Background and Rationale 
Current guidance concerning administration of U.S.-approved or -authorized COVID-19 
vaccines indicates that these vaccines may be given with influenza vaccines.1 
In accordance with general best practices, routine administration of all age-appropriate doses of 
vaccines concomitantly is recommended for children, adolescents, and adults for whom no 
specific contraindications exist at the time of the healthcare visit.  Providers should be aware of 
the potential for increased reactogenicity with concomitant administration and should consult 
updated CDC guidance as more information becomes available.   
Extensive experience with non-COVID-19 vaccines has demonstrated that immunogenicity and 
adverse event profiles are generally similar when vaccines are administered concomitantly as 
when they are administered alone. 
Studies that compared coadministration of COVID-19 vaccines and seasonal influenza vaccines 
with separate administration of these vaccines found similar levels of immunogenicity and 
similar or slightly higher reactogenicity;2-4 no specific safety concerns were identified. 
During the 2023-2024 influenza season, CDC plans a randomized influenza vaccine 
immunogenicity study to compare immune responses to concomitant vs sequential 
administration of licensed or authorized vaccines for COVID-19 and influenza in select age 
groups.  For future seasons, immunogenicity studies may enroll different age groups each 
season depending on the vaccines to be compared.  To study immune responses to 
vaccination, each study site will recruit and enroll up to 250 participants who are eligible to 
receive specific influenza and COVID-19 vaccines and for whom the vaccines are 
recommended.  Study sites will implement common protocols developed in collaboration with 
CDC.  Data will be combined across sites so that all sites will contribute to a total anticipated 
sample size of 800 participants for comparisons of immune responses by vaccine administration 
schedule.  Investigators will collect, process, store, and ship sera, plasma, and PBMC 
specimens to a designated CDC laboratory for testing. Some additional specimen testing may 
be performed by a CDC-approved site.  
Investigators will participate in cross-site decisions on specific influenza and COVID-19 
vaccines to be administered, populations to be examined, and interpretation of influenza 
vaccine response findings.  Enrolled participants may either reside or work in settings that 
facilitate active weekly surveillance for ARI and influenza virus and SARS-CoV-2 infection.  To 
improve representation of specific population groups, study sites may use enrollment quotas for 
participant demographics including race and ethnicity.  To identify influenza virus and SARS-
CoV-2 infections, participants will respond to weekly automated electronic surveys to report new 
ARI symptoms and test results, if applicable.  Participants may have the opportunity to provide 
specimens for standard point of care respiratory testing and up to 2 additional blood draws (at 
acute illness and 28 days after PCR-confirmed influenza or SARS-CoV-2 infection) if they report 
qualifying ARI symptoms during weekly surveillance.  Participants will provide scheduled blood 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
14 
 
samples at enrollment (before vaccination) and at several timepoints after vaccination to 
measure and monitor humoral and cell-mediated immune (CMI) responses to vaccination.  
Participants will respond to an automated electronic survey 3 days following each vaccination 
visit to report the occurrence of fever, injection site reactions, systemic reactions, the severity of 
reactions and their impact on health.  The weekly automatic electronic survey will also request 
participants to report the occurrence of any change in health requiring hospitalization. 
3.3 
Vaccines Background and Rationale 
3.3.1 COVID-19 Vaccines 
COVID-19 vaccines available in the United States are effective at protecting people from getting 
seriously ill, being hospitalized, and dying from SARS-CoV-2.  U.S.-licensed or authorized under 
EUA mRNA COVID-19 vaccine used in this study will contain the recommended composition to 
be used as a booster dose at the time of enrollment.  Currently, CDC recommends one bivalent 
vaccine dose for everyone aged 6 years and older that have not previously been vaccinated or 
have only received monovalent doses if it has been at least 2 months since their last dose.5   
Two COVID-19 vaccine manufacturers, Pfizer and Moderna, have developed updated (bivalent) 
COVID-19 boosters.  The updated (bivalent) boosters are called “bivalent” because they protect 
against both the original or ancestral virus that causes COVID-19 and the Omicron variant BA.4 
and BA.5.  Primary series doses and previous boosters are called “monovalent” because they 
were designed to protect against the original virus that causes COVID-19.  They also provide 
some protection against Omicron, but not as much as the updated (bivalent) boosters.  The 
virus that causes COVID-19 has changed over time.  The different versions of the virus that 
have developed over time are called variants.  As variants continue to evolve it remains unclear 
at this time what the composition of future booster doses of COVID-19 vaccine.  The latest 
Moderna COVID-19 vaccine will be administered in this trial. 
3.3.2 Influenza Vaccines 
U.S.-licensed 2023-2024 cell culture-based influenza vaccine used in this study will contain 
components from four influenza viruses antigenically similar to those recommended by FDA for 
cell culture–based vaccines: an influenza A(H1N1)pdm09 virus; an influenza A(H3N2) virus; an 
influenza B/Victoria lineage virus; and an influenza B/Yamagata lineage virus.  Most commonly, 
seasonal influenza vaccines are produced using egg-grown viral isolates; however, this 
approach could lead to individual’s receiving a boost in antibody protection against egg-adapted 
antigens which may vary from circulating influenza viruses.1-4  Previous work in adults has 
suggested that vaccination with cell culture-based derived vaccines could improve immune 
response to circulating influenza viruses compared to traditional egg-based vaccines.6-8 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
15 
 
A cell culture-based influenza vaccine, Flucelvax Quadrivalent (ccIIV4), will be administered in 
this trial.9 ccIIV4 may be administered concomitantly or sequentially with other inactivated 
vaccines.  Injectable vaccines that are given concomitantly should be administered at separate 
anatomic sites. 
Standard-dose, nonadjuvanted cell culture-based vaccine, Flucelvax Quadrivalent (ccIIV4) 
contains 15 µg of HA per vaccine virus in a 0.5-mL dose.  ccIIV4 is approved for persons aged 
≥6 months.  Egg-based and cell culture-based vaccines differ in the substrate in which 
reference vaccine viruses supplied to the manufacturer are propagated in quantities sufficient to 
produce the needed number of doses of vaccine.  For Flucelvax Quadrivalent, reference 
vaccine viruses are propagated in Madin-Darby canine kidney cells instead of eggs.   
4 
STUDY OBJECTIVES AND OUTCOME MEASURES 
OBJECTIVE 
OUTCOME 
4.1 
Primary 
Determine baseline and post-vaccination 
influenza antibody titers by 
hemagglutination inhibition (HAI), in a 
subset of samples, against vaccine antigens 
following concomitant administration with 
mRNA vaccine or alone  
i. 
Proportion of participants in each 
vaccination group with a seroprotective 
HAI (in a subset of samples) titer 
(≥1:40) pre- and post-ccIIV4 
immunization for each 2023-24 
influenza vaccine antigen  
ii. Proportion of participants in each 
vaccination group achieving HAI (in a 
subset of samples) seroconversion (a 
titer ≥1:40 following ccIIV4 if the 
baseline titer is <1:10 or a four-fold rise 
in titer if the baseline titer is >1:10) 
following ccIIV4 vaccination for each 
2023-24 influenza vaccine antigen 
iii. HAI (in a subset of samples) GMT for 
each 2023-24 influenza vaccine 
antigen pre- and post-ccIIV4 in each 
vaccination group 
iv. GMFR in HAI (in a subset of samples) 
titers for each 2023-24 influenza 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
16 
 
vaccine antigen in each vaccination 
group 
4.2 
Exploratory 
Determine baseline and post-vaccination 
influenza antibody titers by 
microneutralization (MN), in a subset of 
samples, against vaccine antigens following 
concomitant administration with mRNA 
vaccine or alone 
i. 
Proportion of participants in each 
vaccination group with a seroprotective 
MN (in a subset of samples) titer 
(≥1:40) pre- and post-ccIIV4 
immunization for each 2023-24 
influenza vaccine antigen  
ii. Proportion of participants in each 
vaccination group achieving MN (in a 
subset of samples) seroconversion (a 
titer ≥1:40 following ccIIV4 if the 
baseline titer is <1:10 or a four-fold rise 
in titer if the baseline titer is >1:10) 
following ccIIV4 vaccination for each 
2023-24 influenza vaccine antigen 
iii. HAI (in a subset of samples) MN for 
each 2023-24 influenza vaccine 
antigen pre- and post-ccIIV4 in each 
vaccination group 
i. 
GMFR in MN (in a subset of samples) 
titers for each 2023-24 influenza 
vaccine antigen in each vaccination 
group 
Determine baseline and post-vaccination 
SARS-CoV-2 antibody binding titers and 
ACE2 binding inhibition and viral 
neutralization, on a subset of specimens, 
against mRNA COVID-19 vaccine antigens 
following concomitant administration with 
ccIIV4 or alone 
i. 
GMT for SARS-CoV-2 binding antibody 
titer and viral neutralization titer (for a 
subset of samples) pre- and post-
vaccination with mRNA COVID-19 
vaccine 
Determine duration and rate of waning of 
influenza HAI and MN antibody titers 
i. 
HAI and MN GMT for each 2023-24 
influenza vaccine and circulating 
influenza virus antigens at visit 1, visit 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
17 
 
