Clinical trial NCT04782323
Conditions: Influenza
Sponsor: Seqirus
Start date: 2021-04-13; Completion date: 2022-03-03
Enrollment: 839 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This Phase 2, randomized, observer-blind, antigen and adjuvant dose-ranging Clinical study is evaluating different formulations of MF59-Adjuvanted Quadrivalent Subunit Inactivated Influenza Vaccine. Approximately 800 subjects are to be randomized into 1 of 8 possible treatment groups. Immunogenicity and safety will be assessed in the overall study population (adults ≥50 years and above) and in the age subgroups ≥50-64 years and ≥65 years. Data from this study will be used to select the optimal dose to be tested in the pivotal Phase 3 immunogenicity and safety study in older adults.

Disclosure Statement: This is a parallel-group dose-ranging study with 8 arms that is participant, investigator and observer-blinded.

Detailed description:

Arms: arm 1: None arm 2: None arm 3: None arm 4: None arm 5: None arm 6: None arm 7: None arm 8: None
Interventions: intervention 1: aQII-1 intervention 2: aQII-3 Investigational intervention 3: aQII-6 Investigational intervention 4: aQII-7 Investigational intervention 5: aQII-9 Investigational intervention 6: aQII-10 Investigational intervention 7: aQII-11 Investigational intervention 8: Licensed QII Active Comparator
Intervention descriptions: intervention 1: Biological/Vaccine: Investigational aQII-1 Investigational MF59 Adjuvanted Quadrivalent Influenza vaccine, containing four influenza virus strains (A/H1N1, A/H3N2, B/Yamagata and Victoria lineage) recommended by the WHO (World Health Organization) for quadrivalent vaccines for the respective season. intervention 2: Biological/Vaccine: Investigational aQII-3 Investigational MF59 Adjuvanted Quadrivalent Influenza vaccine, containing four influenza virus strains (A/H1N1, A/H3N2, B/Yamagata and Victoria lineage) recommended by the WHO (World Health Organization) for quadrivalent vaccines for the respective season. intervention 3: Biological/Vaccine: Investigational aQII-6 Investigational MF59 Adjuvanted Quadrivalent Influenza vaccine, containing four influenza virus strains (A/H1N1, A/H3N2, B/Yamagata and Victoria lineage) recommended by the WHO (World Health Organization) for quadrivalent vaccines for the respective season. intervention 4: Biological/Vaccine: Investigational aQII-7 Investigational MF59 Adjuvanted Quadrivalent Influenza vaccine, containing four influenza virus strains (A/H1N1, A/H3N2, B/Yamagata and Victoria lineage) recommended by the WHO (World Health Organization) for quadrivalent vaccines for the respective season. intervention 5: Biological/Vaccine: Investigational aQII-9 Investigational MF59 Adjuvanted Quadrivalent Influenza vaccine, containing four influenza virus strains (A/H1N1, A/H3N2, B/Yamagata and Victoria lineage) recommended by the WHO (World Health Organization) for quadrivalent vaccines for the respective season. intervention 6: Biological/Vaccine: Investigational aQII-10 Investigational MF59 Adjuvanted Quadrivalent Influenza vaccine, containing four influenza virus strains (A/H1N1, A/H3N2, B/Yamagata and Victoria lineage) recommended by the WHO (World Health Organization) for quadrivalent vaccines for the respective season. intervention 7: Biological/Vaccine: Investigational aQII-11 Investigational MF59 Adjuvanted Quadrivalent Influenza vaccine, containing four influenza virus strains (A/H1N1, A/H3N2, B/Yamagata and Victoria lineage) recommended by the WHO (World Health Organization) for quadrivalent vaccines for the respective season. intervention 8: Biological/Vaccine: Licensed QII Licensed Quadrivalent Influenza vaccine, containing four influenza virus strains (A/H1N1, A/H3N2, B/Yamagata and Victoria lineage) recommended by the WHO (World Health Organization) for quadrivalent vaccines for the respective season.

