Open-Label, Randomized Study of Immune Response to Licensed Influenza Vaccines in Adults 65-74 Years of Age (US Flu Vaccine Effectiveness Serologic Study, 2016-17 and 2017-18)
Phase: NA
Conditions: Immune Response
Sponsor: Marshfield Clinic Research Foundation

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 1 
 
Open-Label, Randomized Study of Immune Response to Licensed Influenza 
Vaccines in Adults 65-74 Years of Age (US Flu VE Serologic Study, 2016-17 and 
2017-18) 
 
Vaccines 
Trivalent Standard Dose Inactivated Influenza Vaccine (Fluvirin, 
Seqirus)(IIV3) 
Trivalent High Dose Inactivated Influenza vaccine (Fluzone 
High-Dose, Sanofi Pasteur)(HD-IIV3) 
Trivalent Inactivated Influenza Vaccine with adjuvant (Fluad, 
Seqirus)(aIIV3) 
Quadrivalent Recombinant Influenza Vaccine (FluBlok, Protein 
Sciences) 
Sponsor 
US Centers for Disease Control and Prevention (CDC) 
1600 Clifton Road 
Atlanta, GA 30329 
CDC Project Officers 
Brendan Flannery, PhD 
Alicia Fry, MD 
Marshfield Clinic Research 
Institute Investigators and 
Key Personnel 
Huong McLean, PhD 
Edward Belongia, MD 
Jennifer Meece, PhD 
Jennifer King, MPH 
Version and Date 
Version 3.0 –  July 2017 
 
 
 
 
 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 2 
 
PROTOCOL CHANGE HISTORY 
 
Date/Version 
Change 
Dec 2016 
Change to illness lockout period (page 10, 19). 
July 2017; 
Version 3.0 
Add new influenza vaccine for Year 2; 
Add 30 new participants for Year 2 
. 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 3 
 
Table of Contents 
 
1 
BACKGROUND AND SIGNIFICANCE ....................................................................................... 13 
2 
STUDY OBJECTIVES ................................................................................................................ 14 
2.1 
Primary Objectives ......................................................................................................... 14 
2.2 
Secondary Objectives ..................................................................................................... 14 
3 
STUDY DESIGN ...................................................................................................................... 15 
3.1 
Design ............................................................................................................................. 15 
3.2 
Study Endpoints ............................................................................................................. 16 
3.2.1 
Primary Endpoints ................................................................................................... 16 
3.2.2 
Secondary Endpoints .............................................................................................. 16 
3.3 
Randomization Procedure .............................................................................................. 16 
3.4 
Study Duration ............................................................................................................... 17 
4 
VACCINES .............................................................................................................................. 17 
5 
SELECTION OF STUDY SUBJECTS ........................................................................................... 17 
5.1 
Inclusion Criteria ............................................................................................................ 17 
5.2 
Exclusion Criteria ............................................................................................................ 18 
6 
STUDY PROCEDURES ............................................................................................................. 18 
6.1 
Enrollment Period .......................................................................................................... 18 
6.2 
Recruitment .................................................................................................................... 18 
6.3 
Study Visit Procedures ................................................................................................... 18 
6.3.1 
Visit 1: Enrollment and Vaccination – Approximately 60 minutes ......................... 19 
6.3.2 
Visit 2: Day 28 Post-Vaccination, Year 1 – Approximately 30 minutes .................. 19 
6.3.3 
Visit 3: 6-month Post-Vaccination, Year 1 – Approximately 30 minutes ................ 19 
6.3.4 
Visit 4: Year 2 vaccination – Approximately 45 minutes ........................................ 20 
6.3.5 
Visit 5: Year 2 1-month Post-Vaccination – Approximately 30 minutes................. 20 
6.3.6 
Visit 6: Year 2 6-month Post-Vaccination Visit – Approximately 30 minutes ......... 21 
6.3.7 
Illness Surveillance .................................................................................................. 21 
6.4 
Data Collection ............................................................................................................... 21 
6.4.1 
Influenza Vaccination Status ................................................................................... 21 
6.4.2 
Medical Record Review ........................................................................................... 22 
7 
SPECIMEN COLLECTION AND HANDLING ............................................................................. 22 
7.1 
Serum ............................................................................................................................. 22 
7.2 
Swabs .............................................................................................................................. 23 
8 
LABORATORY METHODS ....................................................................................................... 23 
8.1 
Hemagglutination Inhibition Antibody Testing .............................................................. 23 
8.2 
Microneutralization Antibody Testing ........................................................................... 23 
8.3 
Neuraminidase Antibody Testing ................................................................................... 24 
8.4 
Antibody Dependent Cellular Cytotoxicity (ADCC) Analysis .......................................... 24 
8.5 
RT-PCR ............................................................................................................................ 24 
9 
PARTICIPANT COMPENSATION ............................................................................................. 25 
10 SHARING OF LABORATORY SPECIMENS AND DATA ............................................................. 25 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 4 
 
11 DATA MANAGEMENT ............................................................................................................ 25 
11.1 
Recording and Collection of Data ............................................................................... 25 
11.2 
Data Quality Assurance .............................................................................................. 25 
12 STATISTICAL CONSIDERATIONS ............................................................................................ 26 
12.1 
General ....................................................................................................................... 26 
12.2 
Analyses Addressing Objectives ................................................................................. 26 
12.2.1 Analyses of Primary Objectives ............................................................................... 26 
12.2.2 Analyses of Secondary Objectives .......................................................................... 26 
12.3 
Power Considerations ................................................................................................. 27 
13 HUMAN SUBJECTS................................................................................................................. 27 
13.1 
Informed Consent ....................................................................................................... 27 
14 REFERENCES .......................................................................................................................... 28 
 
 
 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 5 
 
GLOSSARY OF ABBREVIATIONS 
 
Abbreviation or Term 
Definition 
ADCC 
Antibody dependent cellular cytotoxicity 
aIIV3 
Trivalent inactivated influenza vaccine with adjuvant  
CDC 
US Centers for Disease Control and Prevention 
CI 
Confidence interval 
GMT 
Geometric mean titers 
HD-IIV3 
High-dose trivalent inactivated influenza vaccine 
HI 
Hemagglutination inhibition 
IM 
Intramuscular 
IIV3 
Standard dose trivalent inactivated influenza vaccine 
MFR 
Mean fold rise 
MN 
Microneutralization 
NAI 
Neuraminidase inhibition 
RIV4 
Quadrivalent recombinant influenza vaccine 
SD 
Standard deviation 
SPR 
Seroprotection rate 
SRR 
Seroresponse rate 
 
 
 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 6 
 
CLINICAL PROTOCOL SYNOPSIS 
 
Study Sponsor: CDC 
Title: Open-Label, Randomized Study of Immune Response to Licensed Influenza Vaccines in 
Adults 65-74 Years of Age 
Study short name: Open-Label Influenza Vaccine Evaluation (OLIVE) 
Vaccines: Fluvirin, Fluzone High-Dose, Fluad, FluBlok 
Regimen and Dosing: Subjects will receive a 0.5 mL intramuscular (IM) injection of one of three 
licensed 2016-17 influenza vaccines at study visit 1 and either 2017-18 Fluzone High-Dose (HD-
IIV3), Fluad (aIIV3) or FluBlok (RIV3) at study visit 4 (approximately 1 year after enrollment).  
Study Rationale: 
Influenza vaccination is recommended annually for all persons aged ≥6 months. Several newer 
vaccines have been developed and are recommended for persons aged ≥65 years, including 
vaccines with increased amounts of antigen (high dose) or adjuvant, to increase immune 
response. The recombinant influenza vaccine has also shown high effectiveness in older adults. 
Immunologic response to influenza vaccination is dependent on age and pre-existing antibody 
levels from prior exposure either to vaccination or disease. Recent observational studies of 
influenza vaccine effectiveness have suggested that prior season vaccination status may be an 
important factor in vaccine protection, but studies have been inconsistent. Limited data suggest 
that repeat high-dose vaccination does not interfere with immune response while there are no 
data on immune response to repeat vaccination with adjuvanted vaccines. 
 
