The Impact of Imprinting and Repeated Influenza Vaccination on Adaptive Immunity, Transcriptomics, and Metabolomics
Phase: PHASE4
Conditions: Influenza
Sponsor: Emory University

 
1
The impact of imprinting and repeated influenza vaccination on 
adaptive immunity, transcriptomics, and metabolomics. 
 
Protocol Number: FLU2 
 
 
Sponsored by: 
 
Emory Institutional Funds 
 
 
Principal Investigator: 
 
Nadine Rouphael, MD  
 
 
Draft or Version Number:  
 
3.0 
 
13 SEP 2019 
 
 
NCT Number:  
 
NCT03686514 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
2
 
 
 
Statement of Compliance 
 
The trial will be carried out in accordance with Good Clinical Practices (GCP) as required by the 
following: 
 
• 
United States Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR 
Part 46; 21 CFR Part 50, 21 CFR Part 54, 21 CFR Part 56, and 21 CFR Part 312); 
• 
International Conference on Harmonization (ICH) E6; 62 Federal Register 25691 (1997); 
 
Compliance with these standards provides public assurance that the rights, safety and wellbeing 
of study subjects are protected, consistent with the principles that have their origin in the 
Declaration of Helsinki. 
 
All key personnel (all individuals responsible for the design and conduct of this trial) have 
completed Human Subjects Protection Training. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
3
 
 
 
SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable US federal regulations and ICH guidelines. 
 
 
Signed: 
 
Date: 
 
 
 
 
    Nadine Rouphael, MD 
 
      
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
4
 
Protocol Summary 
 
Title:  
The impact of imprinting and repeated influenza vaccination  
on adaptive immunity, transcriptomics, and metabolomics.  
 
 
Population: 
 
60 participants, age greater than 40 years of age, who are in 
good health and meet the eligibility criteria. 
 
Clinical Site: 
 
The Hope Clinic of the Emory Vaccine Center 
 
Study Duration: 
 
Approximately 3 years  
 
Subject Duration:  
Approximately 6 months 
 
Description of Agent: 
One dose delivered intramuscularly of the FDA-approved 
seasonal influenza vaccine 
 
Objectives: 
 
Primary:  
• 
To characterize the magnitudes and specificities of the hemagglutination inhibition (HAI) 
antibody response to each influenza A subtype or influenza B lineage included in the 
seasonal quadrivalent influenza vaccine in adults who had “imprinted” against either 
influenza A/H3N2 or influenza A/H1N1 and who have been either repeatedly vaccinated 
or unvaccinated 
 
Secondary:  
• 
To characterize the magnitudes and specificities of the neutralizing antibody (NAb) 
responses to each influenza A subtype or influenza B lineage included in the seasonal 
quadrivalent influenza vaccine in adults who had “imprinted” against either influenza 
A/H3N2 or influenza A/H1N1 and who have been either repeatedly vaccinated or 
unvaccinated   
• 
To characterize the duration of the immune responses to each subtype of the seasonal 
quadrivalent influenza vaccination in adults who have “imprinted” against H3N2 or H1N1 
and who have been either repeatedly vaccinated or unvaccinated   
• 
To evaluate adaptive cellular immune responses and cytokines before and following 
vaccination     
 
Exploratory: 
• 
To evaluate systems biology measures (transcriptional and metabolic changes) after 
seasonal influenza vaccination in subjects from two distinct birth cohorts who have been 
either repeated vaccinated or unvaccinated   
• 
To evaluate ELISA binding antibodies to the highly conserved HA stalk, NA, and M2 
ectodomain regions of the influenza virus   
• 
To evaluate the influences on HAI antibody titers of age, gender, ethnicity, vaccination 
history, and/or birth cohort   

 
5
• 
 To evaluate the temporal dynamics of the humoral immune response to influenza 
vaccination in oral fluid compared to serum.    
 
Endpoints:  
Primary: 
• 
The proportion of subjects achieving seroprotection (titer of ≥ 40) or seroconversion 
(four-fold rise in HAI post-influenza vaccination compared to pre-vaccination, or 
achieving a titer of >40 if the pre-vaccination titer was <10), against each strain 
contained in the seasonal quadrivalent influenza vaccine, as measured by HAI antibody 
response approximately 28 days after vaccination 
• 
Geometric Mean Titers (GMTs) of serum HAI and neutralizing antibodies against each 
strain of the seasonal quadrivalent influenza vaccine, approximately 28 days after 
vaccination (Study Day 29)     
Secondary: 
• 
The proportion of subjects achieving seroprotection (titer of ≥ 40) or seroconversion 
(four-fold rise in HAI post-influenza vaccination compared to pre-vaccination, or 
achieving a titer of >40 if the pre-vaccination titer was <10), against each strain 
contained in the seasonal quadrivalent influenza vaccine, as measured by NAb 
responses approximately 28 days after vaccination   
• 
Geometric Mean Titers (GMT) of serum NAb against each strain of the seasonal 
quadrivalent influenza vaccine, approximately 28 days after vaccination 
• 
For HAI and NAb responses, the proportions of subjects achieving seroprotection (titer 
of ≥ 40) and seroconversion (four-fold rise in HAI post-influenza vaccination compared to 
pre-vaccination) against each strain of the seasonal quadrivalent influenza vaccine 
approximately 180 days after vaccination (Study Day 181) 
• 
Geometric Mean Titers (GMTs) of serum HAI and neutralizing antibodies against each 
strain of the seasonal quadrivalent influenza vaccine approximately 180 days after 
vaccination (Study Day 181) 
• 
Level of circulating follicular helper T cells (cTFH) at days 1, 8, and 15 
• 
Level of plasmablasts (Ag-specific) by ELISpot at days 1, 8, and 15 
• 
Level of CD4+ T-cells at days 1, 8, 15 and 29   
• 
Level of cytokine and chemokine levels (including IL-2, IFN-γ, IL-21, CD40L/154, IL10) at 
days 1, 3, 8 and 15     
Exploratory:  
• 
Differentially expressed genes (DEG) as measured by transcriptomics comparing 
baseline to Study Days 3 and 8       