against ccIIV4 vaccine antigens and 
circulating influenza viruses 
2, visit 3, and visit 4 for each 
vaccination group  
ii. Geometric mean fold change in HAI 
and MN titers for each vaccination 
group from visit 1 to visit 2, visit 3, and 
visit 4 for each 2023-24 influenza 
vaccine and circulating influenza virus 
antigen 
Determine duration of and rate of waning of 
SARS-CoV-2 antibody titers and viral 
neutralization titers, on a subset of samples, 
against mRNA COVID-19 vaccine antigens 
and circulating SARS-CoV-2 variants 
i. 
SARS-CoV-2 binding antibody and viral 
neutralization GMT for each mRNA 
COVID-19 vaccine and circulating 
SARS-CoV-2 virus antigens at visit 1, 
visit 2, visit 3, and visit 4 for each 
vaccination group  
ii. Geometric mean fold change in SARS-
CoV-2 and viral neutralization (in a 
subset of samples) titers for each 
vaccination group from visit 1 to visit 2, 
visit 3, and visit 4 for each COVID-19 
Moderna mRNA COVID-19 vaccine 
and circulating SARS-CoV-2 virus 
antigen 
Determine influenza HAI and MN antibody 
titers against circulating influenza viruses 
during baseline and post-vaccination  or 
post-infection 
i. 
Proportion of participants in each 
vaccination group with a seroprotective 
titer (≥1:40) pre- and post-ccIIV4 
immunization (visits 1-3) and/or pre- 
and post-influenza infection (acute and 
convalescent visits), if applicable, for 
each 2023-24 circulating influenza virus 
antigen  
ii. Proportion of participants in each 
vaccination group achieving 
seroconversion (a titer ≥1:40 following 
ccIIV4 if the baseline titer is <1:10 or a 
four-fold rise in titer if the baseline titer 
is >1:10) following ccIIV4 vaccination 
(visit 1 to visits 2-3) and/or influenza 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
18 
 
infection (acute to convalescent visits), 
if applicable, for each 2023-24 
circulating influenza virus antigen 
iii. HAI and MN GMT for each 2023-24 
circulating influenza virus antigen pre- 
and post-ccIIV4 (visits 1-3) and/or pre- 
and post-influenza infection (acute and 
convalescent visits), if applicable, in 
each vaccination group 
iv. GMFR in HAI or MN titers for each 
vaccination group following ccIIV4 
vaccination (visit 1 to visits 2-3) and/or 
influenza infection (acute to 
convalescent visits), if applicable, for 
each 2023-24 circulating influenza virus 
antigen 
Determine SARS-CoV-2 antibody binding 
titers and viral neutralization titers (in a 
subset of samples) against circulating 
SARS-CoV-2 variants during baseline and 
post-vaccination 
i. 
SARS-CoV-2 binding antibody and viral 
neutralization (for a subset of samples) 
titers GMT at pre- and post-vaccination 
(visits 1-3) and/or pre- and post-SARS-
CoV-2 infection (acute and 
convalescent visits), if applicable, 
against 2023-24 circulating SARS-CoV-
2 virus antigens 
ii. GMFR in SARS-CoV-2 binding 
antibody and viral neutralization (for a 
subset of samples) titers following 
mRNA COVID-19 vaccination (visit 1 to 
visits 2-3) and/or SARS-CoV-2 infection 
(acute to convalescent visits), if 
applicable, against 2023-24 circulating 
SARS-CoV-2 virus antigens 
Compare the frequencies of antigen-specific 
T and B cells utilizing conventional 
ELISPOT assays, high-dimensional 
flowcytometric analysis of innate and 
adaptive immune cell phenotype 
i. 
The frequency of antigen-specific T and 
B cells to influenza and SARS-CoV-2 at 
visit 1, visit 2, visit 3, visit 4, and acute 
and convalescent visits, if applicable  

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
19 
 
characterization using fluorochrome-
conjugated antigen tags, and intracellular 
cytokine staining to 2023-24 ccIIV4 and 
mRNA COVID-19 vaccines administered 
either concomitantly or sequentially, 28 
days apart 
ii. High-dimensional flowcytometric 
analyses of innate and adaptive 
immune cells 
iii. ELISPOT evaluation for SARS-CoV-2 
memory B cells 
Compare transcriptome, B cell receptor 
(BCR) and T cell receptor (TCR) diversity of 
antigen-specific cells and assess serological 
antibody repertoire (clonotypes) using 10X 
genomics platform, and liquid 
chromatography/mass spectroscopy in a 
subset of participants 
i. 
BCR and TCR diversity and clonal 
dominance (antibody clonotypes) of 
serological repertoire in a subset of 
participants  
Investigate the waning of antigen-specific B 
cells and antibody clonotypes in a subset of 
participants 
i. 
The frequency of antigen-specific B 
cells and clonal dominance (antibody 
clonotypes) to influenza and SARS-
CoV-2 at visit 1, visit 2, visit 3, visit 4, 
and acute and convalescent visits (if 
applicable) in a subset of participants 
Describe safety of concomitant versus 
sequential administration of ccIIV4 and 
mRNA COVID-19 vaccination 
ii. Number and percent of participants 
reporting fever or injection site and/or 
systemic reactions during the first 3 
days following each vaccination visit 
stratified by severity and, for those 
reporting symptoms, their impact on 
health 
iii. Number and percent of participants 
reporting a change in health status 
requiring hospital admission (including 
a description of the event) during the 
study period 
 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
20 
 
5 
METHODS 
5.1 
Main study design 
This study is a randomized comparative immunogenicity study in an enrolled cohort. During this 
study, participants will be randomly assigned to receive an approved quadrivalent cell culture-
based influenza vaccine (ccIIV4) and mRNA COVID-19 vaccine (Moderna) either concomitantly 
or sequentially, 28 days apart.  Participants (aged 6-11 years and 18-64 years) will be enrolled 
in the 2023-2024 influenza season. Only two of the sites (Washington University in St. Louis 
and University Hospitals Cleveland Medical Center) will enroll children aged 6-11 years. 
Individuals will be enrolled who have not received their 2023-2024 influenza vaccine and have 
not received any COVID-19 vaccine in the preceding 6 months (i.e.  at least 6 months have 
passed since their last COVID-19 vaccination).  
Participants will be enrolled if one dose of 2023-2024 influenza vaccine and a single booster 
dose of mRNA COVID-19 vaccine are indicated for current vaccination according to ACIP 
recommendations.  To qualify for the study, children aged 8 years and younger must only need 
to receive a single dose of ccIIV4 per ACIP recommendations for the 2023-2024 season.  
mRNA COVID-19 vaccines recommended by CDC and/or authorized or approved by the FDA at 
the time of enrollment may include updated SARS-CoV-2 viral components which may be 
included in this study if authorized or approved by the FDA and recommended by the CDC. 
Demographic and health data (including influenza and COVID-19 vaccination and infection 
history) will be collected upon enrollment and verified after enrollment via electronic medical 
record extraction.  Enrolled participants will be randomized to one of the following interventions 
(2:1:1) using a permuted block method stratified by age group (aged 6-11 years and 18-64 
years) and site: (i) concomitant administration of the mRNA COVID-19 vaccine (Moderna) and 
quadrivalent influenza vaccine (ccIIV4); (ii) sequential administration of the quadrivalent 
influenza vaccine (ccIIV4) at Visit 1 (day 0) and the mRNA COVID-19 vaccine (Moderna) at Visit 
2 (day 28); (iii) sequential administration of the mRNA COVID-19 vaccine (Moderna) at Visit 1 
(day 0) followed by the quadrivalent influenza vaccine (ccIIV4) at Visit 2 (day 28).  Whole blood 
samples to isolate sera, PBMC and plasma (target to collect PBMC and plasma samples at 
every visit from a subset of 250 participants; 200 adults and 50 children) will be collected prior to 
vaccination administration at Visit 1 (day 0) and Visit 2 (day 28).  Participants will not be blinded 
to vaccine group and will receive two vaccines in separate limbs (for the concomitant 
vaccination [group i]) or one vaccine at Visit 1 (day 0) and a second vaccine at Visit 2 (day 28) 
(for sequential vaccination [groups ii and iii]) in an unblinded manner. 
Participants will provide a blood sample for isolation of serum, PBMC, and plasma (target to 
collect PBMC and plasma samples at every visit from a subset of 250 participants; 200 adults 
and 50 children) at Visit 3 (day 56; post-vaccination 2) and Visit 4 (day 180; end of local flu 
circulation).  If participants exhibit ARI during the study period, they may be asked to present for 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
21 
 