Given the universal recommendation for annual vaccination for all adults, increased risk of 
complications from influenza infection in older adults, and the availability of vaccines 
specifically formulated to increase immune response to vaccination for persons aged ≥65 years, 
a better understanding of the humoral immune response to repeat influenza vaccination and 
vaccine type in this population is needed.  There is currently no preferential recommendation 
for a specific vaccine for the population aged ≥65 years. Understanding immune responses to 
these newer vaccines compared to standard dose vaccines upon vaccination and revaccination 
will help to guide vaccine policy for this age group. 
 
Study Objectives: 
Primary: 
 
Compare immune responses to vaccine and drifted viruses as measured by 
hemagglutination inhibition (HI) among persons aged 65-74 years vaccinated with one of 
two licensed influenza vaccines: high-dose trivalent inactivated influenza vaccine (HD-IIV3) 
or trivalent inactivated influenza vaccine with adjuvant (aIIV3) approximately 28 days, 6 
months, and 1 year after receipt of the 2016-17 vaccine 
 
Compare immune responses to vaccine and drifted viruses as measured by HI among 
subjects after repeat influenza vaccination (2016-17 and 2017-18) with HD-IIV3 and aIIV3 
approximately 28 days and 6 months after receipt of the 2017-18 vaccine.  
 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 7 
 
Study Sponsor: CDC 
Title: Open-Label, Randomized Study of Immune Response to Licensed Influenza Vaccines in 
Adults 65-74 Years of Age 
Study short name: Open-Label Influenza Vaccine Evaluation (OLIVE) 
Vaccines: Fluvirin, Fluzone High-Dose, Fluad, FluBlok 
Secondary: 
 Compare immune responses as measured by virus neutralizing antibody titers, 
neuraminidase antibody mediated inhibition titers, and anti-human CD107a antibody titers 
among persons aged 65-74 years vaccinated with one of two licensed influenza vaccines: 
HD-IIV3 or aIIV3 approximately 28 days, 6 months, and 12 months after receipt of the 2016-
17 vaccine. 
 Compare immune responses as measured by virus neutralizing antibody titers, 
neuraminidase antibody mediated inhibition titers, and anti-human CD107a antibody titers 
among subjects after repeat influenza vaccination (2016-17 and 2017-18) with HD-IIV3 and 
aIIV3 with subjects vaccinated approximately 28 days and 6 months after receipt of the 
2017-18 vaccine. 
 Compare immune responses as measured by HI by prior receipt of 2015-16 influenza 
vaccine among persons aged 65-74 years vaccinated with one of two licensed influenza 
vaccines: HD-IIV3 or aIIV3 approximately 28 days, 6 months, and 1 year after receipt of the 
2016-17 vaccine. 
 Compare immune responses as measured by HI among persons aged 65-74 years who 
received standard dose trivalent inactivated influenza vaccine (IIV3) in 2016-17 and 
vaccinated with one of three licensed influenza vaccines in 2017-18: HD-IIV3, aIIV3, or RIV4 
approximately 28 days, and 6 months after receipt of the 2017-18 vaccine. 
 Compare immune responses as measured by HI among persons who received IIV3 in 2016-
17 and vaccinated with one of two licensed influenza vaccines in 2017-18: HD-IIV3 oraIIV3 
with subjects who received repeat influenza vaccination (2016-17 and 2017-18) with HD-
IIV3 and aIIV3 approximately 28 days and 6 months after receipt of the 2017-18 vaccine. 
 Conduct influenza surveillance to determine factors associated with vaccine failure 
(laboratory confirmed influenza) among study participants if it occurs during the 
surveillance period. 
Study Endpoints: 
Primary: 
 Hemagglutinin inhibition (HI) antibody levels specific for vaccine strains and representative 
strains of circulating influenza viruses that are antigenically or genetically different. 
Endpoints based on these data will include geometric mean titers (GMT), mean fold rise 
(MFR), seroresponse rate (SRR), and seroprotection rate (SPR).  
 Seroresponse rate defined as the proportion of subjects with paired samples with 
prevaccination HI titer <1:10 and postvaccination (day 28) HI titer ≥1:40 or at least a 
4-fold increase in HI from prevaccination HI titer ≥1:10. 
 Seroprotection rate defined as the proportion of subjects with postvaccination HI 
titer ≥1:40.  

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 8 
 
Study Sponsor: CDC 
Title: Open-Label, Randomized Study of Immune Response to Licensed Influenza Vaccines in 
Adults 65-74 Years of Age 
Study short name: Open-Label Influenza Vaccine Evaluation (OLIVE) 
Vaccines: Fluvirin, Fluzone High-Dose, Fluad, FluBlok 
 
Secondary: 
 Virus neutralizing antibody titers, neuraminidase antibody mediated inhibition titers, and 
anti-human CD107a antibody titers specific for vaccine strains and representative strains of 
circulating influenza viruses that are antigenically or genetically different. Endpoints based 
on these data will include geometric mean titers (GMT) and mean fold rise (MFR). 
 RT-PCR confirmed influenza 
Study Design: 
This is an open-label, randomized trial with enrollment of up to 180 adults aged 65-74 years in 
the first year (2016-17) and an additional 30 adults in the second year (2017-18) for a total of 
210 across two years.  
Randomization to a vaccine group in the first year (2016-17) will be stratified by receipt of 
2015-16 influenza vaccine. The randomization will target enrollment of approximately 25% of 
subjects in the unvaccinated in 2015-16 stratum. It is anticipated that a percentage of the 
randomized study subjects may not complete the study; subjects who withdraw or are 
discontinued may be replaced at the beginning of the 2017-18 influenza season. 
Vaccines administered will include a single IM dose of one of three licensed 2016-17 influenza 
vaccines at study visit 1. At study visit 4, subjects who received HD-IIV3 and aIIV3 at visit 1 will 
receive the same vaccine type for the 2017-18 influenza season. Subjects who received IIV3will 
be randomized to receive HD-IIV3, aIIV3 or RIV4. An additional 30 individuals who received IIV3 
in the 2016-17 season will be recruited for study visit 4 (the beginning of the 2017-18 influenza 
season) and randomized to receive HD-IIV3, aIIV3 or RIV4. 
 
Group 
2016-17 Vaccine 
2017-18 Vaccine 
Total 
65-74 
years 
A 
2016-17 HD-IIV3 
2017-18 HD-IIV3 
60 
B 
2016-17 aIIV3 
2017-18 aIIV3 
60 
C1 
2016-17 IIV3  
2017-18 HD-IIV3 
30 
C2 
2016-17 IIV3 
2017-18 aIIV3 
30 
C3 
2016-17 IIV3 
2017-18 RIV4 
30 
 
Total 
 
210 
 
For each subject enrolled in the first year, study follow-up will span approximately two years 
from the first dose. Study follow-up will span approximately 6 months from their enrollment 
date for study subjects recruited in Fall 2017. 
Eligibility Criteria: 
 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 9 
 
Study Sponsor: CDC 
Title: Open-Label, Randomized Study of Immune Response to Licensed Influenza Vaccines in 
Adults 65-74 Years of Age 
Study short name: Open-Label Influenza Vaccine Evaluation (OLIVE) 
Vaccines: Fluvirin, Fluzone High-Dose, Fluad, FluBlok 
Inclusion: 
Subjects must meet all of the following criteria to be eligible to participate: 
1) Age 65-74 years at the time of study enrollment and are ambulatory and live in 
Marshfield, Wisconsin or surrounding communities. 
2) Willing and able to give informed consent prior to study enrollment. 
3) Able to comply with study requirements. 
 