Protocol documents:
Prot_000.pdf: 
Protocol Number: V201_01
Product Name: aQIVc
Document Status: Version 2.0, Document Date: 22 Jan 2021
Document #: FORM1-000107830  Ed: 1.0 
Source Document #: SOP-000056619
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CLINICAL STUDY PROTOCOL
Study Number
V201_01
Protocol Version
VERSION 2.0: 22 JAN 2021
Study Title
A Phase 2, Randomized, Observer-Blind, Antigen and Adjuvant 
Dose-Ranging Clinical Study to Evaluate Safety and 
Immunogenicity of Different Formulations of MF59-Adjuvanted 
Quadrivalent Subunit Inactivated Cell-derived Influenza Vaccine 
(aQIVc) in Older Adults ≥50 Years of Age
Study Phase
Phase 2
Product Name
MF59-Adjuvanted Quadrivalent Subunit Inactivated Cell-derived 
Influenza Vaccine (aQIVc)
Regulatory Agency 
Identifying Number(s)
IND 029805
Sponsor
SEQIRUS UK LIMITED.
The Point, 29 Market Street, Maidenhead, UK
Previous version, if 
applicable
VERSION 1.0
Date, if applicable
29 SEPTEMBER 2020
Property of Seqirus*
Confidential
May not be used, divulged, published or otherwise disclosed without the written consent 
of the Sponsor
*“Seqirus” includes all legal entities under which the company operates


Protocol Number: V201_01
Product Name: aQIVc
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Table of Contents
Table of Contents .................................................................................................................. 2
List of Tables ........................................................................................................................ 7
List of Figures....................................................................................................................... 7
PROTOCOL SYNOPSIS ...................................................................................................... 8
Definition of Terms ............................................................................................................. 19
List of Abbreviations........................................................................................................... 20
1
BACKGROUND AND RATIONALE ........................................................................ 22
1.1
Background............................................................................................................... 22
1.2
Rationale................................................................................................................... 24
1.3
Potential Risks and Benefits ...................................................................................... 24
2
STUDY OBJECTIVES ............................................................................................... 26
2.1
Primary Objective ..................................................................................................... 26
2.1.1 Primary Immunogenicity Objective ..................................................................... 26
2.1.2 Primary Safety Objective:.................................................................................... 26
2.2
Secondary Objectives................................................................................................ 26
2.2.1 Secondary Immunogenicity Objective(s) ............................................................. 26
2.2.2 Secondary Safety Objectives ............................................................................... 27
2.3
Exploratory Objectives.............................................................................................. 27
2.3.1 Exploratory Immunogenicity Objectives.............................................................. 27
3
STUDY DESIGN......................................................................................................... 27
3.1
Overview of Study Design ........................................................................................ 27
3.2
Scientific Rationale for Study Design........................................................................ 30
3.3
Justification for Dose ................................................................................................ 31
3.4
Study Period.............................................................................................................. 31
3.5
Blinding Procedures.................................................................................................. 31
3.6
Data Collection ......................................................................................................... 32
3.6.1 Data Collected from Subjects .............................................................................. 32
3.6.2 Tools Used for Data Collection............................................................................ 32