an acute (<10 days after symptom onset) and convalescent (28 days after acute visit) visit to 
provide a nasal swab for viral testing (acute visit only) and blood sample for isolation of serum, 
PBMC, and plasma (acute and convalescent visit for those with positive viral molecular test for 
influenza or SARS-CoV-2).  Influenza virus and SARS-CoV-2 serologic and cellular immunity 
testing will be conducted at CDC and/or a CDC-approved site. 
5.2 
Laboratory Studies 
Blood samples from participants will be collected for measurement of biomarkers of immune 
response at Visit 1 (day 0; baseline), Visit 2 (day 28; post-vaccination 1), Visit 3 (day 56; post-
vaccination 2), and Visit 4 (day 180; post-season).  Sera, PBMC, and plasma samples will be 
tested for biomarkers of vaccine immunogenicity, duration of antibody responses, and immune 
response to circulating SARS-CoV-2 variants and influenza virus strains. 
5.2.1 Influenza Antibody Assays 
5.2.1.1   Influenza Hemagglutination Inhibition and Microneutralization Assays 
Sera collected from participants at Visit 1 (day 0), Visit 2 (day 28), Visit 3 (day 56), Visit 4 (day 
180), acute visit (<10 days after symptom onset) and convalescent visit (28 days after Acute 
Visit) will be stored at -20 C or colder (collection and storage described in detail in Lab Appendix 
B) and tested for influenza immunologic assays including hemagglutination inhibition (HAI) and 
microneutralization (MN) antibody titers.  HAI and MN antibody titers will be compared between 
groups receiving COVID-19 and ccIIV4 concomitantly or alone for each of the four 2023-2024 
recommended influenza vaccine strains for the cell culture-based vaccine (ccIIV4), as well as 
against cell culture-based propagated circulating influenza viruses identified during the 2023-
2024 influenza season.  Participants will not receive individual HAI or MN antibody titer results; 
these are not routinely used in clinical practice. 
5.2.1.2   Influenza Cell Mediated Immunity Assays 
PBMCs and plasma will be isolated from whole blood samples collected from participants at 
Visit 1 (day 0), Visit 2 (day 28), Visit 3 (day 56), Visit 4 (day 180), Acute Visit (<10 days after 
symptom onset) and Convalescent Visit (28 days after Acute Visit) to be stored for influenza 
immunologic assays.  PBMCs will be stored in liquid nitrogen and plasma will be stored at -80 C 
(collection and storage described in detail in Lab Appendix C).  PBMC and plasma isolates will 
be shipped to CDC and the CDC influenza immunology laboratory will transfer the samples to 
the identified testing laboratories.  PBMCs will be tested to assess the frequencies of H1-, H3-, 
B- (Yamagata and Victoria) specific memory B cells as well as Groups 1, 2 and B stem-specific 
memory B cells using fluorochrome-conjugated influenza HA and Stem tags.  In addition, the 
frequencies of follicular helper T cells and antigen-specific T cells post-stimulation with influenza 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
22 
 
viral antigens or influenza viruses will be assessed by high-dimensional flowcytometry and 
intracellular cytokine staining.  PBMCs and plasma from a subset of participants will be tested 
for transcriptome, BCR, TCR and antibody repertoire using single cell genomics with 10X 
Genomic platform and plasma antibody repertoire with LC/MS.   
5.2.2 SARS-CoV-2 Antibody Assays 
5.2.2.1   Quantitative SARS-CoV-2 antibody binding assessment 
Sera isolated from whole blood samples collected from participants at Visit 1 (day 0), Visit 2 
(day 28), Visit 3 (day 56), Visit 4 (day 180), acute Visit (<10 days after symptom onset) and 
convalescent Visit (28 days after Acute Visit) will be stored and tested for SARS-CoV-2 binding 
antibody using the Meso Scale Discovery (MSD) assay.  Sera will be assayed for the presence 
of SARS-CoV-2 antibody at CDC. Participants will not receive individual COVID-19 antibody 
assay results; these are not routinely used in clinical practice. 
5.2.2.2   Quantitative SARS-CoV-2 assessments 
Sera will be isolated from whole blood samples collected from participants at Visit 1 (day 0), 
Visit 2 (day 28), Visit 3 (day 56), Visit 4 (day 180), Acute Visit (<10 days after symptom onset) 
and Convalescent Visit (28 days after Acute Visit) to be stored for sera COVID-19 neutralization 
assays. ACE2 binding inhibition in sera will be measured using MSD and percent inhibition 
compared between groups receiving COVID-19 and ccIIV4 simultaneously or sequentially for 
each variant tested. COVID-19 neutralizing antibody titers will be compared in a subset of 
samples between groups receiving COVID-19 and ccIIV4 simultaneously or sequentially for 
each variant tested.  Participants will not receive individual COVID-19 antibody titer results; 
these are not routinely used in clinical practice. 
5.2.2.3   SARS-CoV-2 Cell Mediated Immunity Assays 
PBMCs and plasma isolated from whole blood samples collected from participants at Visit 1 
(day 0), Visit 2 (day 28), Visit 3 (day 56), Visit 4 (day 180), Acute Visit (<10 days after symptom 
onset) and Convalescent Visit (28 days after Acute Visit) will be stored and tested for SARS-
CoV-2 immunologic assays to evaluate the memory response to SARS-CoV-2. PBMCs will be 
evaluated to detect the presence of SARS-CoV-2 specific CD4+ and CD8+ memory T cells by 
high-dimensional flowcytometry and intracellular cytokine staining. The memory B cell 
population specific to SARS-CoV-2 antigens will be evaluated by ELISPOT. Participants will not 
receive individual COVID-19 antibody results; these are not routinely used in clinical practice.    

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
23 
 
5.2.3 Future studies 
In addition to the specified analyses described thus far, there may be other tests or assays, 
including antibody binding assays, that have yet to be identified that may be important for 
interpreting our study findings or of relevance to vaccine outcomes.  Additional laboratory 
assays may test for antibodies against other bacteria or viruses, markers of inflammation, or 
used in research on the health of the participants.  Specimens banked for use in other studies 
will be linked to information (including identifying information) that participants provided to the 
study.  Participants/participant’s legally authorized representative (LAR) must agree to potential 
future use of samples in order to be in the study.  No human genetic tests will be performed on 
samples collected in this study.  Because it is unknown if future testing will be of any utility, 
results of future testing will not be provided.   
5.3 
Study Enrollment and Withdrawal  
5.3.1 Participant Inclusion Criteria 
Participants who meet all of the following criteria and consent to randomization to one of three 
vaccine scheme groups will be eligible to participate in this interventional study: 
i. 
Healthy children aged 6-11 years and healthy adults aged 18-64 years that have not 
received the current season’s influenza vaccine or a mRNA COVID-19 vaccine in the 
past 6 months and have previously received at least a single dose of any US approved 
or authorized  COVID-19 vaccine 
ii. 
English or Spanish literate 
iii. 
Email or text message capability for weekly follow-up 
iv. 
Intention of receiving influenza vaccine and mRNA COVID-19 vaccine based on ACIP-
CDC guidelines 
v. 
Willing to provide written/electronic informed consent 
vi. 
Intention of being available for entire study period and able to complete all relevant study 
procedures, including follow-up phone calls and clinic visits 
5.3.2 Participant Exclusion Criteria 
Participants who meet any of the following criteria or do not consent to receiving both vaccines 
at enrollment, or one 28 days later, will not be eligible to participate in this study: 
i. 
Self-reported COVID-19 infection within 3 months prior to enrollment 
ii. 
Received COVID-19 vaccine within 6 months prior to enrollment 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
24 
 
iii. 
Received influenza vaccine during the respective influenza season in which they are 
being enrolled 
iv. 
< 9 years of age and recommended to receive two doses of IIV4 during the respective 
influenza season in which they are being enrolled  
v. 
History of severe allergic reaction after a previous dose of any influenza or COVID-19 
mRNA vaccine; or to an influenza or COVID-19 mRNA vaccine component 
vi. 
Receipt of any licensed vaccine within 6 weeks prior to enrollment in this study or 
planning receipt of any vaccines within 4 weeks after the receipt of the second vaccine 
dose administered during study procedures 
vii. 
Has an immunocompromising condition or taking immunosuppressive medication* 
* Received oral, intramuscular or intravenous systemic immunosuppressants, or immune 
modifying drugs for >14 days in total within 6 months prior to any study vaccine dose (for 
corticosteroids ≥ 20 mg/day of prednisone equivalent).  
** Note: Topical medications are allowed 
viii. 
Received immunoglobulin, SARS-CoV-2 immunoglobulin, SARS-CoV-2 monoclonal 
antibody, or blood-derived products, within 3 months prior any study vaccine dose. 
ix. 
History of Guillain-Barré syndrome  
x. 
History of myocarditis or pericarditis  
xi. 
History of multisystem inflammatory syndrome in children (MIS-C) or adults (MIS-A) 
xii. 
Currently pregnant, planning to become pregnant within the first three months of the 
study per participant self-report or likely to be pregnant per screening criteria 
xiii. 
Bleeding disorder diagnosed by a healthcare provider or bleeding difficulties with 
intramuscular injections or blood draws. 
xiv. 
Has injury or other reason why deltoid site on both arms cannot be used for vaccinations 
xv. 
Any condition which, in the opinion of the investigators, may pose a health risk to the 
participant or interfere with the evaluation of the study objectives 
5.3.3 Temporary Delay Criteria (Visits 1 and 2) 
Participants who meet any of the following criteria will be subject to temporary delay in protocol: 
i. 
History of febrile illness (> 100.0°F or 37.8°C) within the past 72 hours prior to vaccine 
administration 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
25 
 
