Clinical trial NCT03603509
Conditions: Influenza
Keywords: Fluad, MF59, Fluzone, HDFlu, Influenza, Vaccine
Sponsor: Mayo Clinic
Start date: 2018-08-27; Completion date: 2019-01-02
Enrollment: 241 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this research study is to better understand the immune response to the Adjuvanted Subunit flu vaccine (MF59) and the High Dose flu vaccine (HDFlu) in people 65 years of age and older. The research team will be studying why immune response diminishes as people get older in both men and women. The ultimate goal is to understand how flu immunity develops after vaccination. This information may lead to the development of more effective flu vaccines in the future.

Detailed description:
The overall goal of this proposal is to determine how vaccine type, sex, and gene expression influence both innate and T helper cell immune responses using systems biology and bioinformatics as tools to comprehensively assess the human transcriptome. We will evaluate sex-dependent immune responses to two unique influenza vaccines in a population of older adults; the recently FDA-licensed MF59-adjuvanted influenza subunit vaccine and the high-dose split virion influenza virus vaccine. The influence of sex on immune response to vaccination has been observed across multiple vaccines (including standard dose influenza vaccines, but the mechanisms are unknown, it affects all age groups regardless of hormonal status, and existing studies focus almost exclusively on humoral immune responses. Relatively little is known about the effect of sex on innate and T helper responses following vaccination and we are unaware of any studies evaluating the effect of sex on immune responses to adjuvanted influenza vaccine. The presence of adjuvant (MF59Flu) or higher antigen (Ag) dose leads to greater immunogenicity through mechanisms that have not been fully deciphered and are likely to be different. Further, a direct comparison of innate and T helper immune responses between adjuvanted and high dose influenza vaccines has not been reported.

The study design will include 200 generally healthy individuals (ages ≥65) who meet all inclusion criteria. 100 subjects will receive each vaccine with equal sex representation in each subgroup (a factorial design for sex by vaccine type). Subjects will undergo venipuncture for blood samples (\~100 mL each, sufficient for the proposed assays) before vaccination and at three timepoints after vaccination (Day 1, Day 8, Day 28).

The clinical characterization of our study subjects will include demographic information, height, weight, BMI, waist circumference, medications, and medical conditions that do not meet exclusion criteria (see Protection of Human Subjects). We will also quantify blood leukocyte populations (CBC, WBC differential).

Immunosenescence and cytomegalovirus (CMV) infection can affect influenza vaccine-induced immune responses. We will evaluate whether CMV seropositivity or other measures of immunosenescence are associated with immune response and whether they interact with vaccine type/sex.

We will monitor/characterize transcriptional changes (mRNA and miRNA) as well as measures of immune function (cytokine secretion, leukocyte surface phenotype, hemagglutination inhibiting antibody titer, and memory B cell ELISPOT) at each time point.

Arms: arm 1: Subjects receive a single dose of the Fluad influenza vaccine. arm 2: Subjects receive a single dose of the Fluzone High-Dose influenza vaccine.
Interventions: intervention 1: Fluad Vaccine intervention 2: Fluzone High-Dose
Intervention descriptions: intervention 1: FLUAD is an inactivated influenza vaccine indicated for active immunization against influenza. disease caused by influenza virus subtypes A and type B contained in the vaccine. FLUAD is. approved for use in persons 65 years of age and older. intervention 2: FLUZONE® HIGH-DOSE vaccine is indicated for people 65 years of age and older to help prevent influenza disease caused by influenza A and B strains contained in the vaccine.

Protocol documents:
SAP_003.pdf: 
Transcriptomic Signatures of Influenza Vaccine 
Responses 
 
NCT03603509  
 
May 19, 2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Analysis Approach. Subjects will be randomly assigned to vaccine type. For all laboratory assays, 
randomized complete or incomplete block designs that include technical replicates and laboratory 
controls will be utilized to ensure sample and assay processing are balanced with respect to collection 
date, vaccine, and sex, keeping specimens from the same subject adjacent to one another. Data will be 
scrutinized to identify potential biases, batch effects, or failed assays. We will normalize mRNA-
sequence data by Conditional Quantile Normalization (CQN), adjusting for library size, gene length, and 
GC content. Our primary aim is to test the hypotheses that vaccine type (Fluad vs Fluzone) is associated 
with the change in immune response as measured by hemagglutination inhibition (HAI) Antibody titer. 
To test this hypothesis, we will use the Wilcoxon Rank Sum test to test for differences between the 
vaccine types in the change from baseline HAI to Day 28 HAI Ab titers. 