Protocol Number: V201_01
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3.7
Collection of Clinical Specimens............................................................................... 34
3.8
Stopping/Pausing Guidelines and Internal Safety Team ............................................. 35
3.9
Data Monitoring Committee...................................................................................... 36
3.10
End of Study ............................................................................................................. 36
4
SELECTION OF STUDY POPULATION................................................................. 37
4.1
Inclusion Criteria....................................................................................................... 37
4.2
Exclusion Criteria ..................................................................................................... 38
4.3
Criteria for Delay of Vaccination............................................................................... 39
4.4
Criteria for Repeat Vaccination in the Study.............................................................. 39
4.5
Premature Withdrawal from Study ............................................................................ 39
5
STUDY PROCEDURES ............................................................................................. 42
5.1
Pre-vaccination Procedures: Screening/Randomization ............................................. 43
5.1.1 Informed Consent................................................................................................ 43
5.1.2 Screening ............................................................................................................ 44
5.1.3 Enrolment............................................................................................................ 46
5.1.4 Randomization .................................................................................................... 46
5.1.5 Blood Draw......................................................................................................... 46
5.2
Vaccination Clinic Visit............................................................................................. 47
5.3
Post-Vaccination Procedures ..................................................................................... 47
5.3.1 eDiary Reminder Alerts....................................................................................... 49
5.4
Post -Vaccination Visits............................................................................................. 49
5.4.1 Follow-up Clinic Visit ......................................................................................... 49
5.4.2 Safety Follow-up Calls ........................................................................................ 50
5.5
Unscheduled Visits.................................................................................................... 51
5.6
Study Completion Visit ............................................................................................. 51
5.6.1 Early Termination Visit........................................................................................ 52
6
TREATMENT OF SUBJECTS .................................................................................. 52
6.1
Study Vaccine(s) ....................................................................................................... 53
6.2
Non-Study Vaccines .................................................................................................. 56
6.3
Vaccine Preparation and Administration .................................................................... 56


Protocol Number: V201_01
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6.4
Vaccine Administration Error or Overdose of Vaccine ............................................... 58
6.5
Prior and Concomitant Medications and Vaccines ..................................................... 58
6.6
Vaccine Supply, Labeling, Storage and Tracking ....................................................... 60
7
ASSESSMENTS.......................................................................................................... 62
7.1
Safety Assessments ................................................................................................... 62
7.1.1 Solicited Adverse Events..................................................................................... 63
7.1.2 Unsolicited Adverse Events................................................................................. 65
7.1.3 Evaluation of Adverse Events.............................................................................. 65
7.1.4 Serious Adverse Events ....................................................................................... 67
7.1.4.1 Adverse Events of Special Interest................................................................. 69
7.1.5 Methods for Recording Adverse Events and Serious Adverse Events................... 69
7.1.5.1 Post-Study Events.......................................................................................... 70
7.1.6 Pregnancies ......................................................................................................... 70
7.1.7 Safety Laboratory Measurements......................................................................... 71
7.2
Efficacy Assessment.................................................................................................. 71
7.3
Immunogenicity Assessment ..................................................................................... 71
8
STATISTICAL CONSIDERATIONS......................................................................... 72
8.1
Endpoints.................................................................................................................. 72
8.1.1 Primary Endpoint(s) ............................................................................................ 72
8.1.1.1 Primary Immunogenicity Endpoints............................................................... 72
8.1.1.2 Primary Safety Endpoints .............................................................................. 73
8.1.2 Secondary Endpoints........................................................................................... 73
8.1.2.1 Secondary Immunogenicity Endpoints........................................................... 73
8.1.2.2 Secondary Safety Endpoints .......................................................................... 74
8.1.3 Exploratory Endpoints......................................................................................... 74
8.2
Success Criteria......................................................................................................... 74
8.3
Analysis Sets............................................................................................................. 75
8.3.1 All Enrolled Set................................................................................................... 75
8.3.2 All Exposed Set................................................................................................... 75
8.3.3 Safety Set............................................................................................................ 75
8.3.4 Full Analysis Set (FAS) Immunogenicity............................................................. 75
8.3.5 Per Protocol Set (PPS) Immunogenicity .............................................................. 76