5.3.4 Recruitment 
Physical distancing and other COVID-19 control measures will be incorporated into all study 
activities according to state and local guidance.  The study will use multiple recruitment methods 
to reach potential participants within the study catchment area.  These recruitment methods will 
be informed by site experience with their local populations.  Sites will be asked to make efforts 
to engage, partner with, and recruit from communities of color to recruit a participant population 
whose self-reported race and ethnicity distribution approximates the racial and ethnic 
composition of the United States based on US Census data.  Each site will aim to enroll 250 
participants. 
5.3.4.1   University of Pittsburgh 
Directly approaching potential participants (in-person) 
Medical records will be reviewed by staff who have legitimate access to this information.  For 
example, pediatric GAP nurses already have privileges to see and access data at Pediatric care 
sites.  The study script would be read to a potentially eligible participant who could decide if they 
want to hear more about the study. 
Email/Listserv/Electronic Mailing List, Letters sent to potential participants, telephone 
scripts, Registries (remote initial contact) 
Recruitment will include participants from prior research studies (STUDY19040242, 
STUDY22040177, STUDY22100188) or registries (STUDY20040088 and Pitt+Me) who agreed 
to be contacted regarding research participation as well as mailing lists through the University of 
Pittsburgh employees (Read Green employee newsletter email, for example). 
If contact is by mail (prior research studies), a study recruitment letter will be sent to potential 
participants meeting the inclusion criteria.  In the recruitment letter, we will briefly describe study 
activities and provide contact information for individuals interested in potential participation. 
If contact is made by text, email, phone, or voicemail (these three methods will be used for 
those both previously enrolled and from the registry who have not previously enrolled), a brief 
message will be used and a script is attached. 
Those who agree to be screened after the study is described to them can be transferred to the 
research assistant for screening and potential enrollment.  All potential participants referred to 
our study will undergo standard screening by the study team using the REDCap screening 
survey and will not require medical record review to determine eligibility. 
Information collected during the screening process on participants who are deemed ineligible, 
regardless of the recruitment method, will be discarded. 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
26 
 
5.3.4.2   Washington University in St. Louis 
Washington University in St. Louis will recruit participants from their study registry, Volunteers 
for Health, via emails and social media posts. 
5.3.4.3   University Hospitals Cleveland Medical Center 
University Hospitals Cleveland will recruit from participants that were enrolled in previous 
vaccine trials, employees, and via Facebook advertising and brochures. 
5.3.4.4   Arizona State University, Tempe 
Arizona State University will recruit local student and faculty population through study flyers. 
Valleywise Health will recruit employees via study flyers and the intranet.  Phoenix Children’s 
Hospital will approach and recruit patients presenting for seasonal influenza vaccination or 
mRNA COVID-19 vaccination and via study flyers. 
5.3.5 Enrollment 
Enrollment in this study will begin once 2023-24 seasonal influenza vaccinations become 
available, anticipated August 2023.  Enrolled participants will be randomized to one of the 
following three interventions (2:1:1) using a permuted block method stratified by site: (i) 
concomitant administration of the mRNA COVID-19 vaccine and quadrivalent influenza vaccine 
(ccIIV4); (ii) sequential administration of the quadrivalent influenza vaccine (ccIIV4) at Visit 1 
(day 0) and the mRNA COVID-19 vaccine at Visit 2 (day 28); (iii) sequential administration of 
the mRNA COVID-19 at Visit 1 (day 0) followed by the quadrivalent influenza vaccine (ccIIV4) at 
Visit 2 (day 28). 
Individuals will be recruited to participate in the study using site-specific methods which may 
include virtual and/or in-person recruitment.  Potentially eligible individuals will be directed to 
complete the eligibility screening and consent processes to determine whether 
inclusion/exclusion criteria are met.  All individuals who are eligible for the study will view a 
study description (purposes and procedures) electronically and will be asked to read, sign, and 
date the written/electronic informed consent and assent, if applicable, forms .  The 
written/electronic informed consent form will explain study details, risks, and benefits and 
emphasize the voluntary nature of participation.  Participants will be directed to contact study 
staff with questions. 
Individuals will be enrolled in the study if they meet inclusion criteria and provide 
written/electronic informed consent and assent, if applicable.  After completing the 
written/electronic informed consent/assent process, participants will complete an enrollment 
survey that collects information about demographic characteristics, medical history, and 
influenza and COVID-19 vaccination history.  Enrolled participants will then provide whole blood 
to isolate sera, PBMC and plasma samples (target to collect PBMC and plasma samples at 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
27 
 
every visit from a subset of 250 participants; 200 adults and 50 children) and receive a mRNA 
COVID-19 vaccine and/or quadrivalent influenza vaccine (ccIIV4) based on their intervention 
randomization.  Study staff will schedule remaining study visits for sample collection (Visits 2, 3, 
and 4) and second vaccination (Visit 2; groups ii and iii). 
5.3.6 Participant Withdrawal 
Participants may withdraw from the study at any time for any reason.  If a participant withdraws 
from the study after randomization but prior to receipt of the first vaccine dose administered 
during study procedure, sites will replace that participant in their randomization group to keep a 
2:1:1 randomization scheme.  Participants who wish to withdraw from the study will contact the 
study site principal investigator or coordinator to inform them of their decision.  Upon withdrawal 
from the study, the participant’s specimens will be destroyed and discarded if requested by 
study participant.  The participant’s data (including responses to the enrollment questionnaire, 
follow-up survey, and any extracted data) will be removed from all study site databases if 
requested by study participant. 
5.4 
Study Schedule, Procedures, & Evaluations 
5.4.1 Schedule of events and data collection 
Persons meeting the proposed eligibility criteria (Section 5.3.1) will be recruited.  
Written/electronic informed consent will be obtained from study participants/participant’s legally 
authorized representative(s) (LAR) prior to conducting any study procedures.   

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
28 
 
Table 1: Schedule of Events 
Procedure 
Visit 1 
Follow-up 
Survey 
(Reactogenicity) 
Visit 2 
Follow-up 
Survey 
(Weekly ARI) 
Visit 3 
Visit 4 
 
Clinic 
Phone/Email 
Clinic 
Phone/Email 
Clinic 
Clinic 
Estimated Study Day 
(Relative to Visit 1) 
0 
3 and 31 
28 
Weekly 
56 
180 
Time Following Visit 
1 (Days) [Window] 
0 
3 [± 3] 
28 [28-42] 
Weekly 
 
 
Time Following Visit 
2 (Days) 
 
3 [± 3] (groups ii 
and iii only) 
 
 
28 [28-92] 
152 [± 30] 
Informed consent & 
Medical Release of 
Information 
X 
 
 
 
 
 
Review Eligibility 
Criteria 
X 
 
 
 
 
 
Review Temporary 
Delay Criteria 
X 
 
X 
 
 
 
Demographic and 
Health History 
X 
 
 
 
 
 
Influenza and 
COVID-19 
Vaccination History 
X 
 
 
 
 
 
Randomization 
X 
 
 
 
 
 
Administer ccIIV4 
vaccine 
Groups i and 
ii 
 
Group iii 
 
 
 
Administer Moderna 
COVID-19 vaccine 
Groups i and 
iii 
 
Group ii 
 
 
 
Whole blood 
collection for sera 
sample 
Up to 10 mL 
 
Up to 10 mL 
 
Up to 10 mL 
Up to 10 mL 
Whole blood 
collection for PBMC 
and plasma sample 
Up to 24 mL 
[adults] or 8 
mL [children] 
 
Up to 24 mL 
[adults] or 8 
mL [children] 
 
Up to 24 mL 
[adults] or 8 
mL [children] 
Up to 24 mL 
[adults] or 8 
mL [children] 
Obtain solicited 
adverse events at 3 
days following Visits 
1 and 2 
 
X 
 
 
 
 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
29 
 
Table 1: Schedule of Events 
Procedure 
Visit 1 
Follow-up 
Survey 
(Reactogenicity) 
Visit 2 
Follow-up 
Survey 
(Weekly ARI) 
Visit 3 
Visit 4 
 
Clinic 
Phone/Email 
Clinic 
Phone/Email 
Clinic 
Clinic 
Estimated Study Day 
(Relative to Visit 1) 
0 
3 and 31 
28 
Weekly 
56 
180 
Time Following Visit 
1 (Days) [Window] 
0 
3 [± 3] 
28 [28-42] 
Weekly 
 
 
Time Following Visit 
2 (Days) 
 
3 [± 3] (groups ii 
and iii only) 
 
 
28 [28-92] 
152 [± 30] 
Obtain change in 
health status 
requiring 
hospitalization 
 
 
 
X 
 
 
Obtain ARI 
information 
 
 
 
X 
 
 
 
Visit 1; study day 0 – Screening, Enrollment, Specimen Collection and Vaccination – 
Clinic Visit 
• 
Review and confirm study eligibility  
• 
Obtain written/electronic informed consent and assent (if applicable, children age 6-11 
years) and release of medical record information  
• 
Obtain information on preferred method of contact for follow-up (text or email) 
• 
Obtain demographic and medical history including history of influenza and COVID-19 
vaccination via enrollment questionnaire  
• 
Take temperature and review temporary delay criteria 
• 
Collect whole blood for sera sample (Section 5.5.4.1) and PBMC and plasma samples 
(in subset of participants, target n=250; 200 adults and 50 children) (Section 5.5.4.2) 
• 
Randomize study participant to intervention (2:1:1) using a permuted block method 
stratified by site: 
i. 
concomitant administration of the mRNA COVID-19 vaccine and quadrivalent 
influenza vaccine 
ii. sequential administration of the quadrivalent influenza vaccine at Visit 1 (day 0) 
and the mRNA COVID-19 vaccine at Visit 2 (day 28) 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
30 
 