Prot_001.pdf: 
 
 
Transcriptomic Signatures of Influenza Vaccine 
Responses 
 
NCT03603509 
 
May 19, 2019 


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IRB Minimal Risk Protocol Template  
 
General Study Information 
 
Principal Investigator: Richard Kennedy, PhD       
 
 
 
 
 
 
 
 
Study Title: Transcriptomic Signatures of Influenza Vaccine Responses 
 
Protocol version number and date:  Version 7, 05/19/2019    
 
IRB #17-010601 
 
Research Question and Aims 
 
Specific aims and Hypotheses: 
 
Specific Aim 1: Identify Innate Cell Transcriptomic Signatures Associated with Sex and Immune 
Response to Influenza Vaccines in the Elderly. To do this we will:  
• Aim 1.1 Test the hypotheses that vaccine type (MF59Flu vs HDFlu) and/or sex are associated with variations 
in innate cell immune outcomes (e.g., APC activation phenotype and cytokine/chemokine secretion).  
• Aim 1.2 Test the hypothesis that the increased Ag dose in HDFlu results in greater activation/suppression of 
the innate genes/genesets previously associated with immune responses to SDFlu. We will also test the 
hypothesis that the MF59 adjuvant results in activation/suppression of additional specific innate genes/genesets 
compared to HDFlu (see Overview and Rationale of Specific Aim 1).  
• Aim 1.3 Test the hypothesis that innate immune cell transcriptomic signatures mediate the association of 
vaccine type (or sex) with innate cell immune outcomes.  
• Aim 1.4 Test the hypothesis that the innate cell immune outcomes and transcriptomic signatures from Aim 1.3 
will predict markers of humoral immunity (HAI Ab and B cell ELISPOT response).  
 
Specific Aim 2: Identify CD4+ T Helper (Th) Cell Transcriptomic Signatures Associated with Sex and 
Immune Response to Influenza Vaccines in the Elderly. To do this we will:  
• Aim 2.1 Test the hypotheses that vaccine type (MF59Flu vs HDFlu) and/or sex are associated with variations 
in Th cell immune outcomes (e.g., Th phenotype and cytokine production).  
• Aim 2.2 Test the hypothesis that the increased Ag dose in HDFlu results in greater activation/suppression of 
the Th cell genes/genesets previously associated with immune responses to SDFlu. We will also test the 
hypothesis that the MF59 adjuvant results in activation/suppression of additional specific genes/genesets in 
CD4+ Th cells compared to HDFlu (see Overview and Rationale of Specific Aim 2).  
• Aim 2.3 Test the hypothesis that Th cell transcriptomic signatures mediate the association of vaccine type (or 
sex) with Th cell immune outcomes.  
• Aim 2.4 Test the hypothesis that the Th cell immune outcomes and transcriptomic signatures from Aim 2.3 
will predict markers of humoral immunity (HAI Ab and B cell ELISPOT response).  
 
Background: 


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Scientific Premise of the Proposed Research. While older populations are at the highest health risk from 
seasonal influenza, these populations have poor immune responses to standard-dose seasonal influenza vaccine 
(SDFlu).1, 2 Our study is focused on two influenza vaccines for use in subjects ≥65 years of age: MF59Flu, an 
adjuvanted subunit vaccine (licensed in late 2015);3 and HDFlu, a split-virus vaccine containing 4x the 
hemagglutinin protein (licensed in 2009).4, 5 These vaccines have demonstrated greater immunogenicity than 
SDFlu in older populations, but little is known about the underlying mechanisms.5-16 For these two vaccines, 
systems biology studies designed to identify the mechanisms for improved immune response have never 
been conducted in older individuals, nor have sex-based differences been adequately studied. The goals of 
this proposed project are to examine inter-individual variations in innate and T helper (Th) responses, and 
identify transcriptomic signatures associated with immune responses (including correlates of protective 
immunity) to influenza vaccines in older individuals.  
 
This is important for the following reasons:  
A. Public Health Importance. Seasonal influenza A is believed to kill between 3,000–49,000 annually,17 
resulting in over 250,000 excess hospitalizations and annual costs of >$90 billion in the US.18 Influenza 
morbidity and mortality increase significantly with age.19, 20 More than 90% of influenza-associated deaths 
occur in individuals ≥65 years of age, and are predominantly associated with influenza A/H3N2.21 During 
the 2014–2015 influenza season, adults age ≥65 years had an influenza-related hospitalization rate of 314 per 
100,000 people—a record high since surveillance began in 2005.19, 20 The efficacy of influenza vaccines is 
diminished in older adults and varies widely (15% to 75%), averaging <50%.1, 2, 22, 23 Considering the 
unprecedented population growth in persons ≥65 years of age and the significant public health impact of 
influenza, it is imperative that influenza vaccine-induced immunity in older adults be better 
understood.24-26 Due to in-creased mortality in the elderly and cognizant of budgetary limitations, we will 
focus on influenza A/H3N2.  
 