 
6
• 
Differentially abundant metabolites (DAM) comparing baseline to Study Days 3 and 8 
• 
Levels of ELISA binding antibodies to the highly conserved HA stalk, NA, and M2 
ectodomain, before and approximately 28 days after vaccination  
• 
Multivariate analyses of HAI and neutralizing antibody titers with regards to age, gender, 
ethnicity, vaccination history, and birth cohort    
• 
Levels of IgA and IgG binding to a panel of influenza antigens and HAI titers to each 
component of the quadrivalent influenza vaccine in the crevicular fluid vs serum at days 
1, 8, 15, 29, and 180 
    
Figure 1. Schematic of Study Design 
 
Per each influenza season:  

 
7
 

 
8
1 
KEY ROLES 
Principal Investigators:  Nadine Rouphael, MD 
Co-investigator: Amy Sherman, MD 
 
Institution:  
The Hope Clinic of the Emory Vaccine Center  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
9
2 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
 
2.1 Background Information 
 
 
Seasonal influenza outbreaks continue to cause substantial disease burden, with 
an estimated 3-5 million cases of severe illness, and 250,000 to 500,000 deaths 
worldwide each year.i  In the United States, the CDC reports that influenza has 
resulted in 9.2-35.6 million illnesses with 12,000-56,000 deaths annually since 2010.ii  
There is an urgent need to better understand the immunologic responses to current 
licensed vaccines in order to develop a more effective vaccine that does not rely on 
annual updates, provides broad protection, and is durable; i.e., a universal influenza 
vaccine.  
In 1960, Francis described the “doctrine of original antigenic sin.”iii  He observed 
that the antibodies produced by a child to the first influenza A sub-type infection 
continued to dominate throughout his or her life, and governed the immune response 
to all subsequent influenza exposures thereafter. Gostic et al. provided evidence that 
individuals indeed have a lifelong “imprint” from his or her first influenza A virus (IAV) 
exposure, and thus have a reduced risk of suffering from severe disease due to 
infection with a novel strain of IAV within the same phylogenetic group (HA group 1 
contains subtypes H1, H2, and avian H5, while HA group 2 contains seasonal H3 and 
avian H7).  Amino acid homology for the conserved HA stem region is significantly 
higher within, as opposed to between groups (see Figure 1)iv.   

 
10
 
Figure 1: Gostic et. al 
Fonville et al. also demonstrated imprinting in a large Vietnamese cohort of 
unvaccinated individuals, and showed that the highest serologic titers were for 
influenza viruses that had circulated when the individuals were around six years of age 
(likely the time frame of their first infection).v  Thus, birth-year cohorts can be defined 
based on an individual’s initial childhood exposure to H1N1, H3N2, or another IAV sub-
type.  Other studies have also alluded to the significance of imprinting in terms of 
patterns seen during influenza outbreaks.  Gagnon et al. reported that individuals born 
during the H2N2 pandemic had higher mortality in 2009 and 2013-2014 to pandemic 
H1N1.vi  A recent retrospective study by Flannery et al. examined data from the Flu VE 
Network study, and demonstrated that patients’ initial infections with specific A(H1N1) 
virus clades did influence vaccine efficacy after exposure to A(H1N1).  Birth cohorts 
were based upon estimated immunologic priming with A(H1N1) viral clades circulating 

 
11
from 1918-1957 and from 1977-2015,vii similar to Gostic above.   
Several reports have described decreased vaccine efficacy for H3N2 in patients 
who have received repeated seasonal vaccination.  Petrie et al. described a cohort 
from 2014-2015 who had received influenza vaccination in the preceding season and 
had overall a minimal response to vaccination in terms of efficacy.viii  Similar findings 
were described in previous years (2012-2013 and 2013-2014).ix,x  Our research group 
recently completed the FLU1 study, which demonstrated a dramatic impact of repeated 
seasonal influenza vaccination on the post-vaccination T and B cell responses, and a 
fold-increase in HAI antibodies (reductions in all), in repeated vaccinated persons 
relative to subjects who had not been repeatedly vaccinated (Figure 2; manuscript in 
preparation).   
 

 
12
These findings underscore the importance of several factors that shape host 
immunity to influenza, and highlight a need for further studies employing newer 
methods to explore the complicated interplay of pre-existing circulating antibodies, 
imprinting, vaccination, and responses to novel influenza strains.  There is a need for a 
prospective evaluation to better elucidate the immunological mechanisms involved.   
 
2.2 Rationale 
 
Study Question:   
The immune response to the influenza vaccine is affected by many parameters, 
including prior imprinting to a specific influenza strain based on birth cohort, as well as 
prior influenza vaccination.  The FDA-approved, quadrivalent seasonal influenza vaccine 
that will be administered contains four distinct strains, two IAVs and two influenza B 
viruses (IBVs).  The approved seasonal influenza vaccine will be given for each season 
of influenza: 2018-2019, 2019-2020, and 2020-2021. 
We propose that the A(H1N1) birth cohort (individuals born between 1948-1957) 
will produce more robust HAI antibody and cellular responses to the H1N1 component of 
the vaccine as compared to the other three vaccine components, while the H3N2 birth 
cohort (individuals born between 1968-1977) will produce more robust HAI antibody and 
cellular responses to the H3N2 component of the vaccine.   
This study is not powered to test a formal null hypothesis.  Rather, it is a 
hypothesis-generating investigation that will hopefully lead to larger trials based on the 
findings.  The study will be conducted over the course of three years to increase the total 
sample population size and to validate our findings over different influenza seasons. 