Protocol Number: V201_01
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8.3.6 Other Analysis Sets ............................................................................................. 76
8.3.7 Subgroups ........................................................................................................... 76
8.3.8 Protocol Deviations ............................................................................................. 76
8.4
Statistical Analysis Plan ............................................................................................ 77
8.4.1 Analysis of Demographic and Baseline Characteristics........................................ 77
8.4.2 Analysis of Primary Objectives ........................................................................... 77
8.4.2.1 Analysis of Primary Safety Objectives........................................................... 77
8.4.2.1.1 Statistical Hypothesis............................................................................... 77
8.4.2.1.2 Analysis Sets ........................................................................................... 77
8.4.2.1.3 Statistical Methods .................................................................................. 77
8.4.2.2 Analysis of Primary Immunogenicity Objectives ........................................... 78
8.4.2.2.1 Statistical Hypothesis............................................................................... 78
8.4.2.2.2 Analysis Sets ........................................................................................... 78
8.4.2.2.3 Statistical Methods .................................................................................. 78
8.4.3 Analysis of Secondary Objectives........................................................................ 80
8.4.3.1 Analysis of Secondary Safety Objectives....................................................... 80
8.4.3.1.1 Analysis of Extent of Exposure................................................................ 80
8.4.3.1.2 Analysis of Solicited Local, Systemic and Other Adverse Events............. 80
8.4.3.1.3 Analysis of Unsolicited Adverse Events................................................... 80
8.4.3.1.4 Statistical Hypotheses.............................................................................. 81
8.4.3.1.5 Analysis Sets ........................................................................................... 81
8.4.3.1.6 Statistical Methods .................................................................................. 81
8.4.3.2 Analysis of Secondary Immunogenicity Objective(s)..................................... 82
8.4.3.2.1 Statistical Hypotheses.............................................................................. 82
8.4.3.2.2 Analysis Sets ........................................................................................... 82
8.4.3.2.3 Statistical Methods .................................................................................. 82
8.4.4 Analysis of Other Objectives............................................................................... 82
8.4.4.1 Analysis of Other Safety Objectives .............................................................. 82
8.4.4.2 Analysis of Other Immunogenicity Objectives............................................... 82
8.5
Sample Size and Power Considerations of Primary Objectives .................................. 83
8.6
Interim Analysis........................................................................................................ 84
9
SOURCE DOCUMENTATION, STUDY MONITORING, AND AUDITING......... 84
9.1
Source Documentation .............................................................................................. 85


Protocol Number: V201_01
Product Name: aQIVc
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9.2
Study Monitoring, Auditing and Source Data Verification ......................................... 86
10 DATA MANAGEMENT ............................................................................................. 87
10.1
Data Entry and Management ..................................................................................... 87
10.2
Data Clarification...................................................................................................... 87
10.3
Data Protection.......................................................................................................... 88
11 RECORD RETENTION............................................................................................. 88
12 USE OF INFORMATION AND PUBLICATION...................................................... 89
13 ETHICAL CONSIDERATIONS ................................................................................ 89
13.1
Regulatory and Ethical Compliance........................................................................... 89
13.2
Informed Consent Procedures.................................................................................... 90
13.3
Responsibilities of the Investigator and IRB/IEC....................................................... 91
13.4
Protocol Amendments ............................................................................................... 92
14 REFERENCE LIST.................................................................................................... 93
APPENDIX 1 – COMORBIDITY RISK SCORE................................................................ 95
APPENDIX 2 – LIST OF ADVERSE EVENTS OF SPECIAL INTEREST........................ 96
APPENDIX 3 – SPONSOR AND INVESTIGATOR SIGNATURE PAGES........................ 99
APPENDIX 4 – GENERAL INFORMATION .................................................................. 101


Protocol Number: V201_01
Product Name: aQIVc
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List of Tables
Table 0-1: Time and Event Schedule ................................................................................... 17
Table 3-1: Overview of study vaccines................................................................................ 28
Table 3-2: Alert Rules for Ad-hoc IST Review .................................................................... 36
Table 3-3: Pause Rules for Planned IST Review.................................................................. 36
Table 5-1: Study Procedures................................................................................................ 43
Table 6-1: aQIVc Vaccine Composition............................................................................... 54
Table 6-2: QIVc Vaccine Composition ................................................................................ 55
Table 6-3: MF59 Composition............................................................................................. 55
Table 6-4: Treatment Arms.................................................................................................. 57
Table 7-1: Severity Grading for Solicited Local and Systemic Adverse Events.................... 63
Table 8-1: Power Calculations............................................................................................. 84
List of Figures
Figure 3-1: Design Space .................................................................................................... 29