Exclusion: 
Subjects will be excluded if they fulfill any of the following criteria: 
1) Prior receipt of 2016-17 (or 2017-18 for participants recruited in Fall 2017) influenza 
vaccine. 
2) Current participation in another clinical trial. 
3) Presence of a contraindication to influenza vaccination. 
Study Visit Procedures: 
Subjects recruited in Fall 2016 will complete all the following study visits and acute respiratory 
illness during the 2016-17 and 2017-18 influenza seasons. Subjects recruited in Fall 2017 will 
complete visits 4 through 6, and surveillance for acute respiratory illness during the 2017-18 
influenza season. 
 
Visit 1 – Screening 
Adult volunteers aged 65-74 years who have provided written informed consent and are able to 
comply with study requirements will have the following procedures performed: inclusion and 
exclusion criteria review; measurement of height and weight; review and recording of 
concomitant medications. Potential subjects who meet all inclusion criteria and none of the 
exclusion criteria may be enrolled.  
Visit 1 – Vaccination 
All subjects who have eligibility confirmed will have blood draw (20 mL) for baseline serologic 
evaluation (including hemagglutination inhibition and other assays).  
Subjects will then be randomized to one of three vaccine group and administered the 2016-17 
influenza vaccine using standard procedures for IM injection of the licensed vaccines. 
The remaining study visits will be scheduled. 
Visit 2 – Follow-Up 
Subjects will return to the clinic approximately 28 days (±5 days) following receipt of the 2016-
17 influenza vaccine. A blood sample (20 mL) will be obtained for serologic evaluation of 
influenza vaccine responses. Instructions for illness monitoring will be given to subjects and the 
next study visit will be confirmed. 
Visit 3 – Follow-Up 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 10 
 
Study Sponsor: CDC 
Title: Open-Label, Randomized Study of Immune Response to Licensed Influenza Vaccines in 
Adults 65-74 Years of Age 
Study short name: Open-Label Influenza Vaccine Evaluation (OLIVE) 
Vaccines: Fluvirin, Fluzone High-Dose, Fluad, FluBlok 
All subjects will return to the clinic approximately 182 days (±10 days) following receipt of the 
2016-17 influenza vaccine. A blood sample (20 mL) will be obtained for serologic evaluation of 
influenza vaccine responses. The study visit will be confirmed. 
Visit 4 – Follow-up and Vaccination 
Subjects will return approximately 1 year following receipt of the 2016-17 influenza vaccine. A 
blood sample (20 mL) will be obtained for serologic evaluation (including hemagglutination 
inhibition and other assays). Subjects who received high-dose inactivated influenza vaccine and 
inactivated vaccine with adjuvant in 2016-17 will receive the same vaccine type for the 2017-18 
season. Subjects who received standard dose inactivated vaccine in 2016-17 will be randomized 
to receive high-dose vaccine, vaccine with adjuvant, or the recombinant vaccine. The vaccines 
will be administered using standard procedures for IM injection of the licensed vaccines. The 
next study visits will be confirmed. 
 
An additional 30 subjects will be added to the study. Adult volunteers aged 65-74 years who 
have provided written informed consent and are able to comply with study requirements will 
have the following procedures performed: inclusion and exclusion criteria review; 
measurement of height and weight; review and recording of concomitant medications. All 
subjects who have eligibility confirmed will have blood draw (20 mL) for baseline serologic 
evaluation (including hemagglutination inhibition and other assays). Subjects will then be 
randomized to receive high-dose vaccine, vaccine with adjuvant, or the recombinant vaccine 
and administered the 2017-18 influenza vaccine using standard procedures for IM injection of 
the licensed vaccines. The remaining study visits will be scheduled. 
Visit 5 – Follow-Up 
Subjects will return to the clinic approximately 28 days (±5 days) following receipt of the 2017-
18 influenza vaccine. A blood sample (20 mL) will be obtained for serologic evaluation of 
influenza vaccine responses. Instructions for illness monitoring will be given to subjects and the 
next study visit will be confirmed. 
Visit 6 – Follow-Up 
All subjects will return to the clinic approximately 182 days (±10 days) following receipt of the 
2017-18 influenza vaccine. A blood sample (20 mL) will be obtained for serologic evaluation of 
influenza vaccine responses.  
 
Time and Events Schedule 
Visit 
1 
2 
3 
4 
5 
6 
Approximate study day 
0 
28 
182 
365/0* 393/28* 547/182* 
Informed consent 
X 
 
 
 
 
 
Inclusion/exclusion criteria review 
X 
 
 
 
 
 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 11 
 
Study Sponsor: CDC 
Title: Open-Label, Randomized Study of Immune Response to Licensed Influenza Vaccines in 
Adults 65-74 Years of Age 
Study short name: Open-Label Influenza Vaccine Evaluation (OLIVE) 
Vaccines: Fluvirin, Fluzone High-Dose, Fluad, FluBlok 
Influenza serologic evaluation 
X 
X 
X 
X 
X 
X 
Influenza vaccination 
X 
 
 
X 
 
 
Respiratory illness surveillance 
 
11/1/16-4/1/17 
 
11/1/17-4/1/18 
*For participants recruited and enrolled in Fall 2017 
Surveillance for Acute Respiratory Illness: 
From approximately November 1, 2016 through April 1, 2017 and November 1, 2017 through 
April 1, 2018, subjects will enter passive and active surveillance for acute respiratory illness. 
Surveillance will include: 
 Instructions to contact the study site with onset of new acute respiratory illness. 
 Weekly calls from study staff to solicit respiratory illness 
“New onset” will require a period free of symptoms, or with baseline symptoms, and at least 14 
days from onset of previous illness to differentiate an episode from any prior illness. When a 
new acute respiratory illness is reported and confirmed, the subject will be invited to return to 
the clinic, or study staff will make a home visit to collect a nasal and throat swab within 7 days 
of illness onset for influenza testing. 
Subjects will continue to receive weekly telephone calls for ascertainment of respiratory 
symptoms. Subsequent weekly calls will document resolution, or return to baseline, of 
symptoms. 
Statistical Methods: 
General 
Demographic and other baseline characteristics will be summarized by treatment group for all 
subjects. Continuous variables will be presented by summary statistics (e.g., mean, and 
standard deviation (SD)) for non-immunogenicity endpoints, and geometric means and their 
95% confidence interval for the immunogenicity endpoints. Categorical variables will be 
presented by frequency distributions; i.e., frequency counts and percentages for the non-
immunogenicity endpoints, and the percentages and their 95% confidence intervals (CI) for the 
immunogenicity endpoints. 
 
Analyses of Primary Objectives 
Geometric mean HI antibody titers at each time point will be compared by vaccine group.  For 
the primary aim, adults receiving high dose trivalent inactivated influenza vaccine will be 
compared with those receiving aIIV3. Linear mixed-effects models will be used to adjust for 
variables such as age, given the potential for small differences in age and the known difference 
in GMT by age, baseline antibody titer and prior season vaccination status. The mean fold rise 
(MFR), defined as the mean of the ratio of post vaccination GMT and pre-vaccination GMT for 
each subject, between the groups will be computed from mixed-effects model along with its 
associated 95% CI. 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 12 
 
Study Sponsor: CDC 
Title: Open-Label, Randomized Study of Immune Response to Licensed Influenza Vaccines in 
Adults 65-74 Years of Age 
Study short name: Open-Label Influenza Vaccine Evaluation (OLIVE) 
Vaccines: Fluvirin, Fluzone High-Dose, Fluad, FluBlok 
 
Analyses of Secondary Objectives 
Analyses of virus neutralizing antibody titers, neuraminidase antibody mediated inhibition 
titers, and anti-human CD107a antibody titers will be similar to HI data described above. 
 
If vaccine failures (RT-PCR confirmed influenza infection) occur, we will assess factors 
associated with failure using logistic regression, including vaccine type. 
 