 
13
The goal of this study, and the subsequent studies it will lead to, is to understand 
the impact on the human immune system’s response to the quadrivalent vaccine in 
individuals who have “imprinted” on specific influenza strains.  We will also consider the 
effects of repeated prior annual influenza vaccination on the immune system.  Collection 
of crevicular fluid, which may contain high concentrations of pathogen-specific IgA and 
IgG, will also be performed to investigate the relative dynamics between serum and 
saliva influenza-specific humoral immune responses.  
2.3 Potential Risks and Benefits 
 
The study utilizes FDA-approved influenza vaccines.  See package insert for full 
risks and benefits of the vaccine.   
2.3.1 Potential Risks 
 
The potential harms to participants are those associated with intramuscular 
injection of the FDA-approved influenza vaccine, possible reactions to the vaccine, 
having blood drawn, and breach of confidentiality.  
The potential harms of receiving the influenza vaccine include, but are not limited 
to: pain and redness at the injection site, muscle aches, fatigue, and headache. These 
risks are taken directly from the vaccine package insert from the FDA-approved 
influenza vaccine, with serious adverse reactions including anaphylaxis and Guillain-
Barre Syndrome.  For further details, see the attached package insert. The risks of these 
potential harms are considered to be outweighed by the potential benefits gained by 
vaccination, as well as the value of the new information yielded from the blood samples 
collected following vaccination.  

 
14
Blood sample collection and intramuscular injection involve transient discomfort 
and may cause fainting, the risk of which is mitigated by having the subject lie down prior 
to these procedures if needed, and remain seated in the clinic for 20 minutes after 
vaccination. The blood draw site may bruise, and this can be alleviated by holding 
pressure to this site following the blood draw. The sites of blood draw and intramuscular 
injection are potential sites of infection, but this risk is made very unlikely by the use of 
sterile technique.  Saliva collection is low risk but has the potential to cause minimal gum 
bleeding; a sponge connected to a plastic stick will be used to collect saliva from the 
area where the gums meet the teeth (crevicular fluid).    
2.3.2 Known Potential Benefits 
Participants will receive the FDA-approved influenza vaccine recommended by 
the United States Advisory Committee on Immunization Practices (ACIP) for anyone 6 
months and older, known to help protect against infection by influenza viruses. 
Participants will also receive financial reimbursement to account for time, travel, missed 
work, and inconvenience.   
 

 
15
3 
OBJECTIVES 
3.1 Study Objectives 
 
Primary:  
• 
To characterize the magnitudes and specificities of the hemagglutination inhibition (HAI) 
antibody response to each influenza A subtype or influenza B lineage included in the 
seasonal quadrivalent influenza vaccine in adults who had “imprinted” against either 
influenza A/H3N2 or influenza A/H1N1 and who have been either repeatedly vaccinated 
or unvaccinated 
 
Secondary:  
• 
To characterize the magnitudes and specificities of the neutralizing antibody (NAb) 
responses to each influenza A subtype or influenza B lineage included in the seasonal 
quadrivalent influenza vaccine in adults who had “imprinted” against either influenza 
A/H3N2 or influenza A/H1N1 and who have been either repeatedly vaccinated or 
unvaccinated   
• 
To characterize the duration of the immune responses to each subtype of the seasonal 
quadrivalent influenza vaccination in adults who have “imprinted” against H3N2 or H1N1 
and who have been either repeatedly vaccinated or unvaccinated.   
• 
To evaluate adaptive cellular immune responses and cytokines before and following 
vaccination     
 
Exploratory: 
• 
To evaluate systems biology measures (transcriptional and metabolic changes) after 
seasonal influenza vaccination in subjects from two distinct birth cohorts who have been 
either repeated vaccinated or unvaccinated   
• 
To evaluate ELISA binding antibodies to the highly conserved HA stalk, NA, and M2 
ectodomain regions of the influenza virus   
• 
To evaluate the influences on HAI antibody titers of age, gender, ethnicity, vaccination 
history, and/or birth cohort     
• 
To evaluate the temporal dynamics of the humoral immune response to influenza 
vaccination in oral fluid compared to serum.    
 
3.2 Study Outcome Measures 
Primary: 
• 
The proportion of subjects achieving seroprotection (titer of ≥ 40) or seroconversion 
(four-fold rise in HAI post-influenza vaccination compared to pre-vaccination, or 
achieving a titer of >40 if the pre-vaccination titer was <10), against each strain 
contained in the seasonal quadrivalent influenza vaccine, as measured by HAI antibody 
response approximately 28 days after vaccination 
• 
Geometric Mean Titers (GMTs) of serum HAI and neutralizing antibodies against each 
strain of the seasonal quadrivalent influenza vaccine, approximately 28 days after 
vaccination (Study Day 29)     

 
16
Secondary: 
• 
The proportion of subjects achieving seroprotection (titer of ≥ 40) or seroconversion 
(four-fold rise in HAI post-influenza vaccination compared to pre-vaccination, or 
achieving a titer of >40 if the pre-vaccination titer was <10), against each strain 
contained in the seasonal quadrivalent influenza vaccine, as measured by NAb 
responses approximately 28 days after vaccination   
• 
Geometric Mean Titers (GMT) of serum NAb against each strain of the seasonal 
quadrivalent influenza vaccine, approximately 28 days after vaccination 
• 
For HAI and NAb responses, the proportions of subjects achieving seroprotection (titer 
of ≥ 40) and seroconversion (four-fold rise in HAI post-influenza vaccination compared to 
pre-vaccination) against each strain of the seasonal quadrivalent influenza vaccine 
approximately 180 days after vaccination (Study Day 181) 
• 
Geometric Mean Titers (GMTs) of serum HAI and neutralizing antibodies against each 
strain of the seasonal quadrivalent influenza vaccine approximately 180 days after 
vaccination (Study Day 181) 
• 
Level of circulating follicular helper T cells (cTFH) at days 1, 8, and 15 
• 
Level of plasmablasts (Ag-specific) by ELISpot at days 1, 8, and 15 
• 
Level of CD4+ T-cells at days 1, 8, 15 and 29   
• 
Level of cytokine and chemokine levels (including IL-2, IFN-γ, IL-21, CD40L/154, IL10) at 
days 1, 3, 8 and 15     
Exploratory:  
• 
Differentially expressed genes (DEG) as measured by transcriptomics comparing 
baseline to Study Days 3 and 8       
• 
Differentially abundant metabolites (DAM) comparing baseline to Study Days 3 and 8 
• 
Levels of ELISA binding antibodies to the highly conserved HA stalk, NA, and M2 
ectodomain, before and approximately 28 days after vaccination  
• 
Multivariate analyses of HAI and neutralizing antibody titers with regards to age, gender, 
ethnicity, vaccination history, and birth cohort       
• 
Levels of IgA and IgG binding to a panel of influenza antigens and HAI titers to each 
component of the quadrivalent influenza vaccine in the crevicular fluid vs serum at days 
1, 8, 15, 29, and 180 
 