Protocol Number: V201_01
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PROTOCOL SYNOPSIS
Name of Sponsor:
Seqirus Ltd UK
Protocol 
number:
V201_01
Generic name of study vaccines:
Investigational MF59-adjuvanted Quadrivalent 
Subunit Inactivated Cell-derived Influenza 
Vaccine (aQIVc) 
Quadrivalent Subunit Inactivated Cell-derived 
Influenza Vaccine (QIVc)
Title of Study: 
A Phase 2, Randomized, Observer-blind, Antigen and Adjuvant Dose-Ranging Clinical 
Study to Evaluate Safety and Immunogenicity of Different Formulations of 
MF59-adjuvanted Quadrivalent Subunit Inactivated Cell-derived Influenza Vaccine 
(aQIVc) in Older Adults ≥50 Years of Age
Study Period: Approximately 6 months 
for each study participant
Clinical Phase: Phase 2
Background and Rationale:
Vaccination is the primary method for preventing influenza and its potentially severe 
complications. Efficacy of the conventional influenza vaccines in the adult population is 
demonstrated to be high. In contrast, the efficacy in elderly individuals is significantly 
lower due to the aging of the immune system (i.e. immunosenescence) and underlying 
medical conditions that can both increase the risk of influenza complications as well as 
interfere with immune responses (Sasaki and Sullivan 2011). In view of the limitations of 
conventional influenza vaccines in older adults, there continues to be an unmet need for a 
new generation of influenza vaccines that provides more consistent and broader coverage 
against all seasonal virus subtypes and variants (Wong and Webby 2013; Reber et al. 
2012).
The MF59-Adjuvanted Quadrivalent Subunit Inactivated Cell-derived Influenza Vaccine 
(aQIVc) offers important advantages over conventional influenza vaccines. The 
cell-derived antigen provides a better match to circulating strains, therefore addressing 
mismatch due to egg adaptation (Hedge 2015; Lambert et al. 2010). The MF59 adjuvant in 
the vaccine is expected to enable increased magnitude of immune response by stimulating 
higher antibody responses at standard doses; increased breadth of immune response by 
expanding antibody repertoire, and increased duration of immune response with higher 


Protocol Number: V201_01
Product Name: aQIVc
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Name of Sponsor:
Seqirus Ltd UK
Protocol 
number:
V201_01
Generic name of study vaccines:
Investigational MF59-adjuvanted Quadrivalent 
Subunit Inactivated Cell-derived Influenza 
Vaccine (aQIVc) 
Quadrivalent Subunit Inactivated Cell-derived 
Influenza Vaccine (QIVc)
antibody response at 6 months after vaccination, which is important in case of prolonged 
influenza seasons and/or late season outbreaks (Frey et al. 2014).
Results of a dose-finding study with different dosages of antigen and adjuvant for the egg-
based adjuvanted trivalent influenza vaccine (aTIV) indicate that the increase of the 
antigen and MF59 dose may be associated with a higher antigen-specific immune response 
(Otten et al. 2020). As such, optimization of the aQIVc vaccine composition may provide 
additional benefits compared to the currently licensed egg-based standard-dose adjuvanted 
influenza vaccine (Fluad Quadrivalent).
The primary aim of this study is to assess immunogenicity and safety of different 
formulations of the adjuvanted quadrivalent cell-based (aQIVc) vaccine versus the non-
adjuvanted quadrivalent cell-based influenza (QIVc) vaccine. 
Immunogenicity and safety will be assessed in the overall study population (adults ≥50 
years and above) and in the age subgroups ≥50-64 years and ≥65 years to determine the 
appropriate age population for this vaccine. Data from this study will be used to select the 
optimal dose to be tested in the pivotal Phase 3 immunogenicity and safety study in older 
adults.
Disclosure Statement: This is a parallel-group dose-ranging study with 8 arms that is 
participant, investigator and observer-blinded. 