Power Considerations 
A sample size of 60 per group will provide over 90% power using a one-sided, two-sample t-test 
to conclude that one vaccine group is superior as defined by rejecting a null hypothesis of 
equality if the GMT values differ by approximately 20%. 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 13 
 
1 BACKGROUND AND SIGNIFICANCE 
Influenza is an important cause of death and serious illness among persons aged ≥65 years in 
the United States. Recommendations for routine annual influenza vaccination have traditionally 
targeted persons aged ≥65 years because most influenza deaths occur in this age group.1-7  
However, despite high vaccination coverage in this population, high rates of influenza-
associated hospitalizations continue to occur.  One explanation may be poor response to 
traditional, standard dose inactivated influenza vaccines.  Several newer vaccines have been 
developed and are recommended for persons aged ≥65 years, including vaccines with increased 
amounts of antigen (high dose) or adjuvant, to increase immune response.  One advantage of 
adjuvanted vaccines may be cross-protection against antigenically drifted influenza viruses,8 
while cross-protection provided by high-dose vaccine is unknown.  Additionally, recombinant 
influenza vaccine, licensed for all adults aged ≥18 years, has been shown to provide better 
protection than standard dose influenza vaccine against influenza A among adults aged ≥50 
years during a season with an antigenic mismatch between circulating and vaccine influenza 
strains (relative efficacy: 36; 95% CI 14 to 53){Dunkle, 2017 #2603}.  There is currently no 
preferential recommendation for a specific vaccine for the population aged ≥65 years, and all 
influenza vaccines that will be used in this study are licensed and recommended for use in this 
population. Understanding immune responses to these newer vaccines compared to standard 
dose vaccines upon vaccination and revaccination will help to guide vaccine policy for this age 
group. 
 
The immune response to influenza vaccine in older adults, particularly those with chronic 
disease, is less well studied than that of younger, healthier adults and children.  
Immunosenescence likely increases the risk of disease in older adults while at the same time, 
decreases the desired response to vaccination.9 Moreover, while hemagglutinin inhibition (HAI) 
titers have typically been used to evaluate immune response to vaccination, with a 4-fold or 
greater increase in titers associated with a 50% reduction in infection,10 very few studies have 
applied newer, state-of-the-art technologies to also measure cell-mediated immune response 
to vaccination. An improved understanding of the magnitude and breadth of the full immune 
response to vaccination is needed.   
  
Immunologic response to influenza vaccination is dependent on age and pre-existing antibody 
levels from prior exposure either to vaccination or disease. Recent observational studies of 
influenza vaccine effectiveness have suggested that prior season vaccination status may be an 
important factor in vaccine protection. Serologic studies examining antibody response to 
vaccination among persons with and without prior history of vaccination have been 
inconsistent. Some serologic studies have found stronger responses to vaccination in previously 
unvaccinated subjects compared to those who were revaccinated,11-13 some found stronger 
responses in those revaccinated,14 and some found no differences in responses.15,16 Limited 
data suggest that repeat high-dose vaccination does not interfere with immune response while 
there are no data on immune response to repeat vaccination with adjuvanted vaccines.17 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 14 
 
 
Given the universal recommendation for annual vaccination for all adults and the availability of 
vaccines specifically formulated to increase immune response to vaccination for persons aged 
≥65 years, a better understanding of the humoral immune response to repeat influenza 
vaccination and vaccine type in this population is needed. We propose to conduct a prospective 
serologic study with assignment of vaccine type among persons aged 65-74 years to better 
understand the impact on immune response of vaccination and repeat vaccination with 
differing types of vaccines. The findings could have implications on the current vaccine policy if 
there is evidence of differing immune response given the type of vaccine received or the order 
in which specific types are received.  
 
2 STUDY OBJECTIVES 
2.1 Primary Objectives 
 
Compare immune responses to vaccine and drifted viruses as measured by 
hemagglutination inhibition (HI) among persons aged 65-74 years vaccinated with one of 
two licensed influenza vaccines recommended for this age group specifically formulated to 
increase immune response to vaccination: HD-IIV3 or aIIV3 approximately 28 days, 6 
months, and 12 months after receipt of the 2016-17 vaccine. 
 
Compare immune responses to vaccine and drifted viruses as measured by HI among 
subjects after repeat influenza vaccination (2016-17 and 2017-18) with HD-IIV3 and aIIV3 
approximately 28 days and 6 months after receipt of the 2017-18 vaccine.  
2.2 Secondary Objectives 
 
Compare immune responses as measured by virus neutralizing antibody titers, 
neuraminidase antibody mediated inhibition titers, and anti-human CD107a antibody titers 
among persons aged 65-74 years vaccinated with one of two licensed influenza vaccines: 
HD-IIV3 or aIIV3 approximately 28 days, 6 months, and 12 months after receipt of the 
2016-17 vaccine. 
 
Compare immune responses as measured by virus neutralizing antibody titers, 
neuraminidase antibody mediated inhibition titers, and anti-human CD107a antibody titers 
among subjects after repeat influenza vaccination (2016-17 and 2017-18) with HD-IIV3 and 
aIIV3 with subjects vaccinated with IIV3 in 2016-17 and either HD-IIV3 and aIIV3 in 2017-18 
approximately 28 days and 6 months after receipt of the 2017-18 vaccine.  
 
Compare immune responses as measured by HI by prior receipt of 2015-16 influenza 
vaccine among persons aged 65-74 years vaccinated with one of two licensed influenza 
vaccines: HD-IIV3 or aIIV3 approximately 28 days, 6 months, and 1 year after receipt of the 
2016-17 vaccine. 
 Compare immune responses as measured by HI among persons aged 65-74 years who 
received IIV3 in 2016-17 and vaccinated with one of three licensed influenza vaccines in 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 15 
 
2017-18: HD-IIV3, aIIV3 or RIV4 approximately 28 days and 6 months after receipt of the 
2017-18 vaccine. 
 Compare immune responses as measured by HI among persons who received IIV3 in 2016-
17 and vaccinated with one of two licensed influenza vaccines in 2017-18: HD-IIV3 or aIIV3 
with subjects who received repeat influenza vaccination (2016-17 and 2017-18) with HD-
IIV3 and aIIV3 approximately 28 days and 6 months after receipt of the 2017-18 vaccine. 
 Conduct influenza surveillance to determine factors associated with vaccine failure 
(laboratory confirmed influenza) among study participants if it occurs during the 
surveillance period. 
 
3 STUDY DESIGN 
3.1 Design  
This is a randomized, open-label serologic study with enrollment of up to 180 adults aged 65-74 
years in the first year (2016-17) and an additional 30 adults in the second year (2017-18) for a 
total of 210 across two years, at Marshfield Clinic Research Institute (MCRI). Subjects will be 
recruited from community-dwelling individuals who receive their inpatient and outpatient care 
from Marshfield Clinic facilities.   
 
Randomization to one of three licensed influenza vaccine groups in the first year (2016-17) will 
be stratified by prior receipt of the 2015-16 influenza vaccine. The randomization algorithm will 
target enrollment of approximately 25% of subjects in the unvaccinated in 2015-16 stratum. It 
is anticipated that a percentage of the randomized study subjects may not complete the study. 
 
Participants will be randomized to receive IIV3, HD-IIV3, or aIIV3 at their baseline study visit to 
ensure an appropriate minimum number of subjects receive high dose and adjuvanted vaccines 
in 2016-17 for analyses. Participants will be grouped according to vaccination type (standard 
dose, high dose or adjuvanted vaccine) received during the 2016-17 season (Table 1).  All 
subjects enrolled in Fall 2016 will have a baseline serum blood draw, a second serum blood 
draw 28 (±5) days after vaccination and 6 months after vaccination in each season (2016-17 and 
2017-18).  In Fall 2017, participants who received HD-IIV3 and aIIV3 in Fall 2016 will receive the 
same vaccine type. Participants who received IIV3 will be randomized to receive HD-IIV3, aIIV3, 
or RIV4 for the 2017-18 season. 
 