B. Knowledge Gap—Immune Response Differences to Adjuvanted vs. High-Dose Influenza Vaccines.  
HDFlu contains 60ug HA per influenza strain, and has been the focus of high-quality studies, including a study 
of 31,989 older individuals (≥ 65 years of age) that demonstrated higher immunogenicity (ratio of GMT 1.8) 
and 24% greater protection against influenza illness than SDFlu (15ug HA/strain/dose).5, 13, 14  
MF59Flu also induces higher immunogenicity and longer persistence of Ab titers than non-adjuvanted SDFlu.6-
12, 15, 27 A recent large study in 7,082 individuals (≥ 65 years of age) demonstrated significantly higher 
immunogenicity (p<0.001, seroconversion and HAI GMT) of MF59Flu vaccine compared to SDFlu 
(particularly against A/H3N2).15 A single small systems biology study comparing MF59Flu and SDFlu in 
immunoimmature children (14- to 24-months-old, n=90) identified significantly higher transcriptional responses 
to MF59Flu and identified early innate response signatures correlated with Day 28 Ab titers.28 These include 
M16 (a module associated with TLR and inflammatory signaling); M11 (a module regulating monocyte 
function); M75 (a module controlling IFN-induced antiviral response); M156 (a module associated with Ab 
secreting cells); and S3 (a module with genes involved in immunoglobulin production). These genesets and 
those identified in other systems biology studies of influenza vaccine response will be evaluated for their 
influence on immune responses to MF59Flu and HDFlu in this proposal.  
 
The underlying immunologic mechanisms for the improved immunogenicity of MF59Flu and HDFlu are 
largely unknown for older adults. Our proposal will answer key questions (as illustrated in Figure 1) 
regarding innate and Th immune responses to these vaccines, the underlying transcriptomic signatures, 
and their impact on humoral immunity in older adults. At a minimum, this proposal will fill the knowledge 


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gap by examining whether the higher immunogenicity of these vaccines is a result of greater 
activation/suppression of genes/genesets (mRNA and miRNA) previously associated with immune responses to 
SDFlu, or additional unsuspected genes/genesets controlling immune responses.  
 
C. Knowledge Gap—Sex-Based Differences in Immune Responses to Influenza Vaccines. Studies of 
Multiple vaccines (influenza, yellow fever, MMR, hepatitis A and B, herpes simplex [HSV] 2, rabies, 
smallpox, and dengue) demonstrate significantly higher Ab responses in females than males, as reported in a 
series of high-quality papers.16, 29-38 Sex-based differences in humoral immune responses are observed prior 
to puberty, during the reproductive years, and after reproductive senescence,29-38 suggesting that sex hormones 
are not the necessary—or sole—mediators of sex differences in humoral immune responses to vaccines.39, 40 
Despite significant evidence of immune response differences between the sexes, most vaccine studies do 
not analyze immune response outcome data by sex.41, 42  
Across a cohort of 556 older (ages 50–64) and 558 younger (ages 18–49) subjects, the SDFlu vaccine induced 
>1.5-fold higher A/H3N2-specific HAI Ab titers in women than men.29 Similarly, a study of SDFlu and HDFlu 
vaccine responses in 414 elderly subjects (ages 65–95) demonstrated higher rates of seroconversion in females 
than in males (p<0.05).31 However, no significant differences in Ab measures were found in cohorts of 494 and 
158 older adults after receiving SDFlu,43, 44 and sex was also not reliably associated with seroprotection 
and/or Ab titers in children and young adults receiving MF59Flu.45 These publications demonstrate in-
consistent—and sometimes conflicting—findings regarding sex-based effects on immune responses to 
influenza vaccine. Further research is needed, which is a stated priority for NIH research.  
Additionally, new vaccines containing the MF59 adjuvant are likely to alter the transcriptomic signatures 
associated with vaccine responses in elderly populations, and potentially alter the influence of sex on those 
responses. The current lack of knowledge is a critical barrier to understanding poor vaccine responses in the 
elderly, and such knowledge is foundational to the future development of new influenza vaccines.  
 
D. Impact of this Work. Given the substantially diminished efficacy of influenza vaccines with age and the 
importance of developing improved influenza vaccines,46 data from our studies could be used to inform directed 
and rational development of next-generation influenza vaccines and/or therapeutics47-57—although the 
development of such a vaccine is not the purpose of this proposal. Age-related immune dysfunction 
(immunosenescence) might be overcome by adjuvant stimulation of innate and/or Th cell-specific genes, which 
may be different in males and females (see Expected Results). For example, a TLR4 agonist GLA-SE has been 
shown to enhance Th1 responses to influenza vaccine in older adults.58 The identification of critical chemokine 
pathways involving CCR5 and its ligands (CCL5) (see Preliminary Data #7), predictive of influenza vaccine-
induced humoral immunity, may support the usage of CCR5 small molecule agonists/antagonists approaches in 
vaccines to modulate inflammatory response and T cell chemotaxis/activation for optimal Ab response.44, 59-62 
 
Study Design and Methods 
 
Methods:   
 
Subject Recruitment, Enrollment and Screening  
 
We will screen and recruit 400 generally healthy males and females ages 65 and older who meet all inclusion 
and exclusion criteria and are willing to receive a flu vaccine.  
 


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We will also screen and recruit 20 generally healthy males and females ages 18-40 as a control group. These 
subjects will only have a baseline blood draw (no flu vaccine administration and no follow-study visits). 
 
Eligible subjects who consent and enroll into the study will undergo a baseline blood draw (up to 108 mL) and 
then be randomly assigned to receive either the MF59 (Fluad) flu vaccine (N=200) or the High Dose (Fluzone) 
flu vaccine (N=200).  Subjects in the control group will undergo the baseline blood draw only (up to 108 mL).  
They will NOT receive a flu vaccine. The following sections describe the remaining study visits for the subjects 
who are ages 65 and older. 
 