 
 
 

 
17
4 
STUDY DESIGN 
This study will be a prospective pilot study conducted over the course of three years (with three 
specific influenza seasons studied).  For each year (2018-2019, 2019-2020, and 2020-2021), 
two cohorts of 10 participants each, who are in good health and meet all eligibility criteria, will be 
recruited. The H3N2 cohort (N=30 total, 10 per year) will consist of participants born between 
1968-1977, and the H1N1 cohort (N=30 total, 10 per year) will consist of participants born 
between 1948-1957.  The H3N2 cohort and the H1N1 cohort will consist of participants who 
have received the influenza vaccine <2 of the past 5 years, and also participants who have 
received the influenza vaccine >3 of the past 5 years.  The participants in the two cohorts will be 
similar with regard to prior seasonal vaccination history.  Enrollments will be stratified based on 
vaccination history to ensure balance between the two birth cohorts.   
 
Each participant will make a total of six visits to the Hope Clinic. Day 1 will include the informed 
consent process, and screening to ensure the subject meets all inclusion criteria and meets no 
exclusion criteria.  For the consenting and eligible subject, the visit will also include pre-
vaccination phlebotomy for baseline immunogenicity laboratory assays, which will include 
assays for: HAI Ab and NAb, plasmablasts, memory B cells, cTFH cells, T cell responses, 
cytokine/chemokine levels (i.e., IL-2, IFN-γ, IL-21, CD40L/154, IL10), and systems biology 
analysis.  Baseline oral fluid (approximately 1-2cc) will also be collected for evaluation of levels 
of IgA and IgG (See Appendix B)xi.  After phlebotomy and saliva collection, the participants will 
receive the FDA-approved seasonal influenza vaccine.  Subsequent study visits on Days 3, 8, 
15, 29, and 180 will include collection of blood and saliva for immunogenicity assays.  Immune 
response assay results will be charted for each participant and summarized for each study arm.  
Statistical tests for differences in responses between study arms will be performed.   
 
 

 
18
5 
STUDY POPULATION 
 
5.1 Selection of the Study Population 
 
The target sample size will be 60 participants total, 20 participants per year.  Up to 100 
participants will potentially be screened for study enrollment over the course of the three 
years.  Males and females who were born between 1968-1977 (H3N2 birth cohort) and 
1948-1957 (H1N1 birth cohort) will be recruited.  Prior vaccination history will be obtained 
by vaccination records, if available, and by participant report.  The participants in the two 
cohorts will be similar with regard to prior seasonal vaccination history, and enrollments 
will be stratified based on vaccination history to ensure balance between the two birth 
cohorts.  Participants will be screened for eligibility according to the inclusion/exclusion 
criteria. Informed consent will be obtained for study participation. 
5.2 Inclusion/Exclusion Criteria 
 
Subjects eligible to participate shall meet all of the following inclusion criteria: 
 
1. Capable of informed consent and provision of written informed consent before 
any study procedures. 
 
2. Capable of attending all study visits according to the study schedule. 
 
3. Males or females born between 1968-1977 or 1948-1957. 
 
4. Are in good health, as determined by medical history and targeted physical exam 
related to this history. 
 
5. Oral temperature is less than 38°C. 
 
6. Resting pulse rate is between 50 and 100 beats per minute. 
 
7. Female subjects of childbearing age must have a negative urine pregnancy test 
within 24 hours before study vaccination. 
 

 
19
8. Have either received the influenza vaccine at least 3 of the past 5 years or 
received the influenza vaccine 2 or less of the past 5 years. 
 
a. For the 2018-2019 influenza season: received the influenza vaccine at 
least 3 of the 5 past years between September 2013-June 2018 or have 
received the influenza vaccine in 2 or less of the past 5 years  
b. For the 2019-2020 influenza season: received the influenza vaccine at 
least 3 of the 5 past years between September 2014-June 2019 or have 
received the influenza vaccine in 2 or less of the past 5 years  
c. For the 2020-2021 influenza season: received the influenza vaccine at 
least 3 of the 5 past years between September 2015-June 2020 or have 
received the influenza vaccine in 2 or less of the past 5 years  
 
 
 
Subjects eligible to participate shall not meet any of the following exclusion 
criteria: 
 
1. Have an acute illness within 72 hours before vaccination. 
 
2. Have any condition that, in the opinion of the principal investigator, would place 
the subject at an unacceptable risk of harm or confound the interpretation of the 
study results. 
 
3. Have any acute or chronic medical condition that, in the opinion of the principal 
investigator, would make vaccination unsafe or interfere with the evaluation of 
immune response to study vaccination. 
 
4. Have a suppressed immune system as a result of illness, immunosuppressive 
medication, chemotherapy, or radiation therapy within 3 years prior to study 
vaccination. 
 
5. Have known HIV, hepatitis B, or hepatitis C infection. 
 
6. Have a known history of autoimmune disease.  
 
7. Have taken oral or parenteral corticosteroids of any dose within 30 days before 
study vaccination. 
 
8. Have taken high-dose inhaled corticosteroids within 30 days before study 
vaccination. 
 
9. Have received, or plan to receive, any licensed live vaccine within 30 days, or 
any licensed inactivated vaccine within 14 days, prior to, or after, study 
vaccination. 
 
10. Have planned receipt of any unlicensed or investigational medications, biologics, 
or vaccines for the duration of subject study participation. 
 