iii. sequential administration of the mRNA COVID-19 at Visit 1 (day 0) followed by 
the quadrivalent influenza vaccine at Visit 2 (day 28). 
• 
Administer mRNA COVID-19 vaccine and/or quadrivalent influenza vaccine (Flucelvax 
[ccIIV4]) based on randomization scheme  
• 
Confirm date of next appointment at Visit 2 (day 28) of study 
Follow-up Survey (Reactogenicity); 3 days after vaccine administration (Visits 1 and 2) – 
Vaccination Reactogenicity and Safety Data – Online Questionnaire 
• 
Automated reminders sent to all participants to complete electronic survey via 
participant’s preferred contact method approximately 3 days after receipt of either 
vaccine dose administered according to study procedure (day 0 [groups i-iii] and day 
28 [groups ii and iii]) 
• 
Participants/participant’s LAR(s) complete brief online survey regarding vaccine 
reactogenicity and safety data in the three days after receipt of either vaccine dose 
administered according to study procedure 
Follow-up Survey (ARI); weekly following Visit 1 – Weekly ARI Surveillance and Change 
in Health Status – Online Questionnaire 
• 
Automated reminders sent to all participants to complete electronic survey via 
participant’s preferred contact method once per week beginning approximately one 
week after Visit 1 until completion of Visit 4 
• 
Participants/participant’s LAR(s) complete brief online survey regarding new ARI 
episodes and change in health status requiring hospitalization throughout the entire 
study period  
• 
Participants that report new ARI may provide respiratory specimens for standard point 
of care respiratory testing and up to 2 additional blood draws (see Table 2: Schedule 
of Qualifying ARI Events) 
Visit 2; study day 28 (28-42) – Specimen Collection and Vaccination 2 (groups ii and iii) – 
Clinic Visit  
Group i: Concomitant administration of mRNA COVID-19 vaccine and quadrivalent 
influenza vaccine 
• 
Take temperature and review temporary delay criteria 
• 
Collect whole blood for sera sample (Section 5.5.4.1) and PBMC and plasma samples 
(in subset of participants, target n=250; 200 adults and 50 children) (Section 5.5.4.2)  
• 
Confirm date of next appointment on day 56 of study 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
31 
 
Group ii: Sequential administration of quadrivalent influenza vaccine then mRNA COVID-
19 vaccine 
• 
Take temperature and review temporary delay criteria 
• 
Collect whole blood for sera sample (Section 5.5.4.1) and PBMC and plasma samples 
(in subset of participants, target n=250; 200 adults and 50 children) (Section 5.5.4.2)  
• 
Administer mRNA COVID-19 vaccine  
• 
Confirm date of next appointment on day 56 of study 
Group iii: Sequential administration of mRNA COVID-19 vaccine then quadrivalent 
influenza vaccine 
• 
Take temperature and review temporary delay criteria 
• 
Collect whole blood for sera sample (Section 5.5.4.1) and PBMC and plasma samples 
(in subset of participants, target n=250; 200 adults and 50 children) (Section 5.5.4.2)  
• 
Administer quadrivalent influenza vaccine (Flucelvax [ccIIV4]) 
• 
Confirm date of next appointment on day 56 of study 
Visit 3; study day 56 (56-120) – Specimen Collection – Clinic Visit 
• 
Collect whole blood for sera sample (Section 5.5.4.1) and PBMC and plasma samples 
(in subset of participants, target n=250; 200 adults and 50 children) (Section 5.5.4.2) 
• 
Confirm date of next appointment on day 180 of study 
Visit 4; study day 180 – Specimen Collection – Clinic Visit 
• 
Collect whole blood for sera sample (Section 5.5.4.1) and PBMC and plasma samples 
(in subset of participants, target n=250; 200 adults and 50 children) (Section 5.5.4.2) 
 
Table 2: Schedule of Qualifying ARI Events 
Procedure 
Acute Visit 
Convalescent Visit 
 
Clinic 
Clinic 
Estimated Study Day  
< 10 days after symptom 
onset 
28 days after Acute Visit 
Time Following Acute Visit (Days) [Window] 
0 
28 [28-42] 
Nasal swab collection for viral testing 
X 
 
Whole blood collection for sera sample 
X 
X 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
32 
 
Table 2: Schedule of Qualifying ARI Events 
Procedure 
Acute Visit 
Convalescent Visit 
 
Clinic 
Clinic 
Estimated Study Day  
< 10 days after symptom 
onset 
28 days after Acute Visit 
Time Following Acute Visit (Days) [Window] 
0 
28 [28-42] 
Whole blood collection for PBMC and plasma sample 
X 
X 
Acute Visit; <10 Days after Symptom Onset – Specimen Collection – Clinic Visit  
If a participant reports ARI symptoms at any point during the weekly electronic follow-up survey 
(ARI), they may be asked to complete an acute visit. These specimens may be drawn at the 
same time as scheduled visits 1-4. 
• 
Obtain nasal/throat swab (Section 5.5.4.3) 
• 
Obtain sera sample (Section 5.5.4.1), PBMC, and plasma sample (Section 5.5.4.2) 
• 
Schedule convalescent visit 28 (28-42) days after acute visit for influenza or SARS-CoV-
2 positive participants 
• 
Confirm contact information and preferred method of contact for follow-up (telephone, 
text, or email) 
Convalescent Visit, 28 (28-42) days after acute visit – Specimen Collection – Clinic Visit 
If a participant tests positive for influenza and/or SARS-CoV-2 at any acute clinic visit, a 
convalescent visit is required 28 (28-42) days after their acute clinic visit. These specimens may 
be drawn at the same time as scheduled visits 1-4. 
• 
Obtain sera sample (Section 5.5.4.1), PBMC, and plasma sample (Section 5.5.4.2) 
5.4.2 Randomization 
Permuted block randomization stratified by age group (children aged 6-11 years and adults 
aged 18-64 years) and site will be used at each site to ensure even allocation to each study 
arm.  At each site, participants will be randomized 2:1:1 to receive: (i) concomitant 
administration of a mRNA COVID-19 vaccine and a quadrivalent influenza vaccine (ccIIV4), (ii) 
sequential administration of a quadrivalent influenza vaccine (ccIIV4) at Visit 1 (day 0) and a 
mRNA COVID-19 vaccine at Visit 2 (day 28), and (iii) sequential administration of a mRNA 
COVID-19 vaccine at Visit 1 (day 0) and a quadrivalent influenza vaccine (ccIIV4) at Visit 2 (day 
28).  The project statistician will generate the randomization schemes which will be uploaded to 
REDCap. Participants will be randomized through REDCap with treatment allocation recorded 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
33 
 
on the case report form. In the event that REDCap is unavailable, participants will be manually 
randomized through the use of sealed envelopes with randomization allocations enclosed. The 
project statistician will prepare envelopes that will use the same randomization strategy as the 
primary scheme embedded in REDCap. When an unblinded team member is informed of the 
age group, they will pull the next envelope in order. To capture the allocation per subject, a 
separate form in REDCap will be used by the unblinded study personnel to add the assignment. 
Sites will need to keep a log capturing these instances. The next available sequential study-
number will be assigned to each enrolled participant upon study-entry. 
5.4.3 Deviation from Protocol 
Influenza and COVID-19 vaccine may be administered outside of study window in case of public 
health emergency or personal exposure. 
5.5 
Data Collection 
5.5.1 Participant Demographics and Vaccine History 
5.5.1.1   Enrollment questionnaire administration 
Research staff will administer the enrollment questionnaire or participants may complete 
electronically.  Data will be recorded electronically directly into a data management system such 
as REDCap. 
5.5.1.2   Enrollment questionnaire content 
Each participant/participant’s LAR(s) will be interviewed to collect information on patient 
demographics, influenza and COVID-19 vaccination history, and self-reported lab-confirmed 
influenza or COVID-19 infection in the previous 90 days.  Any modifications or additional 
questions will be submitted for IRB approval before inclusion on enrollment questionnaires. 
Participant height and weight information may be collected at one of the first three study visits.  
5.5.2 Follow-up Surveillance for Vaccine Reactogenicity and Safety and Self-Reported 
ARI or Change in Health Status Resulting in Hospitalization 
5.5.2.1   Follow-up Survey (Reactogenicity) 
Participants will complete a brief survey regarding vaccine reactogenicity and safety data 3 days 
after receipt of either vaccine dose administered according to study procedure (day 0 [groups i-
iii] and day 28 [groups ii and iii]). 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
34 
 
Follow-up surveys will be completed online.  Participants will receive an automated link to an 
electronic survey 3 days after receipt of either vaccine dose administered according to study 
procedure (day 0 [groups i-iii] and day 28 [groups ii and iii]) via their preferred contact method 
(text or email).  Participants will complete the survey a maximum of two times during the study 
period.  The first survey will be sent 3 days after Visit 1 (groups i-iii) and the second survey will 
be sent 3 days after Visit 2 (groups ii and iii).  If the survey has not been completed, up to 3 
reminder notifications will be sent to the preferred contact method each week. 
5.5.2.2   Follow-up Survey (ARI) 
Participants will complete a brief survey regarding absence/presence of new ARI symptoms, 
illness test results (if applicable) and change in health status requiring hospitalization each 
week. 
Weekly surveys will be completed online.  Participants will receive an automated link to an 
electronic survey each week via their preferred contact method (text or email).  Participants will 
complete the survey once per week.  The first survey will be sent 7 days after Visit 1 (day 0) and 
will be sent out each week until the end of the study period (Visit 4; day 180).  If the survey has 
not been completed, a reminder notification will be sent to the preferred contact method each 
week. 
5.5.2.3   Qualifying ARI Definition 
An ARI during follow up is defined as an illness with new onset in the past 7 days and any two 
of the following symptoms: cough, fever or feeling feverish/chills, sore throat, runny/stuffy nose, 
muscle or body aches, headache, fatigue (tiredness), nausea or vomiting, diarrhea, new loss of 
smell or taste, or shortness of breath or difficulty breathing. 
5.5.3 Study Product Supply, Storage, and Administration 
In order to ensure adherence to study randomization assignment, age-appropriate, licensed 
quadrivalent cell culture-based influenza vaccines (ccIIV4) and EUA or approved mRNA 
COVID-19 vaccine will be administered as a study procedure.  Vaccines will be stored per 
manufacturer specifications.   
5.5.3.1   Quadrivalent cell culture-based influenza vaccine 
Flucelvax Quadrivalent (ccIIV4) will be the designated study products for influenza vaccination.  
A standard-dose of ccIIV4 contains 15 μg of HA per vaccine virus in a 0.5-mL dose.  ccIIV4 is 
approved for persons aged ≥6 months. 
Vaccine lot numbers, dose, and site of vaccine administration will be recorded by research staff.  
After administration, used study syringes will be disposed of according to standard operating 
procedure.   