Subjects will then be asked to come back for three additional blood draws after their flu vaccination: Day 1, 
Day 8, and Day 28 after vaccination. Each blood draw will be up to 105mL of blood.  
 
Flu vaccination can occur up to 2 weeks after the baseline blood draw. The Day 1 blood draw will occur the 
following day after vaccination. The Day 8 blood draw will occur 7-10 days after the vaccination. The Day 28 
blood draw will occur 25-31 days after vaccination.   
 
During the Baseline visit we will collect patient demographic information, height, weight, BMI, medication 
history, medical information, and information on alcohol and smoking use. Patients will also be offered the 
CDC’s influenza vaccine-specific Vaccine Information Sheet that is routinely provided to Mayo patients. 
 
During the 3 follow-up visits we will collect weight, medication history and medical information. 
 
Subjects will be remunerated $40 for each blood draw visit they complete. 
 
We will also mail subjects, roughly a year after participation, 3 different sleep questionnaires as a way to assess 
sleep hygiene and immune response.  
 
 
 
 
 
Resources: Potential participants will be identified through routine clinical appointments, advertising with 
flyers, brochures, an EMR search to identify mail-merge candidates, and existing lists/databases of individuals 
interested in being contacted about participation in research studies. Subject recruitment and advertisement will 
occur on Mayo campus and at various sites off campus (e.g., retirement centers). Permission will be obtained 
from each location before recruitment begins. We will also use newspaper and social media advertisements.   
 
Based on a 2016 census by the United States Government, there were between 30,447 - 54,748 people 65 years 
of age or older in Olmsted County.  
 
 This project has been funded through the NIH through an R01 award.  
 
The majority of the laboratory assays will be performed in the Vaccine Research Group Laboratory which 
occupies approximately 3,500 square feet on the sixth floor of the Mayo Clinic Guggenheim Research Building. 
Equipment not in this laboratory is readily available in institutional core laboratories and is freely shared. 


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  (1a)  This is a multisite study involving Mayo Clinic and non Mayo Clinic sites. When checked, describe in 
detail the research procedures or activities that will be conducted by Mayo Clinic study staff. 
 
  (1b)  Mayo Clinic study staff will be engaged in research activity at a non Mayo Clinic site.  When checked, 
provide a detailed description of the activity that will be conducted by Mayo Clinic study staff. 
 
Subject Information 
 
Target accrual: 420.  This includes 400 subjects ages 65 and older (over-recruiting by 10% to account for 
subject drop-out and insufficient cell recovery from biospecimens) and 20 younger subjects (18-40 years of age) 
as a control group. 
 
Subject population:  Potential participants will be identified through routine clinical appointments, advertising 
with flyers, brochures, an EMR search to identify mail-merge candidates, and existing lists/databases of 
individuals interested in being contacted about participation in research studies. Subject recruitment and 
advertisement will occur on Mayo campus and at various sites off campus (e.g., retirement centers). Permission 
will be obtained from each location before recruitment begins.   
Inclusion Criteria:  
• Male or female adults ages 18-40 or  65 and older at the time of enrollment 
• Eligible to receive Fluad® (MF59Flu) or Fluzone® (HDFlu) if age 65 or older 
• No history of anaphylactic reaction to gelatin, neomycin, or other vaccine component 
• Not pregnant 
• No immunosuppression or immunodeficiency 
• No acute illness at time of vaccination 
• Determined by medical history and clinical judgment to be eligible for the study, by being generally 
healthy, with no autoimmune or immunosuppressive conditions and having stable current medical 
conditions (subjects with preexisting stable disease, defined as disease not requiring significant change 
in therapy or hospitalization for worsening disease 12 weeks before receipt of study vaccine, will be 
eligible. A change in dose or therapy within a category (e.g., change from one nonsteroidal anti-
inflammatory drug to another) is allowed. A change to a new therapy category (e.g., surgery or addition 
of a new pharmacological class) is only allowed if it is not caused by worsening disease. A change to a 
new therapy category caused by worsening disease is considered significant and therefore ineligible for 
enrollment. 
 
 
 
 
• Patients with diabetes mellitus are eligible for inclusion if they have had a hemoglobin A1c 
measurement of <8.0 within the past 6 months prior to enrollment. These hemoglobin A1c 
measurements are recommended at least twice yearly by the American Diabetes Association (ADA), and 
the target levels here are representative of the goals of the ADA. These hemoglobin A1c levels will 
ensure that these participants have good glycemic control. (American Diabetes Association. American 