An additional 30 adults who received IIV3 in the 2016-2017 season will be recruited in Fall 2017. 
Newly recruited participants will be randomized to receive HD-IIV3, aIIV3, or RIV4 for the 2017-
18 season and will have serum blood draw before vaccination, 28 (±5) days after vaccination 
and 6 months after vaccination. 
 
 
Table 1. 2016-17 Vaccine Randomization & Blood Draw Scheme with Proposed ID numbering 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 16 
 
 
Group 
2016-17 Vaccine 
2017-18 Vaccine 
Total 
65-74 
years 
A 
2016-17 HD-IIV3 
2017-18 HD-IIV3 
60 
B 
2016-17 aIIV3 
2017-18 aIIV3 
60 
C1 
2016-17 IIV3 
2017-18 high dose trivalent 
inactivated influenza vaccine 
30 
C2 
2016-17 IIV3 
2017-18 aIIV3 
30 
C3 
2016-17 IIV3 
2017-18 RIV4 
30 
 
Total 
 
210 
 
All participants will be contacted weekly throughout the influenza season to monitor for 
influenza-like illness and, if exhibiting symptoms, they will be scheduled for nasal and throat 
swabs to be tested for influenza. 
 
Study follow-up will span approximately two years from the first dose for participants enrolled 
in the first year. Study follow-up will span approximately 6 months from their enrollment date 
for study subjects recruited in Fall 2017. 
3.2 Study Endpoints 
 Primary Endpoints 
3.2.1
 Hemagglutinin inhibition antibody levels specific for vaccine strains and representative 
strains of circulating influenza viruses. Endpoints based on these data will include 
geometric meant titers (GMT), mean fold rise (MFR), seroresponse rate (SRR), and 
seroprotection rate (SPR). 
o Seroresponse rate defined as the proportion of subjects with paired samples 
with prevaccination HI titer <1:10 and postvaccination (day 28) HI titer ≥1:40 or 
at least a 4-fold increase in HI from prevaccination HI titer ≥1:10. 
o Seroprotection rate defined as the proportion of subjects with postvaccination 
HI titer ≥1:40.  
 Secondary Endpoints 
3.2.2
 Virus neutralizing antibody titers, neuraminidase antibody mediated inhibition titers, 
and anti-human CD107a antibody titers specific for vaccine strains and representative 
strains of circulating influenza viruses. Endpoints based on these data will include 
geometric meant titers (GMT) and mean fold rise (MFR). 
 RT-PCR confirmed influenza 
3.3 Randomization Procedure 
We will use stratified block randomization to ensure more even distribution of subjects with 
prior receipt of 2015-16 influenza vaccine. Subjects will be stratified based on receipt of 2015-

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 17 
 
16 influenza vaccine. Within each group (vaccinated or unvaccinated in 2015-16), sequential 
enrollment numbers will be randomly assigned to each vaccine group in random blocks ranging 
from four to eight. This will ensure proper distribution of subjects across vaccine group 
assignment.  
 
In Fall 2017, only participants who received IIV in 2016-17 will be randomized. We will use 
stratified block randomization and subjects will be stratified based on participation in this study 
during the 2016-17 season (previously participated or new recruit).  
 
This is an open-label study, therefore the vaccine type will be known to the participant. Trained 
research staff will prepare and administer the influenza vaccine.  Randomization to vaccination 
type will occur at the participant’s first study visit after eligibility is confirmed and informed 
consent obtained. Those enrolled in year 1 who receive IIV3 will be randomized a second time 
at their fourth visit.   
3.4 Study Duration 
The maximum duration of a subject’s participation in the study is approximately two years. 
 
4 VACCINES 
All subjects will receive an FDA approved and recommended influenza vaccine in each season. 
Any individual with a contraindication to influenza vaccination will be unable to participate in 
the study. The four influenza vaccines that will be used in this study are 1) IIV3 (Fluvirin, 
Seqirus), 2) HD-IIV3 (Fluzone High-Dose, Sanofi Pasteur), 3) aIIV3 (Fluad, Seqirus) and 4) RIV4 
(FluBlok, Protein Sciences). Vaccines will be stored and administered according to the 
manufacturer’s package insert (Fluvirin: 
http://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM123
694.pdf, Fluzone High-Dose: 
http://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM305
079.pdf, Fluad: 
http://www.fda.gov/downloads/BiologicsBloodVaccines/SafetyAvailability/VaccineSafety/UCM
474387.pdf)FluBlok: 
https://www.fda.gov/downloads/biologicsbloodvaccines/vaccines/approvedproducts/ucm5246
84.pdf 
 
5 SELECTION OF STUDY SUBJECTS 
5.1 Inclusion Criteria 
Subjects must meet all of the following criteria to be eligible to participate: 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 18 
 
1) Age 65 through 74 years at the time of enrollment and are ambulatory and live in 
Marshfield, Wisconsin or surrounding communities. 
2) Willing and able to give informed consent prior to study enrollment. 
3) Able to comply with study requirements. 
5.2 Exclusion Criteria 
Subjects will be excluded if they fulfill any of the following criteria: 
1) Prior receipt of 2016-17 (or 2017-18 for participants recruited in Fall 2017) influenza 
vaccine. 
2) Current participation in another clinical trial. 
3) Presence of a contraindication to influenza vaccines as described by the Advisory 
Committee on Immunization Practices. 
 
6 STUDY PROCEDURES 
6.1 Enrollment Period 
Enrollment in this study will begin when influenza vaccine is available through the Marshfield 
Clinic, typically in August or September 2016, and will continue until the target sample size for 
each group is reached or all eligible adults have been contacted with the goal of completing all 
baseline study visits by the end of October 2016. The second enrollment period for the 
additional 30 subjects will follow the same recruitment procedures in Fall 2017. 
6.2 Recruitment 
A recruitment letter that includes information on the FDA approved influenza vaccines used in 
this study will be sent to a sample of individuals aged 65-74 years who receive outpatient care 
from Marshfield Clinic facilities. Additional mailings will be sent, as needed, to additional 
patients who receive outpatient care from Marshfield Clinic facilities in batches until the target 
sample size in each group is reached. Marshfield Clinic patients who do not receive a 
recruitment letter but would like to participate in the study may participate provided they meet 
study eligibility criteria. 
 
The letter will be followed by telephone contact within 10 days to assess interest, screen for 
contraindications to any vaccine, answer questions, and schedule a pre-vaccination 
appointment for those who have no contraindications and give initial verbal consent.  Study 
visits will be scheduled at convenient times for the enrollee and to allow for efficient processing 
of blood samples (see additional detail in laboratory methods).   
6.3 Study Visit Procedures 
Participants enrolled in the first season will have 6 scheduled study visits over an approximately 
18 month period. At each visit, all participants will have a blood draw (20 mL [4 teaspoons]) for 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 19 
 
evaluation of serologic response. Influenza vaccine will be administered at visit 1 and 4, prior to 
each influenza season. Participants will have a maximum of 6 blood draws during the study 
period.  
 
Participants enrolled in Fall 2017 will have 3 scheduled study visits over an approximately 6 
month period. At each visit, participants will have a blood draw (20 mL [4 teaspoons]) for 
evaluation of serologic response. Influenza vaccine will be administered at the enrollment visit 
(Study Visit 4). 
 Visit 1: Enrollment and Vaccination – Approximately 60 minutes  
6.3.1
Research staff will meet with eligible individuals to review study requirements and obtain 
written consent. A brief questionnaire may be administered to obtain additional information 
regarding demographics and relevant exposure and medical history. We will also request 
permission from individuals to access their electronic medical record in order to obtain data 
related to medical history, medication use, and vaccination history.  The remaining study visits 
will be scheduled.    
 