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
35 
 
Emergency management supplies including epinephrine (1:1000) and trained staff will be 
available for initial treatment of an allergic reaction, if needed. 
5.5.3.2   mRNA COVID-19 vaccine 
mRNA COVID-19 vaccine will be administered per FDA and ACIP recommendations.  Study 
sites will work to coordinate the brand of mRNA vaccine administered based on 
recommendations and availability. 
Vaccine lot numbers, dose, and site of vaccine administration will be recorded by research staff.  
After administration, used study syringes will be disposed of according to standard operating 
procedure.   
Emergency management supplies including epinephrine (1:1000) and trained staff will be 
available for initial treatment of an allergic reaction, if needed. 
5.5.4 Biospecimens Collection & Handling 
5.5.4.1   Sera 
Blood specimens for sera collection will be collected from participants at Visit 1 (day 0), Visit 2 
(day 28), Visit 3 (day 56), Visit 4 (day 180), Acute Visit (<10 days after symptom onset), and 
Convalescent Visit (28 days after Acute Visit).  Blood samples will be processed for sera, 
aliquoted, and stored at -20 C or colder at VE network laboratories until shipped to CDC for 
storage and subsequent testing via immunological assays to be performed at CDC and/or CDC-
approved laboratory facilities (Laboratory Appendix B).  We will collect 5-10 mL of whole blood 
for children aged 6-11 years and 10 mL of whole blood for adults aged 18-64 years.  These 
volumes are preferred for sera processing, however, smaller volumes can be accepted if the 
situation does not allow for the collection of these amounts.  The minimum accepted volume of 
whole blood to collect is 3 mL for all participants over the age of 6 years.  Guidelines will be 
followed to ensure the amount drawn meets minimal risk criteria (i.e., 3 ml/kg in any 24-hour 
period, and 7 ml/kg in any eight-week period).   
5.5.4.2   PBMC and Plasma 
Blood specimens for PBMC and plasma collection will be collected from a subset of participants 
(target n=250; 200 adults and 50 children) at Visit 1 (day 0), Visit 2 (day 28), Visit 3 (day 56), 
Visit 4 (day 180), Acute Visit (<10 days after symptom onset), and Convalescent Visit (28 days 
after Acute Visit).  Blood samples will be processed for PBMC and plasma, aliquoted, and 
stored at VE network laboratories until shipped to CDC for immunologic assays (Laboratory 
Appendix C).  We will collect 8 mL (approximately 1 CPT tube) whole blood for children aged 6-
11 years and 24 mL (approximately 3 CPT tubes) whole blood for adults aged 18-64 for PBMC 
and plasma processing.  These volumes are preferred for PBMC and plasma processing, 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
36 
 
however, smaller volumes can be accepted if the situation does not allow for the collection of 
these amounts.  The minimum accepted volume of whole blood to collect for PBMC and plasma 
processing is 16 mL from adults between the ages of 18-64 and 5 mL for children between the 
ages of 6-11 years.  Guidelines will be followed to ensure the amount drawn meets minimal risk 
criteria (i.e., 3 ml/kg in any 24-hour period, and 7 ml/kg in any eight-week period).   
5.5.4.3   Nasal/Throat Swab 
Sites may conduct respiratory testing for those that report ARI via the follow-up survey (ARI) 
during the study period via standard point of care respiratory testing for influenza and COVID-
19.  
5.6 
Vaccine Safety and Reactogenicity 
Participants will respond to an automated electronic survey 3 days following each vaccination 
visit to report the occurrence of fever, injection site reactions (pain, redness, swelling and 
itching) systemic reactions (chills headache, joint pain, muscle ache, body ache, fatigue, 
tiredness, nausea, vomiting, diarrhea, abdominal pain, rash lymph node swelling) the severity of 
reactions and their impact on health. Severity will be rated as mild (symptoms noticeable but 
aren’t a problem) moderate (symptoms limit normal daily activities) or severe (symptoms make 
normal daily activities difficult or impossible). Impact on health will be assessed with respect to 
ability of the participant to attend work or school, ability to do normal daily activities and 
utilization of medical care. 
The weekly automatic electronic survey will also request participants to report the occurrence of 
any change in health requiring hospitalization. 
5.7 
Medical Record Extraction 
5.7.1 Prior Vaccination 
All study sites will provide lot number and manufacturer information for documented influenza 
and COVID-19 vaccination, if recorded, from July 1, 2020 through the end of the study period.  
The following data elements for vaccination verification will be collected for each dose if 
provided: date of vaccination, product name, manufacturer, lot number, and source of 
information, as well as route of administration (e.g., intramuscular, intranasal), valence (e.g., 
quadrivalent) and type of vaccine (e.g., high-dose, recombinant, standard dose), if applicable.  
In addition to data sources described below, vaccination cards may also be used as a verified 
source for COVID-19 vaccination. 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
37 
 
5.7.1.1   In-system sources/electronic medical records 
Each study site will query the health system’s electronic medical record system to obtain prior 
season influenza and COVID-19 vaccination data for all participants regardless of self-reported 
vaccination status.  Prior influenza and COVID-19 vaccination history data may include records 
from multiple prior seasons from electronic immunization records. 
5.7.1.2   External sources 
Each site will, at a minimum, query state and/or local immunization information systems to 
obtain prior influenza and COVID-19 vaccination data for all participants regardless of self-
reported vaccination status.  Prior influenza and COVID-19 vaccination data may include 
records from multiple prior seasons from electronic immunization records.  Sites may seek 
vaccine data from other sources including, but not limited to, occupational health records for 
major employers, retail pharmacies, and insurance plan claims.  A medical release of 
information (MROI) may be required to be obtained from participants to request data from 
sources outside the study site’s medical system. 
5.7.2 Prior Lab-Confirmed Illness 
All study sites will provide information for documented influenza and COVID-19 infection, if 
recorded, from January 1, 2020 through the end of the study period. The following data 
elements for prior infection will be collected for each lab-confirmed test if provided: date of test, 
type of test, result of test, and source of information. 
5.7.2.1   In-system sources/electronic medical records 
Each study site will query the health system’s electronic medical record system to obtain prior 
season influenza and COVID-19 infection data for all participants regardless of self-reported 
prior infection status.  Prior influenza and COVID-19 infection history data may include records 
from multiple prior seasons from electronic health records. 
6 
HUMAN SUBJECTS PROTECTIONS 
6.1 
Institutional Review Board Review 
Participating study sites will rely on the Duke IRB.  The Duke IRB will enter into an IRB 
Authorization Agreement (IAA) that will include a communication plan with each relying 
institution prior to study implementation.  Relying study sites will submit study protocols and any 
amendments and associated consent and assent forms and patient-facing instruments and 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
38 
 
recruitment materials to their local IRB for administrative review of the study site reliance as per 
local IRB policy. 
6.2 
Participant Confidentiality 
According to HIPAA, the information collected by the researchers for this study includes 
protected health information (PHI).  We are requesting a HIPAA waiver of authorization to 
review electronic medical records.  The study meets the requirements of the waiver of HIPAA 
authorization by ensuring that disclosure of protected health information in this study involves no 
more than minimal risk to the privacy of individuals.  The research could not practicably be 
conducted without the waiver or alteration and the research could not practicably be conducted 
without access to and use of the protected health information.  The data security plan outlines 
how identifiers will be protected from improper use or disclosure, how identifiers will be 
destroyed after study completion, and how the protected health information will not be reused 
for other research. 
Consistent with Section 301(d) of the Public Health Service Act, a Certificate of Confidentiality 
(CoC) applies to this research because this research is funded, conducted, or supported by 
CDC and the following is true: 
1. The activity constitutes biomedical, behavioral, clinical, or other research; and 
i. 
Individually identifiable (including coded) information or biospecimens will be 
obtained or used for research purposes or as defined at 45 CFR 46.102(e); 
ii. Biospecimens are collected or used as part of the research, and is there a small 
risk that some combination of the biospecimen and other available data sources 
could be used to deduce the identity of an individual; and 
iii. The research involves information about an individual for which there is at least a 
very small risk, that some combination of the information, a request for the 
information, and other available data sources could be used to deduce the 
identity of an individual. 
Therefore, CDC and any of its collaborators, contractors, grantees, investigators or collaborating 
institutions that receive “identifiable, sensitive information” as defined by subsection 301(d) of 
the Public Health Service Act shall not: 
• 
Disclose or provide, in any Federal, State, or local civil, criminal, administrative, 
legislative, or other proceeding “identifiable, sensitive information” that was created or 
compiled for purposes of the research, unless such disclosure or use is made with the 
consent of the individual to whom the information, document, or biospecimen pertains; or 
• 
Disclose “identifiable, sensitive information” or provide ISI to any other person not 
connected with the research. 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
39 
 