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Diabetes Association Position Statement: Standards of Medical Care in Diabetes— 2015. Diabetes Care 
2015;38(Suppl. 1): S1–S94) 
• Able to follow study procedures in the opinion of the investigator 
• Expected to be available for the duration of the study 
• Weighs >110 lbs 
Exclusion Criteria:   
• Known or suspected immunodeficiency or receiving treatment with immunosuppressive therapy 
including cytotoxic agents (e.g., for cancer, HIV, or autoimmune disease).  
• Subjects on corticosteroids will be excluded if ≥ 20mg of Prednisone (or equivalent drug) has been (or 
will be) administered daily for 2 weeks or more. Subjects will be eligible if corticosteroid therapy  has 
been discontinued for at least 30 days. 
• Serious chronic medical conditions including metastatic malignancy, severe chronic obstructive 
pulmonary disease requiring supplemental oxygen, end-stage renal disease with or without dialysis, 
clinically unstable cardiac disease, or any other disorder that, in the investigator’s opinion, precludes the 
subject from participating in the study. Diabetic patients will be excluded if they do not have a 
hemoglobin A1c measurement within the past 6 months or if they had a hemoglobin A1c measurement 
of an A1c >8.0 
• Receipt of any blood products, including immunoglobulin, within 6 months of study enrollment. 
• Current anticoagulant therapy or a history of bleeding diathesis that would contraindicate intramuscular 
(IM) injection. (Note: antiplatelet drugs such as aspirin and clopidogrel are permitted.) 
• Receipt of any vaccines within the past 30 days prior to enrollment 
• Receipt of the current seasonal influenza vaccine other than in this study 
• Acute illness within the last 30 days 
• Blood donation within the last 56 days prior to study enrollment and within 56 days following the last 
study visit 
• Pregnancy, Nursing or trying to conceive at the time of the study or for 28 days following the baseline 
visit 
• Any condition (e.g. allergic reaction, Guillain-Barre Syndrome) that precludes their receipt of the 
influenza vaccine 
• Currently taking antibiotics to treat a serious infection. Preventative use of antibiotics (i.e. oral surgery) 
is not an exclusion criterion.  
• Diagnosis of a cognitive disorder (e.g. Alzheimer’s, Dementia) 
• Anemia 
• Any medical condition that would, in the opinion of the investigator, interfere with the evaluation of the 
study objectives 
 
Visit Schedule for subjects 18-40 
 
 
Baseline 
Visit - Vaccination 
 
Review of 
Eligibility/Ineligibility 
X 


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Demographics 
X 
Study Questionnaire 
X 
Medication Review 
X 
Height/Weight 
X 
Informed Consent 
X 
Blood Draw – 108ml 
Xb,  
                               bFollowing standard practice, subjects will be provided juice, water and cookies after providing their blood sample 
 
Visit Schedule for subjects 65 and older 
 
 
Baseline 
Visit - 
Vaccination 
Post-Vaccination Visit  
 
Day 1 
Day 8 (-1 
day/+2    days) 
Day 28(±3 
days) 
Review of 
Eligibility/Ineligibility 
X 
 
 
 
Demographics 
X 
 
 
 
Study Questionnaire 
X 
 
 
 
Medication Review 
X 
X 
X 
X 
Height 
X 
 
 
 
Weight 
X 
X 
X 
X 
Informed Consent 
X 
 
 
 
Blood Draw – 108ml 
Xb,  
 
 
 
Blood Draw – 105ml 
 
Xb 
Xb 
Xb 
Flu vaccine 
            X 
 
 
 
Adverse Events 
            X 
X 
X 
X 
                              bFollowing standard practice, subjects will be provided juice, water and cookies after providing their blood sample 
 
Blood will either be collected using a blood collection bag or with tubes. The blood bag collection is the 
preferred method.  
 
Blood bag collection 
• Baseline visit: 108 mL collected in the blood bag. 5 mL will be separated into a redtop tube x1 and 3 mL 
into an EDTA tube x1 
• Day 1 visit:  105 mL collected in the blood bag. 5 mL will be separated into a redtop tube x1  
• Day 8 visit:  105 mL collected in the blood bag. 5 mL will be separated into a redtop tube x1 
• Day 28 visit: 105 mL collected in the blood bag. 5 mL will be separated into a redtop tube x1  
 
Biospecimen tube collection 
• Baseline visit: 5 mL redtop tube x1; 10 mL green top sodium heparin tubes x10; 3 mL EDTA tube x1 
• Day 1 visit: 5 mL redtop tube x1; 10 mL green top sodium heparin tubes x10  
• Day 8 visit: 5 mL redtop tube x1; 10 mL green top sodium heparin tubes x10  
• Day 28 visit: 5 mL redtop tube x1; 10 mL green top sodium heparin tubes x10 
 


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Experimental Design: 
 
Below, we briefly describe the assays we will use to monitor/characterize transcriptional changes and specific 
innate immune response variables at three important time points as in-formed by other studies and our 
preliminary data: Baseline (Day 0) and Days 1 and 8 after vaccination. The baseline blood draw allows us to 
ascertain each subject’s pre-existing immune status and provides a baseline upon which to evaluate 
immunologic changes caused by the vaccine.  The Day 1 blood draw allows us to examine early, innate immune 
responses.  The Day 8 sample will be used to characterize plasmablast responses which are directly responsible 
for humoral immunity and the development of protective Ab titers.  The Day 28 blood draw will allow us to 
examine the peak adaptive immune response. 
The assays will be performed either on T- and B-cell depleted PBMCs isolated at each timepoint (for APC im-
munophenotyping, mRNA, and miRNA) or on T- and B-cell depleted PBMCs stimulated in vitro with influenza 
A/H3N2 (for cytokine/chemokine secretion). This will allow us to capture the in vivo and in vitro effects of 
these two vaccines.  
 