Blood draw 
6.3.1.1
All participants will have a baseline blood draw for measurement of antibody titers (20 mL [4 
teaspoons]), hemagglutination inhibition [HI] and other assays (see below). Serum from the 
samples will be prepared, frozen, and tested per current protocol (see laboratory methods).  
Additional measures of humoral immune function may also be examined.  
 
Influenza vaccine 
6.3.1.2
Individuals will receive either 1) IIV3, 2) HD-IIV3, or 3) aIIV3 according to a pre-assigned 
randomization scheme.  Trained research staff will administer all vaccines. 
 
Individuals with fevers will be rescheduled regardless of the vaccine to be received.  
Vaccinations will be recorded in the electronic immunization registry, and vaccine information 
sheets will be provided.  
 Visit 2: Day 28 Post-Vaccination, Year 1 – Approximately 30 minutes 
6.3.2
All participants will have a visit with blood draw at 28 (±5) days (20 mL [4 teaspoons]) for 
evaluation of serologic response.  Research staff will also ask for the best time to call during the 
illness surveillance phase of the study.   
  Visit 3: 6-month Post-Vaccination, Year 1 – Approximately 30 minutes 
6.3.3
All participants will be scheduled to collect a 6-month post-vaccination serum specimen at 
approximately 182 (±10 days) days following receipt of the 2016-17 influenza vaccine to 
evaluate serologic response.  The blood draw will be 20 mL (4 teaspoons).  

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 20 
 
 Visit 4: Year 2 Vaccination – Approximately 40 minutes/Enrollment and 
6.3.4
Vaccination of New Recruits – Approximately 60 minutes 
 
Year 2 Vaccination 
6.3.4.1
All participants who received the 2016-17 high dose trivalent inactivated influenza vaccine and 
2016-17 aIIV3 at enrollment will receive the same vaccine type at this visit (for the 2017-18 
season). Participants who received 2016-17 standard dose inactivated influenza vaccine at 
enrollment will be randomized to receive high dose trivalent inactivated influenza vaccine, aIIV3 
or RIV4 at this visit (for the 2017-18 season). Prior to vaccination, all participants will have a 
blood draw to evaluate serologic response.  The blood draw will be 20 mL (4 teaspoons).   
  
Individuals with fevers will be rescheduled regardless of the vaccine to be received.  
Vaccinations will be recorded in the electronic immunization registry, and vaccine information 
sheets will be provided.  
 
Enrollment and Vaccination of New Recruits 
6.3.4.2
Research staff will meet with eligible individuals to review study requirements and obtain 
written consent. A brief questionnaire may be administered to obtain additional information 
regarding demographics and relevant exposure and medical history. We will also request 
permission from individuals to access their electronic medical record in order to obtain data 
related to medical history, medication use, and vaccination history.  The remaining study visits 
will be scheduled.    
 
All participants will have a baseline blood draw for measurement of antibody titers (20 mL [4 
teaspoons]), hemagglutination inhibition [HI] and other assays (see below). Serum from the 
samples will be prepared, frozen, and tested per current protocol (see laboratory methods).  
Additional measures of humoral immune function may also be examined. Participants will 
receive either 1) HD-IIV3, 2) aIIV3, or 3)RIV4 according to a pre-assigned randomization scheme.  
Trained research staff will administer all vaccines. 
 
Individuals with fevers will be rescheduled regardless of the vaccine to be received.  
Vaccinations will be recorded in the electronic immunization registry, and vaccine information 
sheets will be provided.  
 
 Visit 5: Year 2, 1-month Post-Vaccination – Approximately 30 minutes 
6.3.5
All participants will have a visit with blood draw at 28 days (20 mL [4 teaspoons]) for evaluation 
of serologic response.   

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 21 
 
 Visit 6: Year 2, 6-month Post-Vaccination – Approximately 30 minutes 
6.3.6
All participants will be scheduled to collect a 6-month post-vaccination serum specimen in year 
2 at approximately 182 (±10 days) days following receipt of the 2017-18 influenza vaccine to 
evaluate serologic response.  The blood draw will be 20 mL (4 teaspoons).  
 Illness Surveillance  
6.3.7
During both influenza seasons, enrolled subjects will be followed prospectively to monitor for 
influenza illness. Participants will receive a weekly call from an interviewer to ask approximately 
5 screening questions regarding presence of influenza like illness or respiratory symptoms 
including cough and duration of illness. A call list will be generated each week.  The timing of 
the influenza season (surveillance phase) will depend on observations of influenza activity in 
the community and state-wide. We anticipate the surveillance period to be approximately 
November 1 through April 1 of each season.  A reminder letter regarding the start of this phase 
of the study will be sent to enrollees approximately two weeks prior to the start of telephone 
calls. 
 
“New onset” will require a period free of symptoms, or with baseline symptoms, and at least 14 
days from onset of previous illness to differentiate an episode from any prior illness. When a 
new acute respiratory illness is reported and confirmed, an appointment to obtain a combined 
nasal and oropharyngeal (throat) swab for influenza testing will be scheduled within 7 days of 
illness onset if a participant reports an acute respiratory illness. Subjects will be asked to come 
to the clinic to obtain the respiratory sample, but home visits by research staff may also be 
available for subjects who are unable to come to the clinic.  
 
Subjects will continue to receive weekly telephone calls for ascertainment of respiratory 
symptoms. Subsequent weekly calls for persons with recently reported acute respiratory illness 
will document resolution, or return to baseline, of symptoms. 
6.4 Data Collection 
In addition to data collected through the enrollment survey, we will also electronically abstract 
data on vaccination and medical history from the immunization registry and electronic medical 
record.   
 Influenza Vaccination Status 
6.4.1
The availability and types of influenza vaccines vary from season to season. Vaccination status 
and vaccine types received will be obtained for prior seasons for all participants.  
 
We have previously validated the use of the local immunization registry at Marshfield Clinic, 
RECIN, to correctly categorize receipt of influenza vaccine.18 Since RECIN exchanges records 
with the Wisconsin Immunization Registry, all vaccines administered by public health 
immunization providers will be identifiable. Immunization records have sufficient detail to 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 22 
 
distinguish inactivated vaccine types, and to distinguish different manufacturers and lot 
numbers.  
 Medical Record Review 
6.4.2
We may abstract medical records for participants. The goal of the abstraction will be to identify 
potential risk factors for no or low immune response to vaccination or vaccine failure. The 
elements that may be extracted electronically from medical records include: dates of medical 
visits for acute respiratory illness, tests and procedures performed, high risk conditions, 
influenza vaccination status in prior seasons, and patient demographics. 
 
7 SPECIMEN COLLECTION AND HANDLING 
All blood samples will be collected at the Marshfield Clinic Central Laboratory by trained staff. 
The blood samples will be collected using Vacutainer tubes, with uniformly colored tops 
according to type of processing (e.g., red tops for serum), and appropriately labeled with a 
uniform scheme (including study ID and collection time point). Up to 20 mL blood will be 
collected at each visit.  
 
Nasal and throat swabs will be collected by research staff who have been trained on the proper 
technique for collection of nasal and throat swabs. Research staff must demonstrate use of 
proper techniques before they are allowed to collect samples by study participants. Sample 
quality (human RNase P) will be monitored by RT-PCR to ensure adequate sample collection 
procedures. 
 