 
20
11. Have received immunoglobulin or other blood products, with the exception of 
Rho D immunoglobulin, within 90 days prior to study vaccination. 
 
12. Have donated blood or blood products within 30 days before study vaccination, 
or within 60 days after study vaccination, or plan to donate blood within 30 days 
of the last blood draw. 
 
13. Have known hypersensitivity or allergy to eggs, egg protein, chicken protein, or 
other compounds of the study vaccine. 
 
14. Have a history of severe reactions following vaccination with influenza virus 
vaccines. 

 
21
6 
STUDY PROCEDURES/EVALUATIONS 
 
6.1 Study Procedures 
 
Complete medical history will be obtained by interview of study subjects on Day 1 (Visit 1) 
prior to the study vaccination. Subjects will be asked about a known history of significant 
medical disorders, cancer, immunodeficiency, allergies, psychiatric illness, substance 
use, and autoimmune diseases. 
Medication history will include a review of all current medications and any medications 
taken in the last 30 days before study vaccination. Medications included in this history will 
include prescription medication, over-the-counter medication, vitamins, supplements, and 
prohibited treatments listed in the above Inclusion/Exclusion Criteria section. 
On Day 1 (Visit 1) and before the study vaccination, a targeted physical examination, if 
indicated by the patient’s medical history, may be performed by the principal investigator 
or a study investigator who is licensed to make medical diagnoses. At Visits 2-6, a 
targeted physical examination may be performed, if indicated.   
Vital signs (oral temperature, pulse rate, and blood pressure) will be measured on Day 1 
(Visit 1) before study vaccination. Subjects must neither smoke nor eat or drink anything 
hot or cold 10 minutes prior to measuring oral temperature. 
Height and weight will be measured on Day 1 (Visit 1) before study vaccination for the 
calculation of body mass index. 
Subjects will be observed in the clinic for at least 20 minutes after study vaccination on 
Day 1 (Visit 1).  

 
22
6.1.1 Laboratory Evaluations/Assays 
 
Urine pregnancy tests will be performed by the site laboratory on the same day as and 
prior to study vaccination (Day 1). Results must be negative and known prior to study 
vaccination. 
6.1.2 Special Assays or Procedures 
The immunologic assays will be performed according to the existing standard operating 
procedures in place at the Hope Clinic laboratory and at Emory Core Laboratories. 
6.1.3 Specimen Collection, Preparation, Handling and Analyses 
Blood and saliva will be collected at each of the six study visits as per the Study 
Schedule in Appendix A. 
The phlebotomy tubes and saliva collection tubes will be transported immediately from 
the research clinic to the research laboratory for sample processing (both are located 
within the Hope Clinic building) or analysis.  The processed specimens will be 
cryopreserved per the laboratory standard operating procedures. 
 
 
 

 
23
7 
STUDY SCHEDULE 
 
7.1 Screening/Enrollment/Baseline, Visit 01, Day 01 
 
Study participants will be given a description of the study and have an opportunity to have 
their questions and concerns addressed by the study principal investigator or designee. 
Subjects will be given the informed consent form to read and discuss with study staff, and 
if they wish to enroll they will sign the document before any study vaccination, 
procedures, or lab draws are performed. 
A medical history and targeted physical exam, if necessary, as outlined in the Study 
Procedures section will be conducted in order to assess for compliance with study 
inclusion and exclusion criteria.  
A list of any medications taken by participants in the past 30 days and those to be taken 
during any time throughout the study duration will be documented on the appropriate 
study form. 
A urine pregnancy test will be conducted for female participants, and a negative result will 
be required and be documented on the appropriate study form prior to any study 
interventions. 
Vital signs, including pulse rate, blood pressure, oral temperature, and height and weight 
will be measured and documented on the appropriate study form. 
Approximately 90 mL of blood will be collected for baseline study labs, and two tubes of 
saliva will be obtained as outlined in the Study Procedures section. 
A single intramuscular injection of the FDA-approved seasonal influenza vaccine will be 
administered in the deltoid muscle of the preferred arm, and subjects will be asked to 

 
24
remain at the clinic for 20 minutes to monitor for any reactions or adverse events. The 
injection site location and any reactions or adverse events will be documented on the 
appropriate study form. 
Subjects will be informed to contact the study clinic should they experience any severe 
reactions to the vaccine, and they will be asked to visit the clinic for evaluation should the 
principal investigator deem the reaction in need of evaluation. 
7.2 Follow-up and Final Visits 
7.2.1 Visit 02 Day 03, Visit 03 Day 08, Visit 04 Day 15, Visit 05 Day 29, Visit 
06 Day 180 
 
Interim medical history and targeted physical exam and vital signs (including pulse rate, 
blood pressure, and oral temperature), if necessary, will be conducted. 
Medications taken during the interim from the previous visit will be documented on the 
appropriate study form. 
On visits 02, 03, 04, 05 and 06 approximately 32 mL, 32 mL, 48 mL, 90 mL and 90 mL of 
blood will be collected, respectively. Two tubes of saliva will also be obtained on visits 
03, 04, 05 and 06. 
7.3 Early Termination Visit (if needed) 
 
The following will be performed at an early termination visit, if necessary, for subjects who 
withdraw or who are withdrawn from the study: 
Interim medical history and targeted physical exam, if necessary, will be conducted. 

 
25
Medications taken during the interim from the previous visit will be documented on the 
appropriate study form. 
Vital signs, including pulse rate, blood pressure, and oral temperature will be measured 
and documented on the appropriate study form. 
Approximately 90 mL of blood will be collected for immune response study assays as 
outlined in the Study Procedures section. 
7.4 Visit Windows 
 
Visit 01 (Day 01) and Visit 02 (Day 03) will not be allowed a window. 
Visit 03 (Day 08) will be allowed a window to include Day 07, Day 08 and Day 09. 
Visit 04 (Day 15) will be allowed a window to include Day 14, Day 15, and Day 16. 
Visit 05 (Day 29) will be allowed a window to include Day 28, Day 29, Day 30 and Day 
31. 
Visit 06 (Day 181) will be allowed a window to include Days 166-180, and Days 181-196. 
 