The clinical characterization of our study subjects will include demographic information, height, weight, 
medications, and medical conditions that do not meet exclusion criteria. We will also run a CBC to quantify 
blood leukocyte populations. 
 
Secreted Cytokine/Chemokine Mediators. Meso Scale Discovery kits will be used to detect cytokines and 
chemokines in culture supernatants before and 24 hours after in vitro stimulation with influenza A/H3N2.  
Detection of Functional Innate Cell Subsets by Flow Cytometry. Cellular phenotypes of cells (before and 24 
hours after in vitro stimulation with influenza virus) will be characterized in Baseline, Day 1, and Day 8 
PBMCs by flow cytometry.259 We will examine the frequency of immune cell types, and characterize their 
activation by detecting the presence of cell surface markers, co-stimulatory molecules, and activation markers.  
Influenza A/H3N2 Ab Assay. The HAI Ab assay will be performed using standard protocols.262, 359, 369-
373 Given the known issues with some A/H3N2 strains, we will utilize influenza antigen matching the vaccine 
strain and test the antigen for neuraminidase binding to RBCs.374, 375  
CMV Serostatus. CMV Ab titers (IgG) will be measured by multiplex flow-based immunoassay (Bio-Rad). 
B Cell ELISPOT. Influenza-specific Ab-secreting cells/plasmablasts and memory IgG-like B cells will be 
quantified in PBMCs using ELISPOTPLUS for Human IgG kits (Mabtech) with (for memory B cells) or 
without (plasmablasts) R848/IL-2 pre-stimulation.262, 361  
mRNA Transcriptomics.141 mRNASeq will be performed in Mayo Clinic’s Medical Genome Facility on 
RNA samples extracted from PBMCs harvested at Baseline, Day 1 and Day 8. RNA libraries will be 
multiplexed with six samples per lane. Flow cells will be sequenced as 51x2 paired-end reads on an Illumina 
HiSeq. Base-calling will be performed using Illumina’s RTA version 1.17.21.3. We will use StringTie376, 377 
to process the resulting BAM files and sQTLseekeR378 to evaluate alternately spliced transcripts.  
miRNA Sequencing. RNA libraries will be prepared with RNA extracted from Baseline, Day 1, and Day 8-
harvested PBMCs (NEBNext Multiplex Small RNA Kit, New England Biolabs). Following multiplex adapter 
ligation (24 samples/lane) and library enrichment by PCR, equimolar amounts of each library will be pooled, 
purified, and loaded onto flow cells for sequencing (Illumina HiSeq 2000, Illumina cBot and cBot Paired end 
cluster kit version 3). Bioinformatics analysis will use adapter trimmed reads and miRDeep2.379 
 
Research Activity 


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Check all that apply and complete the appropriate sections as instructed.  
  
1. 
  Drug & Device:  Drugs for which an investigational new drug application is not required. Device for 
which (i) an investigational device exemption application is not required; or the medical device is 
cleared/approved for marketing and being used in accordance with its cleared/approved labeling. (Specify in 
the Methods section) 
 
2. 
  Blood:  Collection of blood samples by finger stick, heel stick, ear stick, or venipuncture.  
 
3. 
  Biological specimens other than blood:  Prospective collection of human biological specimens by 
noninvasive means that may include: urine, sweat, saliva, buccal scraping, oral/anal/vaginal swab, sputum, 
hair and nail clippings, etc. 
 
4. 
  Tests & Procedures:  Collection of data through noninvasive tests and procedures routinely employed 
in clinical practice that may include: MRI, surface EEG, echo, ultrasound, moderate exercise, muscular 
strength & flexibility testing, biometrics, cognition testing, eye exam, etc.  (Specify in the Methods section) 
 
5. 
  Data (medical record, images, or specimens):  Research involving use of existing and/or prospectively 
collected data. 
 
6. 
  Digital Record:  Collection of electronic data from voice, video, digital, or image recording. (Specify in 
the Methods section) 
 
7. 
  Survey, Interview, Focus Group:  Research on individual or group characteristics or behavior, survey, 
interview, oral history, focus group, program evaluation, etc.  (Specify in the Methods section) 
 
 
 NIH has issued a Certificate of Confidentiality (COC).  When checked, provide the institution and 
investigator named on the COC and explain why one was requested. ________________________ 
 
Biospecimens – Categories 2 and 3 
 
(2)  Collection of blood samples.  
a. From healthy, non-pregnant, adult subjects who weigh at least 110 pounds. For a minimal risk 
application, the amount of blood drawn from these subjects may not exceed 550ml in an 8 week period 
and collection may not occur more frequently than 2 times per week. 
 
Volume per blood draw (Baseline only): ___108__ml   
Volume per blood draw (Follow-up visits): ___105__ml   
 
Frequency of blood draw: Four blood draws total. Blood draw #1 can occur the day of or up to 2 weeks prior to 
receiving the flu vaccination.  Blood draw #2 will occur the following day after vaccination. Blood draw #3 will 
occur 7-9 days after the vaccination. Blood draw #4 will occur 25-31 days after vaccination.   