To support better understanding of the immune response following influenza vaccination, 
specimens will be banked for future studies that might involve respiratory illnesses. 
7.1 Serum 
For serum collection, whole blood drawn in a tube without anticoagulant will be left at room 
temperature for a minimum of 30 minutes and maximum of 2 hours. The samples will then be 
placed at 4ᵒ C. Samples will be processed within 24 hours of collection, and ideally the following 
morning for these afternoon/evening appointments. Tubes will be placed in a table-top 
centrifuge and serum will be clarified by centrifugation at 3000 rpm for 10 minutes at 4ᵒ C. 
Serum will be aliquotted into labeled, aliquot specific, cryovials with an adjustable pipettor. 
Aliquot size will be determined based on cross-site decisions. Serum will then be stored at -80ᵒC 
in labeled fiberboard boxes and stored in the Marshfield Clinic Research Institute Integrated 
Research and Diagnostic Laboratory per current protocol until shipping. Once all participants 
have been enrolled and have completed all required blood draws, batched samples will be 
shared with collaborators at the CDC (or their designee) in order to conduct serologic assays.  

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 23 
 
7.2 Swabs 
Combined nasal and oropharyngeal (throat) swabs for influenza testing will be collected by 
trained research coordinators. The nasal swab is quick and painless, and we have found that it 
is acceptable and well-tolerated. The nasal swab is collected by inserting a large Dacron or 
rayon tipped swab into one nostril to a depth of 1-2 cm. It is rotated gently against the septum 
for 3-5 seconds and then withdrawn. The throat swab is obtained by swabbing both tonsils and 
the posterior oropharynx. A sterile polyester-tipped applicator will be used for swab collection.  
The swab tips are immediately placed in M4-RT viral transport media and labeled. Specimens 
will be refrigerated or held on ice until they are transported to the laboratory on the same day.  
 
8 LABORATORY METHODS 
Blood samples from each study subject will be drawn for influenza serology testing at each of 
the six scheduled study visits. Hemagglutination inhibition (HI) antibody titers will be 
determined for all subjects at all time-points. Testing for virus neutralizing antibody titers, 
neuraminidase antibody mediated inhibition titers, and anti-human CD107a antibody titers will 
depend on HI results. Serologic parameters will include geometric mean titers (GMT), mean fold 
rise (MFR), seroresponse rate (SRR), and seroprotection rate (SPR) by vaccine group. Additional 
measures of humoral immune function may also be examined. 
 
A brief description of each serological assay is provided in the following sections. 
8.1 Hemagglutination Inhibition Antibody Testing 
Serum samples from all subject collected at each of the 6 study visits will be tested by 
hemagglutination inhibition (HI) assays to estimate antibody titers against the 2016-17 vaccine 
strains and representative strains of circulating influenza viruses that are antigenically or 
genetically different during 2016-17 season in year 1 (visits 1, 2, 3 and 4) and 2016-17 and 
2017-18 vaccine strains and representative strains of circulating influenza viruses that are 
antigenically or genetically different during 2017-18 season in year 2 (visits 4, 5, and 6) using 
standard techniques.19 Each serum sample is tested in duplicate and the final titer is the 
geometric mean titer (GMT) of the duplicate titers. HI testing will be conducted at CDC or by a 
CDC-designated laboratory.  
8.2 Microneutralization Antibody Testing 
Virus neutralization titers will be determined by a standard microneutralization (MN) assay, 
which is a highly  sensitive and specific assay for detecting virus-specific neutralizing antibodies 
to influenza viruses.19Testing for MN titers will depend on the circulating virus for each season. 
Each serum sample will be tested in duplicate and the final titer is the geometric mean titer 
(GMT) of the duplicate titers. MN testing will be conducted at CDC. 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 24 
 
8.3 Neuraminidase Antibody Testing 
Testing to assess neuraminidase antibody mediated inhibition of influenza viruses with the 
neuraminidase inhibition assay (NAI) will depend on HI responses. Neuraminidase inhibition 
assays will be conducted at CDC using standard techniques.19 
 
The NAI assay, also known as the enzyme-linked lectin assay, measures antibodies that inhibit 
the enzymatic activity of viral neuraminidase, utilizing a reassortant influenza virus with a 
mismatched hemagglutinin (H6 subtype), to avoid interference by HA-specific antibodies, and 
neuraminidase from vaccine reference viruses. The percent inhibition of neuraminidase 
enzymatic activity at each serum dilution is calculated by comparison with values from virus 
control wells (virus but no serum); end-point NAI titers are calculated as the reciprocal of the 
highest dilution with at least 50% inhibition.  
8.4 Antibody Dependent Cellular Cytotoxicity (ADCC) Analysis 
A modified influenza HA-specific antibody-dependent cell-mediated cytotoxicity natural killer 
(NK) cell activation assay will be quantified using a modified ADCC natural killer (NK) cell 
activation assay.20 Testing for ADCC will depend on baseline responses as measured by HI. 
Serially diluted human serum samples are added to wells containing recombinant HA and mixed 
with a human NK cell line and mouse anti-human CD107a antibody to measure CD107a 
expression on NK cells stimulated by ADCC antibodies present in sera. The results are expressed 
as endpoint titers, for example, the highest serum dilution that achieved the 3% of the arbitrary 
threshold. Each serum sample is tested in duplicate and the final titer is the geometric mean 
titer (GMT) of the duplicate titers. ADCC testing will be conducted at CDC. 
8.5 RT-PCR 
Nasal and oropharyngeal (throat) swabs will be combined and tested using real-time reverse 
transcription polymerase chain reaction (RT-PCR) by staff from Marshfield Clinic Research 
Institute Integrated Research and Diagnostic Laboratory. CDC primers, probes, and procedures 
will be used for this assay. This PCR assay was developed and evaluated by CDC. Standard 
operating procedures for RT-PCR have been developed and will be followed. Total nucleic 
extractions will be performed using the Roche MagNA Pure Total Nucleic Acid Kit (Roche 
Diagnostics, Indianapolis, IN) on 200 μl of clinical samples. This is a high throughput, automated 
magnetic bead technology. Real-time RT-PCR will be performed on nucleic acid extracts using 
the LightCycler® Real-Time PCR System (Roche Diagnostics, Basel, Switzerland). The RT-PCR 
assay is a TaqMan® based real-time detection of the matrix protein (M1) of influenza A and the 
non-structural protein 1 (NS1) of influenza B. Both have been shown to be highly conserved, 
representing effective targets for detection. For each sample, the initial RT-PCR amplifies 
nucleic acid for type A and type B. The human RNase P gene primer and probe set serves as the 
internal positive control for human RNA. Samples positive for influenza A will be retested for 
subtype identification. These include primers for H1N1pdm09, and H3. Samples positive for 
influenza B will be further tested for B lineage.  

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 25 
 
 
Standard laboratory safeguards have been incorporated into the protocol to decrease the risk 
of carryover amplicon contamination. These include the use of uracil glycosylase, three 
separate unidirectional work flow areas, and laboratory surface decontamination products to 
eliminate any residual DNA/RNA.  
 
9 PARTICIPANT COMPENSATION 
Participants will be compensated for the time and effort required to participate in this study. 
Each participant will receive a $50 gift certificate for each blood sample provided (up to $300 
total), and a $20 gift certificate for each combined nasal and oropharyngeal swab sample 
provided.  
 
10 SHARING OF LABORATORY SPECIMENS AND DATA 
Marshfield Clinic Research Institute will share participant samples (serum and swabs) with CDC, 
or a laboratory designated by CDC. Record review and vaccination history data will also be 
shared with CDC, with appropriate privacy measures in place. 
 
11 DATA MANAGEMENT 
11.1 Recording and Collection of Data 
Microsoft Access will be used for enrollment and data management. Research coordinators will 
carry tablet computers to access the Access database on the secure wireless network. Staff will 
use Access to assess eligibility, schedule and enroll patients, and track study visits. Microsoft 
Access has robust procedures to ensure accurate and complete data collection. Access will 
require documentation of informed consent, responses to all inclusion and exclusion criteria, 
specimen collection dates, and vaccination information (date, type, manufacturer, and lot 
number). Radio buttons, pick lists and pre-programmed data checks will be used to ensure valid 
data entry. Access records will be exported to SAS files for final analyses. RECIN immunization 
records will be merged with Access enrollment records to define exposure (vaccination) status.  
 