 
 

 
26
8 
STATISTICAL CONSIDERATIONS 
 
8.1 Introduction 
 
The goal of this study is to assess the difference in immune response to the seasonal 
influenza vaccine between two distinct birth cohort populations, one of which has 
imprinted on H3N2 and the other on H1N1. This is a pilot study designed to occur over 
the course of three years, and demonstrate the potential utility of a subsequent larger 
scale study.  
8.2 Study Question 
 
This study is not powered to test a formal null hypothesis.  Rather, it is a hypothesis 
generating investigation that will hopefully lead to larger trials based on the findings. 
It is proposed that the H1N1 birth cohort (individuals born between 1948-1957) will 
produce a more robust H1N1 HAI antibody response to the H1N1 component of the 
seasonal vaccine, while the H3N2 birth cohort (individuals born between 1968-1977) will 
produce a more robust H3N2 HAI antibody response to the H3N2 component of the 
vaccine.  The quadrivalent seasonal influenza vaccine that will be administered will 
contain four distinct strains.  
8.3 Study Outcome Measures 
Primary: 
• 
The proportion of subjects achieving seroprotection (titer of ≥ 40) or seroconversion 
(four-fold rise in HAI post-influenza vaccination compared to pre-vaccination, or 
achieving a titer of >40 if the pre-vaccination titer was <10), against each strain 
contained in the seasonal quadrivalent influenza vaccine, as measured by HAI antibody 
response approximately 28 days after vaccination 

 
27
• 
Geometric Mean Titers (GMTs) of serum HAI and neutralizing antibodies against each 
strain of the seasonal quadrivalent influenza vaccine, approximately 28 days after 
vaccination (Study Day 29)     
Secondary: 
• 
The proportion of subjects achieving seroprotection (titer of ≥ 40) or seroconversion 
(four-fold rise in HAI post-influenza vaccination compared to pre-vaccination, or 
achieving a titer of >40 if the pre-vaccination titer was <10), against each strain 
contained in the seasonal quadrivalent influenza vaccine, as measured by NAb 
responses approximately 28 days after vaccination   
• 
Geometric Mean Titers (GMT) of serum NAb against each strain of the seasonal 
quadrivalent influenza vaccine, approximately 28 days after vaccination 
• 
For HAI and NAb responses, the proportions of subjects achieving seroprotection (titer 
of ≥ 40) and seroconversion (four-fold rise in HAI post-influenza vaccination compared to 
pre-vaccination) against each strain of the seasonal quadrivalent influenza vaccine 
approximately 180 days after vaccination (Study Day 181) 
• 
Geometric Mean Titers (GMTs) of serum HAI and neutralizing antibodies against each 
strain of the seasonal quadrivalent influenza vaccine approximately 180 days after 
vaccination (Study Day 181) 
• 
Level of circulating follicular helper T cells (cTFH) at days 1, 8, and 15 
• 
Level of plasmablasts (Ag-specific) by ELISpot at days 1, 8, and 15 
• 
Level of CD4+ T-cells at days 1, 8, 15 and 29   
• 
Level of cytokine and chemokine levels (including IL-2, IFN-γ, IL-21, CD40L/154, IL10) at 
days 1, 3, 8 and 15     
Exploratory:  
• 
Differentially expressed genes (DEG) as measured by transcriptomics comparing 
baseline to Study Days 3 and 8       
• 
Differentially abundant metabolites (DAM) comparing baseline to Study Days 3 and 8 
• 
Levels of ELISA binding antibodies to the highly conserved HA stalk, NA, and M2 
ectodomain, before and approximately 28 days after vaccination  
• 
Multivariate analyses of HAI and neutralizing antibody titers with regards to age, gender, 
ethnicity, vaccination history, and birth cohort 
• 
Levels of IgA and IgG binding to a panel of influenza antigens and HAI titers to each 
component of the quadrivalent influenza vaccine in the crevicular fluid vs serum at days 
1, 8, 15, 29, and 180       

 
28
8.4 Sample Size Considerations 
The study is a hypothesis-generating study with a convenient sample size. 
8.5 Participant Enrollment and Follow-Up 
Subjects will be enrolled and followed for 180 days, with six clinic visits. 
8.6 Analysis Plan 
 
The study investigators will perform descriptive statistics to describe the participants 
enrolled in each group, including demographic parameters and influenza vaccination 
history. 
Immune responses assessed in serum samples obtained immediately prior to vaccination 
(Day 1) and after vaccination will be summarized in terms of HAI or neutralizing antibody 
titers, and other humoral and cellular assay results. Individual antibody results are 
reported as a titer with values of 10*2k, where k=0, 1, 2, etc. The lower limit of detection 
of both assays is 1:10; values below the limit of detection are reported as ‘<10’, and for 
analysis are imputed as one-half the limit of detection (10/2 = 5).  
Seroconversion is defined as either a pre-vaccination HAI titer <1:10 and a post-
vaccination HAI titer ≥1:40 or a pre-vaccination HAI titer ≥1:10 and a minimum four-fold 
rise in post-vaccination HAI titer. HAI summaries include tabular and graphical displays of 
GMT, percentage of subjects with seroconversion, and percentage of subjects with titers 
≥1:40. Summaries of antibody data will include tabular and graphical displays of GMT, 
and percentage of subjects with titers ≥1:40 for all visits as well as percentage of subjects 
with a four-fold increase (to titer ≥1:40) for post-vaccination visits.  Seroprotection is 
defined as achieving a day 29 HAI titer of ≥40.  
Group-specific or pair-wise comparisons will be considered for the percentage of subjects 
achieving seroconversion or seroprotection at 28 days after the study vaccination as well 
as the magnitude of HAI titer at the same time point. 
For each comparison of interest, a Chi-square test will be used to compare the 
percentage of subjects with seroconversion, while a non-parametric test (Kruskal-Wallis 
or Wilcoxon Mann-Whitney) will be used for comparisons of magnitude of HAI titer, other 
adaptive immune response measures, and the individual ‘omics (systems biology) data. 
Statistical significance will be considered at a level of p= 0.05, without adjustment for 
multiple comparisons. 
Systems biology outcomes will be assessed and analyzed using standard bioinformatics 
software packages to compare Days 1 (baseline) with Days 03 and 08 for each individual 
subject, and will be summarized for the subjects within each study arm. 