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b. From other adults and children considering age, weight, and health of subject. For a minimal risk 
application, the amount of blood drawn from these subjects may not exceed the lesser of 50 ml or 3 ml 
per kg in an 8 week period, and collection may not occur more frequently than 2 times per week.   
Volume per blood draw: _____ml 
Frequency of blood draw (e.g. single draw, time(s) per week, per year, etc.) ___________  
 
(3)  Prospective collection of biological specimens other than blood: ______________________________ 
 
Review of medical records, images, specimens – Category 5 
 
For review of existing data: provide a date range or an end date for when the data was generated. The end date 
can be the date this application was submitted to the IRB.   
Date Range:  
 
Check all that apply (data includes medical records, images, specimens).  
 
  (5a)  Only data that exists before the IRB submission date will be collected.   
 
 
  (5b)  The study involves data that exist at the time of IRB submission and data that will be generated after 
IRB submission. Include this activity in the Methods section.  
Examples 
• The study plans to conduct a retrospective chart review and ask subjects to complete a questionnaire.  
• The study plans to include subjects previously diagnosed with a specific disease and add newly 
diagnosed subjects in the future.  
 
  (5c)  The study will use data that have been collected under another IRB protocol. Include in the Methods 
section and enter the IRB number from which the research material will be obtained. When appropriate, note 
when subjects have provided consent for future use of their data and/or specimens as described in this protocol.  
 
 
Enter one IRB number per line, add more lines as needed 
 
 Data    
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 Data & Specimens  ______________________________________ 
 
 Data    
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  (5d)  This study will obtain data generated from other sources. Examples may include receiving data from 
participating sites or an external collaborator, accessing an external database or registry, etc.  Explain the source 
and how the data will be used in the Methods section.  
 


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  (6)  Video audio recording: 
 
HIPAA Identifiers and Protected Health Information (PHI) 
Protected health information is medical data that can be linked to the subject directly or through a combination 
of indirect identifiers.  
 
Recording identifiers (including a code) during the conduct of the study allows you to return to the medical 
record or data source to delete duplicate subjects, check a missing or questionable entry, add new data points, 
etc. De-identified data is medical information that has been stripped of all HIPAA identifiers so that it cannot be 
linked back to the subject. De-identified data is rarely used in the conduct of a research study involving a chart 
review.   
 
Review the list of subject identifiers below and, if applicable, check the box next to each HIPAA identifier 
being recorded at the time of data collection or abstraction.  Identifiers apply to any subject enrolled in the 
study including Mayo Clinic staff, patients and their relatives and household members.  
 
Internal refers to the subject’s identifier that will be recorded at Mayo Clinic by the study staff. 
External refers to the subject’s identifier that will be shared outside of Mayo Clinic. 
 
Check all that apply: 
INTERNAL 
EXTERNAL 
Name 
X 
 
Mayo Clinic medical record or patient registration number, lab accession, 
specimen or radiologic image number  
X 
 
Subject ID, subject code or any other person-specific unique identifying 
number, characteristic or code that can link the subject to their medical data   
X 
 
Dates: All elements of dates [month, day, and year] directly related to an 
individual, their birth date, date of death, date of diagnosis, etc.   
Note: Recording a year only is not a unique identifier.  
X 
 
Social Security number 
X 
 
Medical device identifiers and serial numbers 
 
 
Biometric identifiers, including finger and voice prints, full face photographic 
images and any comparable images 
 
 
Web Universal Resource Locators (URLs), Internet Protocol (IP) address 
numbers, email address 
 
 
Street address, city, county, precinct, zip code, and their equivalent geocodes 
X 
 
Phone or fax numbers 
X 
 
Account, member, certificate or professional license numbers, health 
beneficiary numbers 
 
 
Vehicle identifiers and serial numbers, including license plate numbers 
 
 
Check ‘None’ when none of the identifiers listed above will be recorded, 
maintained, or shared during the conduct of this study.  (exempt category 4) 
 None 
 None 
Data Analysis 
Data Analysis Plan:  


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The planned analyses for Aims 1 and 2 will follow the same strategies, so for Aim 2 analyses we point out the 
differences needed to adapt the analyses for Aim 2.  
Aim 2.1 Test the hypotheses that vaccine type (MF59Flu vs HDFlu) or sex is associated with variations in 
Th cell immune outcomes: The analysis plans for Aim 1.1 will be followed, but using the Th phenotype and 
cytokine production outcomes, evaluating change from Baseline to Days 8 and 28.  
Aim 2.2 Test the hypothesis that the expression of specific genes is associated with differences in Th cell 
immune outcomes: Analyses will parallel those for Aim 1.2, but use differential Th cell-specific gene and 
miRNA expression (change from Baseline to Days 8 and 28) as independent variables to evaluate for their 
association with change in Th cell immune responses.  
Aim 2.3 Test the hypothesis that Th cell transcriptomic signatures mediate the association of vaccine type 
(or sex) with Th cell immune outcomes: Analyses will parallel those described in Specific Aim 1.3. 
Aim 2.4 Test the hypothesis that Th cell immune outcomes and transcriptomic signatures from Aim 2.3 
will predict markers of humoral immunity (HAI Ab and B cell ELISPOT response): The analysis plans 
closely follow the mediation analyses described in Aim 1.4. We will test the hypothesis that Th immune 
response and their associated genes (Aim 2.3), predict humoral immunity (HAI Ab titer and B cell ELISPOT 
response) at Day 28. We will also test the hypothesis that additional genes mediate humoral immunity 
independently of Th responses. To achieve this, we will treat Th responses as adjusting covariates, using the 
regression model: By adjusting for Th response, we seek genes/genesets that are only associated with humoral 
immune response.  
 