• 
Disclosure is permitted only when: 
• 
Required by Federal, State, or local laws (e.g., as required by the Food, Drug and 
Cosmetic Act or required by state laws requiring the reporting of communicable diseases 
to State and local health departments), excluding instances of disclosure in any Federal, 
State, or local civil, criminal, administrative, legislative, or other proceeding; 
• 
Made with the consent of the individual to whom the information, document, or 
biospecimen pertains; or 
• 
Made for the purposes of other scientific research that is in compliance with applicable 
Federal regulations governing the protection of human participants in research. 
CDC and its collaborators and contractors conducting this research will establish and maintain 
effective internal controls (e.g., policies and procedures) that provide reasonable assurance that 
the research is managed in compliance with subsection 301(d) of the Public Health Service Act.  
CDC will ensure: 1) that any investigator or institution not funded by CDC who receives a copy 
of identifiable, sensitive information protected by this Certificate, understands that it is also 
subject to the requirements of the Certificate; and 2) that any subrecipient that receives CDC 
funds to carry out part of this research involving a copy of identifiable, sensitive information 
protected by a Certificate understands that it is subject to subsection 301(d) of the PHS Act.  
Therefore, all study staff will receive training on the importance of protecting the confidentiality 
of human research participants and of personal information acquired, including the collection of 
biological specimens. 
All research participants will be informed of the protections and the limits to protections provided 
by this Certificate through the informed consent process.  All study staff who obtain consent 
from study participants will be trained on how the Certificate protects the information collected 
and the limitations of the Certificate’s protections. 
6.3 
Risks and Benefits to Participants 
A description of this clinical trial will be available on http://ClinicalTrials.gov, as required by US 
Law. This website will not include information that can identify subjects. 
The risks to participants for being in this study will be minimal.  Getting respiratory samples may 
cause some discomfort.  There may be brief soreness while the nose and throat swabs are 
taken.  The nose swab may cause the participant to sneeze, and once in a while, a small 
nosebleed could occur.  If this should happen, study staff will treat it right away.  The participant 
may experience discomfort or slight pain during blood draw.  In rare cases, blood draw may 
cause bruising, prolonged bleeding, or infection at the site of the puncture. 
Some people get severe pain in the shoulder and have difficulty moving the arm where a shot 
was given.  This happens very rarely.  Syncope (fainting) can occur in association with 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
40 
 
administration of injectable vaccines.  Sitting or lying down when space is available for about 15 
minutes can help prevent fainting, and injuries caused by a fall, as recommended in the ACIP 
General Recommendations on Immunization.  Participants should inform their doctor if they feel 
dizzy or have vision changes or ringing in the ears. 
As with any licensed or authorized vaccine, protection may not occur in 100% of vaccinated 
persons for either COVID-19 or influenza vaccines.  Participants may be at risk of delayed 
vaccination if randomized to sequential vaccination groups (groups ii and iii).  These participants 
may be exposed to SARS-CoV-2 or influenza virus before fully immunized.  Second vaccine 
may be offered before visit 2 if risk of COVID-19 or influenza is high based on local viral 
surveillance. 
mRNA COVID-19 Vaccine 
Two COVID-19 vaccine received Emergency Use Authorization (EUA) by the FDA in December 
2020.  BNT162b2 received FDA approval for individuals 16 years of age and older and mRNA-
1273 received authorization for those 18 years of age and older.  The Pfizer/BioNTech mRNA 
COVID-19 vaccine received additional EUA for adolescents 12 to 15 years in May 2021 and 
children 5 to 11 years in October 2021; both the Pfizer/BioNTech and Moderna mRNA COVID-
19 vaccines received addition EUA for children aged down to 6 months in June 2022.  The EUA 
was again updated to authorize the use of the updated bivalent vaccines as boosters for 
children aged down to 6 months in December 2022.  These vaccines have been recommended 
at these ages by the ACIP.   
Side effects that have been reported with mRNA COVID-19 vaccines include both injections site 
reactions as well as general side effects.  Injection site reactions include: pain, tenderness and 
swelling of the lymph nodes in the same arm of the injection, swelling (hardness), and redness 
at the injection site.  General side effects include: fatigue, headache, muscle pain, joint pain, 
chills, nausea and vomiting, fever and feeling unwell.  There is a remote chance that an mRNA 
COVID-19 vaccine could cause a severe allergic reaction, usually occurring within a few 
minutes to hours after getting a dose of vaccine.  Signs of a severe allergic reaction can include: 
difficulty breathing, facial and throat swelling, tachycardia, total body rash, dizziness and 
weakness.  There have been rare reports of cases of inflammation of the heart—called 
myocarditis and pericarditis—happening after mRNA COVID-19 vaccination.   The events have 
mainly occurred in adolescents and young adults and more often after the second dose of 
vaccine.   Available data from short-term follow-up suggest that most individuals have had 
resolution of symptoms, but information is not yet available about potential long-term sequelae.  
These may not be all the possible side effects of the mRNA COVID-19 vaccines.  Serious and 
unexpected side effects may also occur.  mRNA COVID-19 vaccines are still being studied in 
clinical trials.   Available data support the safety of dose 3 mRNA COVID-19 vaccine in 
immunocompromised individuals.   Available data support the safety of booster doses of mRNA 
COVID-19 vaccine. 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
41 
 
ccIIV4 Influenza Vaccine 
ccIIV4 is an FDA-licensed vaccine approved for use in persons 6 months of age and older. This 
vaccine is standard clinical practice and recommended by the CDC.   
ccIIV4 risks include minor problems such as soreness, redness, swelling, or pain where the shot 
was given, hoarseness, sore, red or itchy eyes, cough, fever, aches, headache, itching, fatigue, 
all of which usually occur within 1-2 days of vaccination and are self-limiting. More serious 
problems including a small increased risk of Guillain-Barré Syndrome estimated at 1 or 2 
additional cases per million people vaccinated can occur. This is much lower than the risk of 
severe complications from influenza infection, which can be prevented by ccIIV4.  In addition, 
any medication can cause a severe allergic reaction, or anaphylaxis, which is estimated at ~ 1 
in one million doses of ccIIV4 administered. 
Allowing Twilio to be Used for Text Message Reminders 
We use Twilio to send text messages to participants.  Text messaging does not provide a 
completely secure and confidential means of communication, and the messages are 
unencrypted.  Twilio does encrypt participant information on their servers, but no system is 
completely safe.  If they decide to share these data, it may no longer be covered under the 
privacy protections.  Information that identifies participants, such as phone number, may be sent 
to and permanently kept by Twilio and their business associates.  Information disclosed to these 
companies or their business partners, may no longer be covered under the privacy protections.  
Because text messaging does not provide a completely secure and confidential means of 
communication, a participant may wish to keep communication completely private and we will 
communicate with the participant only through regular channels like the telephone or email. 
Loss of Privacy 
There is also the potential risk of loss of confidentiality.  Every effort will be made to keep 
participant’s information confidential, however, this cannot be guaranteed. 
6.4 
Return of Results 
Enrolled participants will be informed of any result of FDA-approved laboratory tests used for 
study purposes.  Results of serologic and cellular immunity testing performed as part of this 
study will not be returned to enrolled patients.  Reporting of research test results to public health 
authorities will adhere to reporting requirements as described in participant terms of consent, 
and public health authorities may follow up with research participants for further actions per 
appropriate guidelines.   

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
42 
 
Reporting of SARS-CoV-2 Results 
State or local health department regulations may require reporting of incident cases of SARS-
CoV-2 infection.  Local investigators at each study site will be responsible for contacting their 
state or local SARS-CoV-2 surveillance coordinators to ensure study procedures comply with all 
reporting requirements.   
All participants will be informed of positive results once they are available.  Results will not be 
shared with participants’ medical providers. 
Molecular diagnostic results will not always be available during the period when participants are 
acutely ill.  Participants will be informed via a COVID Test Results Disclaimer. 
6.5 
Compensation 
Participants will be compensated for their participation in the study.  Study sites will specify 
compensation amount and schedule in written/electronic informed consent form .  
7 
DATA MANAGEMENT, TRANSFER, AND SECURITY  
7.1 
Data Management and Transfer 
Enrollment interview and follow-up survey data will be collected and entered electronically at the 
sites via Duke’s instance of REDCap and stored behind secure institutional firewalls.  Linkage to 
Personally Identifying Information (PII), including names, medical record numbers, postal 
addresses, phone numbers, email addresses, or other identifying information protected by 
HIPAA will be securely stored by study sites.  Duke will receive names, phone numbers, email 
addresses, visit dates, and other necessary identifiers as collected through REDCap. A HIPAA-
limited dataset will be delivered to CDC by the Network coordinating center (NCC) at Duke 
University using a unique study-specific identifier for each individual (ENROLLID).   
Laboratory data will be linked the unique study identifier (ENROLLID) and Specimen ID to each 
participant’s record and will be uploaded into REDCap by laboratory staff at the central/regional 
lab.  In some sites, study site staff may assist with this task if the laboratory is on-site; other 
laboratories off-site may directly upload the data into REDCap with minimal interaction with 
study site staff. 
To ensure uniform and consistent data collection among network sites, CDC and/or the NCC 
will provide data dictionaries and data collection forms/surveys.  Data dictionaries will be 
created based on agreed-upon data elements to be collected through enrollment interview, 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
43 
 