 
29
9 
QUALITY CONTROL AND QUALITY ASSURANCE 
The study will undergo internal quality control and quality assurance per the Hope Clinic 
standard operating procedures.   
 

 
30
10 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
10.1 Ethical Standard 
 
The site principal investigator will ensure that this trial is conducted in full conformity with  
principles of the Belmont Report: Ethical Principles and Guidelines for the Protection of  
Human Subjects of Research of the National Commission for the Protection of Human  
Subjects of Biomedical and Behavioral Research (April 18, 1979) and codified in 45 CFR 
46, 21 CFR 50 and 56, and ICH E6; 62 Federal Regulations 25691 (1997), if applicable.  
10.2 Institutional Review Board 
 
Prior to enrollment of subjects into this trial, the protocol and informed consent form will 
be reviewed and approved by the Emory IRB. The responsible official for the IRB will sign 
the IRB letter of approval of the protocol prior to the start of this study.  
10.3 Informed Consent Process 
 
The site principal investigator and designees will choose subjects in accordance with the 
eligibility criteria detailed in Section 5. Before any study procedures are performed, 
subjects must sign an informed consent form that complies with the requirements of 21 
CFR Part 50 and 45 CFR 46 and the local IRB. 
 
Informed consent is a process that is initiated prior to an individual agreeing to participate 
in a study and continuing throughout the individual’s study participation. Before any study 
procedures are performed, subjects will receive a comprehensive explanation of the 
proposed study procedures and study interventions/products, including the nature and 
risks of the trial, alternate therapies, any known adverse effects, and the other elements 
that are part of obtaining proper informed consent. Subjects will also receive a detailed 
explanation of the proposed use and disclosure of their protected health information, 
including specifically their serum samples. Subjects will be allowed sufficient time to 
consider participation in the study, after having the nature and risks of the trial explained 
to them, and have the opportunity to discuss the trial with their family, friends or legally 
authorized representative or think about it prior to agreeing to participate.  
 
Informed consent forms describing in detail the study interventions/products, study 
procedures, risks and possible benefits are given to subjects. The informed consent form 
must not include any exculpatory statements. Informed consent forms will be IRB-
approved and subjects will be asked to read and review the appropriate document. Upon 
reviewing the appropriate document, the site principal investigator (or designee) will 
explain the research study to subjects and answer any questions that may arise. Subjects 
must sign the informed consent form, and written documentation of the informed consent 
process is required prior to starting any study procedures/interventions being done 
specifically for the trial, including administering study product.  
 
Study personnel may employ IRB-approved recruitment efforts prior to obtaining the 
subjects consent; however, before any study procedures are performed to determine 
protocol eligibility an informed consent form must be signed. Subjects will be given a copy 

 
31
of all informed consent forms that they sign. 
 
By signing the informed consent form, subjects agree to complete all evaluations required 
by the trial, unless the subject withdraws voluntarily, or is withdrawn or terminated from 
the trial for any reason.  
 
The rights and welfare of subjects will be protected by emphasizing to subjects that the 
quality of their medical care will not be adversely affected if they decline to participate in 
or withdraw from this trial. 
10.4 Exclusion of Women, Minorities, and Children (Special 
Populations) 
 
This trial will be inclusive of all adults who meet the Subject Inclusion/Exclusion Criteria, 
regardless of religion, sex, or ethnic background. 
10.5 Subject Confidentiality 
 
Subjects’ records and specimens will have code numbers and will not be identified by 
name. Subject confidentiality is strictly held in trust by the principal investigator and the 
Hope Clinic personnel directly involved in the study. This confidentiality is extended to 
cover testing of biological samples, in addition to the clinical information relating to 
participating subjects. The study protocol, documentation, data, and all other information 
generated will be held in strict confidence. 
10.6 Future Use of Stored Specimens 
 
Subjects participating in this study are agreeing to allow the investigators to keep any 
remaining samples for possible use in future research studies, such as examining 
additional immunological assessments or testing for antibodies against other viruses or 
bacteria. Some samples may be stored at the Hope Clinic. Samples may be shared with 
other investigators at other institutions. The samples will not be sold or used directly for 
production of any commercial product. No human genetic tests will be performed on 
samples. Each sample will be encoded (labeled) only with a barcode and a unique 
tracking number to protect subjects’ confidentiality. There are no benefits to subjects in 
the collection, storage and subsequent research use of specimens. Reports about future 
research done with subjects’ samples will NOT be kept in their health records. 
 
The subject’s agreement to allow future use of their specimens can be changed at any 
time prior to the end of the study by notifying the study doctors or nurses in writing. 
However, if the subject originally consents to future use and subsequently changes 
his/her decision, any data from a previously collected sample may still be used for this 
research. 
 