Power: See Specific Aim 1. We anticipate stronger signals in the transcriptomic data from purified CD4+ T 
cells compared to PBMCs. The increased signal to noise ratio will allow for detection of smaller effects.  
 
Potential Limitations and Alternative Strategies for Aims 1 and 2.  
False-positive associations will be controlled by our clearly defined analysis plan, the use of false-discovery 
rates to assess significance, and geneset/pathway analysis to maximize power and minimize false discovery.  
Statistical power to detect effects is driven by our sample size of 400 subjects, which is larger than most 
systems biology studies, including our prior study of SDFlu in older subjects where we and others were able to 
successfully identify transcriptomic signatures of vaccine response.16, 44, 59-61, 260, 261, 263, 264, 275, 280, 
437 Accumulation of information across multiple genes increases the percent of variability explained by the 
model and hence increases the likelihood of detecting effects. Our analysis approach will also test the 
association of each innate and Th cell immune response with the expression of all genes in a specified geneset 
using kernel methods.438-441 Advantages of kernel methods are the following: 1) they allow for correlations 
among the different genes in a geneset; 2) they are robust to when some genes are positively associated with a 
response, others are negatively associated with a response, and other genes in the geneset are not associated; 3) 
and they allow adjustment for covariates. Finally, we will evaluate data-driven genesets using WGCNA to 
group genes into biological modules,118 which is an approach we have successfully used (see Preliminary 
Data).  
Separate mediation analyses will be performed for mRNA and miRNA. If we find both mRNA and miRNA to 
be separate mediators in our initial analyses, we will evaluate their joint effects as a pathway of mediators using 
techniques developed by VanderWeele.106 Other confounding factors (e.g., immunosenescence, underlying 
medical conditions, obesity, and CMV serostatus) will be evaluated in our analyses as potential confounders.  
 
Expected Results and Impact of Findings for Specific Aims 1 and 2. Our publications44, 59, 258, 259, 263-
265, 442, 443 and preliminary data indicate a high likelihood of identifying not only significant vaccine type- 


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and sex-specific differences in immune responses, but also transcriptomic signatures associated with those 
differences. Furthermore, the preliminary data from our mediation analysis support our hypothesis that changes 
in gene expression influence the effect of sex on immune response. We therefore anticipate identifying 
comparable sex-specific differences along with the genes/genesets mediating those responses (i.e., 
transcriptomic signatures), following receipt of MF59Flu and HDFlu. Pinpointing the innate and/or Th cell 
responses associated with humoral immunity to these vaccines facilitates our understanding of the critical 
pathways vaccines must trigger in order to elicit protective antibody titers.  
Our data will also provide new insights into why men and women respond differently to influenza vaccines, and 
highlight the specific genetic pathways associated with MF59- and sex-based immune response differences. 
Aims 1.1 and 2.1 may provide additional correlates of protection that can be used in conjunction with HAI titer 
to more accurately assess immune status. Understanding what genes and genesets are associated with the 
increased immunogenicity of MF59Flu and HDFlu (Aims 1.2 and 2.2) will inform functional studies verifying 
the mechanistic relationship between gene expression and immune function. For example, if TLR4 and TLR8 
activation are strongly associated with increased APC activation and HAI titers, it suggests the use of a 
combination of GLA-SE (a TLR4 agonist) and CpG (a TLR8/9 agonist) might make an effective adjuvant. As 
another example, MF59 has been shown to enhance APC recruitment to the Ag site and transport of Ag to the 
draining lymph nodes. Due to reasons that are currently poorly understood, those processes are followed by 
enhanced Th cell responses and more robust (in terms of both titer and breadth of targets) Ab responses. 
Evaluating the transcriptomic changes occurring in innate and Th cells after MF59Flu vaccination is likely to 
identify gene expression patterns occurring in the APCs and responding T cells, providing critical clues 
regarding the nature of the interactions between these cell types, and may highlight the receptor-ligand pairs and 
downstream signaling cascades responsible for enhanced T cell responses. Such information allows for directed 
and informed engineering of novel vaccine candidates and adjuvants. In the unlikely event that we do not 
identify vaccine type or sex-dependent differences, this finding will be interesting its own right, suggesting that 
further studies be conducted to find additional variables that do impact innate and/or Th responses to influenza 
A/H3N2. 
 
Endpoints: 
Secreted cytokine levels 
Detection of Functional Th Cell Subsets 
mRNA and miRNA Sequencing 
 
References: 
 
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