Reports will be generated on a regular basis to track recruitment, enrollment, and study visit 
completion and shared with collaborators during bi-weekly conference calls.  
11.2 Data Quality Assurance 
Study data will be entered by trained study staff. The study programmer will monitor and 
review the study databases regularly. The study programmer will run a series of quality checks 
and validation to look for missing, incomplete, or inconsistent data. Any identified data issues 
will be reconciled with study staff prior to finalization of the dataset and analysis. 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 26 
 
 
12 STATISTICAL CONSIDERATIONS 
12.1 General 
Demographic and baseline characteristics will be summarized by vaccine group for all 
subjections. Continuous variables will be presented by summary statistics (e.g., mean, and 
standard deviation (SD)) for non-immunogenicity endpoints, and geometric means and their 
95% confidence interval for the immunogenicity endpoints. Categorical variables will be 
presented by frequency distributions; i.e., frequency counts and percentages for the non-
immunogenicity endpoints, and the percentages and their 95% confidence intervals for the 
immunogenicity endpoints. 
 
12.2 Analyses Addressing Objectives 
 Analyses of Primary Objectives 
12.2.1
After log transformation of the antibody titers, geometric mean antibody titers (GMT) pre- and 
post- vaccination will be compared within subjects by vaccine type.  For the primary aim, adults 
receiving high dose trivalent inactivated influenza vaccine will be compared with those 
receiving aIIV3.  
 
The mean and standard deviation of log titers will be calculated. Linear mixed-effects models 
will be used to adjust for variables such as age, given the potential for small differences in age 
and the known difference in GMT by age, baseline antibody titer and prior season vaccination 
status. The mean fold rise (MFR), defined as the mean of the ratio of post vaccination GMT and 
pre-vaccination GMT for each subject, between the groups will be computed from mixed-
effects model along with its associated 95% confidence interval (CI).  For the declaration of non-
inferiority of GMT between groups, the lower bound of the adjusted 95% CIs for the GMT ratios 
will be determined for each strain.   
 Analyses of Secondary Objectives 
12.2.2
Analyses of virus neutralizing antibody titers, neuraminidase antibody mediated inhibition 
titers, and anti-human CD107a antibody titers will be similar to HI data described above. 
Secondary analyses will also include comparisons of GMT for HI and neuraminidase inhibition 
assays, seroconversion rates, and ADCC measures by prior season vaccination and vaccine type 
in the current season.  
 
If vaccine failures (RT-PCR confirmed influenza infection) occur, we will assess factors 
associated with failure using logistic regression, including vaccine type. 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 27 
 
12.3 Power Considerations 
Study sample size was based on available resources. For the analysis on HI titers, a sample size 
of 60 per group will provide over 90% power using a one-sided, two-sample t-test to conclude 
that one vaccine group is superior as defined by rejecting a null hypothesis of equality if the 
GMT values differ by approximately 20%.   
 
13 HUMAN SUBJECTS 
13.1 Informed Consent 
Participants in this study will provide written consent and/or assent for specimen collection, 
influenza vaccination, and medical record review. Further details regarding data privacy and 
protection of human subjects are provided in the IRB application document. 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 28 
 
14 REFERENCES 
1. 
Assaad FA, Reid D. Some factors influencing mortality from influenza. Bull World Health 
Organ 1971;45:113-7. 
2. 
Lui KJ, Kendal AP. Impact of influenza epidemics on mortality in the United States from 
October 1972 to May 1985. Am J Public Health 1987;77:712-6. 
3. 
Barker WH, Mullooly JP. Influenza vaccination of elderly persons. Reduction in 
pneumonia and influenza hospitalizations and deaths. JAMA : the journal of the 
American Medical Association 1980;244:2547-9. 
4. 
Barker WH, Mullooly JP. A study of excess mortality during influenza epidemics in the 
United States, 1968-1976. Am J Epidemiol 1982;115:479-80. 
5. 
Sprenger MJ, Van Naelten MA, Mulder PG, Masurel N. Influenza mortality and excess 
deaths in the elderly, 1967-82. Epidemiol Infect 1989;103:633-41. 
6. 
Eickhoff TC, Sherman IL, Serfling RE. Observations on excess mortality associated with 
epidemic influenza. JAMA 1961;176:776-82. 
7. 
Thompson WW, Shay DK, Weintraub E, et al. Mortality associated with influenza and 
respiratory syncytial virus in the United States. JAMA 2003;289:179-86. 
8. 
Orsi A, Ansaldi F, de Florentiis D, et al. Cross-protection against drifted influenza viruses: 
options offered by adjuvanted and intradermal vaccines. Human vaccines & 
immunotherapeutics 2013;9:582-90. 
9. 
Aspinall R, Del Giudice G, Effros RB, Grubeck-Loebenstein B, Sambhara S. Challenges for 
vaccination in the elderly. Immun Ageing 2007;11:9. 
10. 
Hobson D, Curry RL, Beare AS, Ward-Gardner A. The role of serum haemagglutination-
inhibiting antibody in protection against challenge infection with influenza A2 and B 
viruses. J Hyg (Lond) 1972;70:767-77. 
11. 
Govaert TM, Sprenger MJ, Dinant GJ, Aretz K, Masurel N, Knottnerus JA. Immune 
response to influenza vaccination of elderly people. A randomized double-blind placebo-
controlled trial. Vaccine 1994;12:1185-9. 
12. 
Nabeshima S, Kashiwagi K, Murata M, Kanamoto Y, Furusyo N, Hayashi J. Antibody 
response to influenza vaccine in adults vaccinated with identical vaccine strains in 
consecutive years. Journal of medical virology 2007;79:320-5. 
13. 
Sasaki S, He XS, Holmes TH, et al. Influence of prior influenza vaccination on antibody 
and B-cell responses. PloS one 2008;3:e2975. 
14. 
Ng S, Ip DK, Fang VJ, et al. The effect of age and recent influenza vaccination history on 
the immunogenicity and efficacy of 2009-10 seasonal trivalent inactivated influenza 
vaccination in children. PloS one 2013;8:e59077. 
15. 
Keitel WA, Cate TR, Couch RB. Efficacy of sequential annual vaccination with inactivated 
influenza virus vaccine. American journal of epidemiology 1988;127:353-64. 
16. 
Keitel WA, Cate TR, Couch RB, Huggins LL, Hess KR. Efficacy of repeated annual 
immunization with inactivated influenza virus vaccines over a five year period. Vaccine 
1997;15:1114-22. 

Open-Label Influenza Vaccine Evaluation (OLIVE) 
Protocol 
Marshfield Clinic Research Institute and Centers for Disease Control and Prevention 
Version 3.0 –July 2017 
 
Page 29 
 
17. 
DiazGranados CA, Dunning AJ, Robertson CA, Talbot HK, Landolfi V, Greenberg DP. Effect 
of Previous-Year Vaccination on the Efficacy, Immunogenicity, and Safety of High-Dose 
Inactivated Influenza Vaccine in Older Adults. Clinical infectious diseases : an official 
publication of the Infectious Diseases Society of America 2016;62:1092-9. 
18. 
Irving SA, Donahue JG, Shay DK, Ellis-Coyle TL, Belongia EA. Evaluation of self-reported 
and registry-based influenza vaccination status in a Wisconsin cohort. Vaccine 
2009;27:6546-9. 
19. 
Manual for the laboratory diagnosis and virological surveilance of influenza. WHO, 2011. 
at http://apps.who.int/iris/bitstream/10665/44518/1/9789241548090_eng.pdf.) 
20. 
Zhong W, Liu F, Wilson JR, et al. Antibody-Dependent Cell-Mediated Cytotoxicity to 
Hemagglutinin of Influenza A Viruses After Influenza Vaccination in Humans. Open 
forum infectious diseases 2016;3.