 
32
11 
LITERATURE REFERENCES 
 
 
i WHO (2016). Influenza (Seasonal), Fact Sheet. 
<http://www.who.int/mediacentre/factsheets/fs211/en/> 
ii “Disease Burden of Influenza | Seasonal Influenza (Flu) | CDC,” May 22, 2018. 
https://www.cdc.gov/flu/about/disease/burden.htm. 
iii Francis T. On the Doctrine of Original Antigenic Sin. Proc Am Philos Soc. 1960; 104:572–578. 
iv Gostic KM et al. Potent protection against H5N1 and H7N9 influenza via childhood 
hemagglutinin imprinting. Science Nov 2016; 722-726 
v Fonville, J.M., Wilks, S.H., James, S.L., Fox, A., Ventresca, M., Aban, M., Xue, L., Jones, T.C., 
Le, N.M., Pham, Q.T., et al. (2014). Antibody landscapes after influenza virus infection or 
vaccination. Science 346, 996–1000. 
vi Gagnon, Alain, Enrique Acosta, Stacey Hallman, Robert Bourbeau, Lisa Y. Dillon, Nadine 
Ouellette, David J. D. Earn, et al. “Pandemic Paradox: Early Life H2N2 Pandemic Influenza 
Infection Enhanced Susceptibility to Death during the 2009 H1N1 Pandemic.” MBio 9, no. 1 
(March 7, 2018): e02091-17. https://doi.org/10.1128/mBio.02091-17. 
vii Flannery, Brendan, Catherine Smith, Rebecca J. Garten, Min Z. Levine, Jessie R. Chung, 
Michael L. Jackson, Lisa A. Jackson, et al. “Influence of Birth Cohort on Effectiveness of 2015–
2016 Influenza Vaccine Against Medically Attended Illness Due to 2009 Pandemic Influenza 
A(H1N1) Virus in the United States.” The Journal of Infectious Diseases 218, no. 2 (June 20, 
2018): 189–96. https://doi.org/10.1093/infdis/jix634. 
viii Joshua G Petrie, Ryan E Malosh, Caroline K Cheng, Suzanne E Ohmit, Emily T Martin, 
Emileigh Johnson, Rachel Truscon, Maryna C Eichelberger, Larisa V Gubareva, Alicia M Fry, 
Arnold S Monto; The Household Influenza Vaccine Effectiveness Study: Lack of Antibody 
Response and Protection Following Receipt of 2014–2015 Influenza Vaccine, Clinical Infectious 
Diseases, Volume 65, Issue 10, 30 October 2017, Pages 1644–1651. 
ix Ohmit SE, Petrie JG, Malosh RE, Fry AM, Thompson MG, Monto AS. Influenza vaccine 
effectiveness in households with children during the 2012–2013 season: assessments of prior 
vaccination and serologic susceptibility. J Infect Dis 2015; 211:1519–28. 
x Ohmit SE, Petrie JG, Malosh RE et al. Substantial influenza vaccine effectiveness in 
households with children during the 2013–2014 influenza season, when 2009 pandemic 
influenza a(H1N1) virus predominated. J Infect Dis 2016; 213:1229–36. 
x McKie, A., Vyse, A. & Maple, C. Novel methods for the detection of microbial antibodies in oral 
fluid. Lancet. Infect. Dis. 2, 18–24 (2002). 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
33
 
 
SUPPLEMENTS/APPENDICES 
Appendix A. 
 
Study Visit Number 
V01 
V02 
V03 
V04 
V05 
V06 
Study Day Relative 
to Study 
Vaccination 
D1 
D3 
D8  
(+/-1D) 
D15  
(+/-2D) 
D29  
(+/-3D) 
D181 
(+/- 14D) 
Obtain Informed 
Consent# 
X* 
  
  
  
  
  
Review Eligibility 
Criteria 
X* 
  
  
  
  
  
Medical History+ 
X* 
X 
X 
X 
X 
X 
Concomitant 
Medications 
X*% 
X 
X 
X 
X 
X 
Vital Signs  
X*&$ 
(X)$ 
(X)$ 
(X)$ 
(X)$ 
(X)$ 
(Oral Temperature, 
Pulse Rate, and BP) 
X*&$ 
(X)$ 
(X)$ 
(X)$ 
(X)$ 
(X)$ 
Height and Weight 
X* 
  
  
  
  
  
Targeted Physical 
Examination 
(X)* 
(X) 
(X) 
(X) 
(X) 
(X) 
Urine Pregnancy 
Test 
X*@ 
  
  
  
  
  
Study Vaccination 
X 
  
  
  
  
  

 
34
 
20-minute 
Evaluation Period 
After Study 
Vaccination 
X 
  
  
  
  
  
 Saliva Collection 
for Study Assays  
(~1-2mL/visit) 
X 
 
X 
X 
X 
X 
Venous Blood 
Collection for Study 
Assays 
X 
X 
X 
X 
X 
X 
Serological Assays 
10 
- 
- 
- 
10 
10 
Cellular 
Immunology and 
'Omics Assays 
64 
16 
16 
32 
64 
64 
Future Research 
16 
16 
16 
16 
16 
16 
Per Visit Blood 
Volume Total (mL)! 
90 
32 
32 
48 
90 
90 
Cumulative Blood 
Volumes! 
90 
122 
154 
202 
292 
382 
 
#Prior to study procedures 
*Prior to study vaccination. 
%All current medications and medications taken within 30 days prior to day 1 of the study. 
&Vital signs assessed on Day 1 (Visit 1) before the study vaccination will be considered as baseline. 
$Subjects must not eat or drink anything hot or cold, or smoke within 10 minutes prior to taking oral temperature. 
() Targeted physical examination if indicated based on review of complete or interim medical history. 
@Must be performed on all female subjects of childbearing potential within 24 hours prior to each study vaccination 
and results must be negative and known prior to each study vaccination. 
+Complete medical history by interview of subjects to be obtained on Day 1 (Visit 01) prior to the first study 
vaccination and interim medical history by interview of subjects to be obtained at follow-up visits after the first 
study vaccination. 
!Blood volumes are approximate and measured in milliliters (mL). 
 
 
 
 
 
 
 
 
 
 

 
35
 
 
 
 
Appendix B. Saliva collection 
 
Saliva will be obtained per the Study Schedule using Oracol Plus (Malvern Medical) devices, 
which are specifically designed to sample crevicular fluid (saliva expressed from the area where 
the teeth meet the gums) as this is the component of saliva that is particularly rich in IgA and 
IgG. Sample will be collected by the subject by gentle brushing of the crevicular region (gum 
line) with an Oracol Plus saliva collection device for 60 sec or until saturation. The sponge will 
be inserted into a labeled plastic test tube, capped, and stored at -80○C until analysis.