follow-up survey, and electronic medical records.  Modification to data collection instruments 
and addition of data requested will be submitted for IRB approval before collection.   
All sites are expected to use secure, encrypted file transfer protocols (FTP) or other secure file 
transfer (upload, download, etc.) to CDC and/or the NCC for data not uploaded directly to the 
NDCC instance of REDCap.  Data will be downloaded by CDC and/or the NCC on a regular 
basis for quality control checks, tracking, and preparation and cleaning of the final complete 
dataset.  Quality control would include checking data for adherence to the common protocol, 
outliers, and missing or incomplete data. 
7.2 
Data Security 
Sites will comply with federal, state, and institutional requirements regarding time horizons for 
retention and/or destruction of research records.  Data privacy will be maintained according to 
local IRB protocols and requirements.  No research participant will be identified by name, 
picture, or any other personally identifying manner if information from this study is presented 
publicly or published in a medical journal. 
Any data stored by each study site prior to upload into REDCap (e.g., medical record and lab 
results data) will be stored on each site’s own servers.  Each site must have security policies 
that comply with all appropriate security requirements.  Once data has been uploaded into the 
NDCC instance of REDCap, it is housed on NCC servers at Duke University.  Forms, lists, 
logbooks, appointment books, and any other listings that link ENROLLID numbers to other 
identifying information must be stored in a separate, locked (or encrypted) file in an area with 
limited access.  If participant names and corresponding ENROLLIDs are entered into a 
computer database, this database must be password protected and must be maintained in a 
directory and in a location that is separate from any study-specific data.  The purpose of this 
requirement is to assure that a breach at one location will not allow the PHI to be associated 
with other study data. 
Deidentified data from this study must be available for release for public use within a year after 
the data are evaluated for quality as stipulated in CDC Policy 385 (“Policy on Public Health 
Research and Nonresearch Data Management and Access”).  Data that cannot be released 
publicly (i.e., where deidentification would preclude the proposed analysis) may be shared 
through a special data-use agreement in accordance with this policy.  Data and associated 
documentation from this study will be available only under a data use agreement developed by 
CDC in coordination with the NCC that provides for (1) a commitment to using the data only for 
research purposes and not to identify any individual participant; (2) a commitment to securing 
the data using appropriate information technology; and (3) commitments for destruction, return, 
or retention of data as stipulated. 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
44 
 
8 
STATISTICAL ANALYSIS 
8.1 
Study Populations 
There will be two study populations: the modified Intent-to-Treat (mITT) and Immunogenicity 
populations. The mITT population will include any participant who was enrolled, randomized, 
and received at least one vaccine. The Immunogenicity Population is a subset of the mITT 
Population that includes only subjects who received at least one vaccine, provide visit 1 and 
visit 2 blood draws available for analysis within the protocol-defined time frame, and with no 
protocol violations affecting immunogenicity.  These protocol violations will be listed in the 
Statistical Analysis Plan. Statistical analyses of safety outcomes will be performed for the mITT 
population, and immunogenic outcomes will be analyzed for the Immunogenicity Population. 
8.2 
Analysis Plan of Year 2 Component D  
HAI antibody titers will be compared between groups receiving the mRNA COVID-19 and ccIIV4 
influenza vaccines concomitantly or sequentially for each of the four influenza vaccine strains 
contained in the respective vaccines for that season as well as circulating influenza viruses and 
SARS-CoV-2 variants. Specifically, we will assess the following objectives:  
Primary Objective 1a: Compare the proportion of participants in each vaccination group 
with a seroprotective HAI titer (≥1:40) pre- and post-ccIIV4 immunization for each ccIIV4 
antigen. These proportions will be compared between vaccination groups using an exact 
Mantel-Haenszel statistic. 
Primary Objective 1b: Compare the proportion of participants in each vaccination group 
achieving seroconversion following ccIIV4 (an HAI titer ≥1:40 following ccIIV4 
immunization if the baseline titer is <1:10 or a four-fold rise in HAI titer if the baseline titer 
is >1:10) for each ccIIV4 antigen. These proportions will be compared between 
vaccination groups using an exact Mantel-Haenszel statistic. 
Primary Objective 1c: Compare the GMT for each ccIIV4 antigen and circulating 
influenza viruses pre- and post-ccIIV4 immunization in each vaccination group. GMT will 
be compared between vaccination groups using a regression model. 
Primary Objective 1d: Compare the GMFR in HAI titer for each vaccination group. 
GMFR will be compared between vaccination groups using a mixed effects regression 
model with a random intercept.  
Exploratory objectives will be assessed using exact Mantel-Haenszel statistics to compare 
proportions between vaccination groups, regression models to compare GMT, and mixed 
effects regression models to compare GMFR. Tables will be provided summarizing counts and 
proportions for categorical outcomes or summary statistics and confidence intervals for 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
45 
 
continuous outcomes. The statistical analysis of each exploratory objective will be fully 
described in the statistical analysis plan (SAP). 
HAI titers will be log transformed prior to analysis and back transformed to estimate GMT and 
GMFR. The same analysis plan for HAI titers will be repeated for MN titers. 
Data analysis will be performed using SAS version 9.4 software (SAS Institute Inc., Cary, NC) or  
R software (R Core Team, Vienna, Austria). 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
46 
 
REFERENCES 
1. 
Rosenblum HG, Wallace M, Godfrey M, et al. Interim recommendations for the advisory 
committee on immunization practices for the use of bivalent booster doses of COVID-19 
vaccines - United States, October 2022. Morbidity and Mortality Weekly Report 
2022;71(45):6. 
2. 
Lazarus R, Baos S, Cappel-Porter H, et al. Safety and immunogenicity of concomitant 
administration of COVID-19 vaccines (ChAdOx1 or BNT162b2) with seasonal influenza 
vaccines in adults in the UK (ComFluCOV): a multicentre, randomised, controlled, phase 
4 trial. Lancet 2021;398(10318):2277-2287. DOI: 10.1016/S0140-6736(21)02329-1. 
3. 
Izikson R, Brune D, Bolduc JS, et al. Safety and immunogenicity of a high-dose 
quadrivalent influenza vaccine administered concomitantly with a third dose of the 
mRNA-1273 SARS-CoV-2 vaccine in adults aged >/=65 years: a phase 2, randomised, 
open-label study. Lancet Respir Med 2022;10(4):392-402. DOI: 10.1016/S2213-
2600(21)00557-9. 
4. 
Toback S, Galiza E, Cosgrove C, et al. Safety, immunogenicity, and efficacy of a 
COVID-19 vaccine (NVX-CoV2373) co-administered with seasonal influenza vaccines: 
an exploratory substudy of a randomised, observer-blinded, placebo-controlled, phase 3 
trial. Lancet Respir Med 2022;10(2):167-179. DOI: 10.1016/S2213-2600(21)00409-4. 
5. 
Centers for Disease Control and Prevention. Interim Clinical Considerations for Use of 
COVID-19 Vaccines Currently Authorized in the United States. 
(https://www.cdc.gov/vaccines/covid-19/clinical-considerations/interim-considerations-
us.html#:~:text=At%20the%20time%20of%20initial,on%20age%20and%20vaccine%20p
roduct). 
6. 
Richards KA, Moritzky S, Shannon I, et al. Recombinant HA-based vaccine outperforms 
split and subunit vaccines in elicitation of influenza-specific CD4 T cells and CD4 T cell-
dependent antibody responses in humans. NPJ Vaccines 2020;5:77. DOI: 
10.1038/s41541-020-00227-x. 
7. 
Yu ED, Grifoni A, Sutherland A, et al. Balanced Cellular and Humoral Immune 
Responses Targeting Multiple Antigens in Adults Receiving a Quadrivalent Inactivated 
Influenza Vaccine. Vaccines (Basel) 2021;9(5). DOI: 10.3390/vaccines9050426. 
8. 
Gaglani M, Kim SS, Naleway AL, et al. Effect of Repeat Vaccination on Immunogenicity 
of Quadrivalent Cell-Culture and Recombinant Influenza Vaccines Among Healthcare 
Personnel Aged 18-64 Years: A Randomized, Open-Label Trial. Clin Infect Dis 2022. 
DOI: 10.1093/cid/ciac683. 
9. 
Grohskopf LA, Blanton L, Ferdinands J, et al. Prevention and control of seasonal 
influenza with vaccines: Recommendations of the advisory committee on immunization 
practices - United States, 2022-23 influenza season. Morbidity and Mortality Weekly 
Report 2022;71(1). 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
47 
 
LAB APPENDICES 
 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
48 
 
Lab Appendix A: Respiratory Specimen Testing for Influenza 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
49 
 
Lab Appendix B: Human Sera Collection Guidelines for Influenza Serology 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
50 
 
Lab Appendix C: Peripheral Blood Mononuclear Cells 

Comparative immunogenicity of concomitant vs sequential mRNA and influenza vaccination  
NCT06020118 
 
 
October 2, 2023 
 
51