Protocol Number: V201_01
Product Name: aQIVc
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Study Objectives:
Primary Objective:
Primary Immunogenicity Objective:

To assess the immunogenicity of the different aQIVc formulations in comparison 
with the non-adjuvanted QIVc vaccine as measured by hemagglutination inhibition 
(HI) assay* at 28 days after vaccination.
* In case of lack of agglutination for a specific strain, immunogenicity for that strain will 
be assessed as measured by microneutralization (MN) assay as an acceptable alternative. 
Primary Safety Objective:

To assess the reactogenicity of all aQIVc formulations and the comparator, QIVc,
for 7 days after vaccination.

To assess the safety of all aQIVc formulations and the comparator, QIVc, for 28 
days after vaccination.
Secondary Objectives:
Secondary Immunogenicity Objective:

To assess the immunogenicity of the different aQIVc formulations in comparison 
with the QIVc vaccine as measured by HI assay* at 180 days after vaccination.
* In case of lack of agglutination for a specific strain, immunogenicity for that strain will 
be assessed as measured by microneutralization (MN) assay as an acceptable alternative.

To assess the immunogenicity of the different aQIVc formulations in comparison 
with the QIVc vaccine as measured by MN assay at 28 and 180 days after 
vaccination.
Secondary Safety Objective:

To assess the safety of all aQIVc formulations and the QIVc comparator for 180 
days after vaccination.
Exploratory Objective(s):
Further details on exploratory objectives are provided in Section 2.3, Exploratory 
Objectives.


Protocol Number: V201_01
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Name of Sponsor:
Seqirus Ltd UK
Protocol 
number:
V201_01
Generic name of study vaccines:
Investigational MF59-adjuvanted Quadrivalent 
Subunit Inactivated Cell-derived Influenza 
Vaccine (aQIVc) 
Quadrivalent Subunit Inactivated Cell-derived 
Influenza Vaccine (QIVc)
Primary and Secondary Endpoints:
A list of primary and secondary endpoints is provided in Section 8.1, Endpoints.
Study Design:
Experimental design: This is a Phase 2, randomized, stratified, controlled, observer-blind 
study in approximately 800 male and female adults aged 50 years and older 
(approximately equally split between two age groups; ≥50-64 and ≥65 years of age) who 
are healthy or have co-morbidities which increase their risk of complications from 
influenza infection.
Duration of the study: The study duration is approximately 6 months for each subject. The 
study will be conducted in one single influenza season.
Vaccination schedule: Single vaccination (Day 1).
Investigational Vaccine: The study will use 7 investigational formulations of aQIVc.
Comparator Vaccine: QIVc, containing 15 µg HA/strain without MF59 adjuvant (0.5 mL)
Treatment groups: All treatment groups will include approximately 100 subjects aged ≥50 
years of age and receive one dose of study vaccine at Day 1. For more details on the 
different treatment groups, see Table 3-1.
Randomization: an Interactive Response Technology (IRT) will be used in the study with 
stratification factors for age (≥50-64 and ≥65 years of age) and history of any influenza 
vaccination within 3 previous years (yes/no) to randomly assign subjects to one of the 8
treatments.
Enrolment: Enrolment will be done in a controlled manner, while the safety data will be 
regularly reviewed by the Internal Safety Team (IST). Upon IST recommendations, 
enrolment may be paused, Further details can be found in Section 3.8 Stopping/Pausing 
Guidelines and Internal Safety Team.


Protocol Number: V201_01
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Name of Sponsor:
Seqirus Ltd UK
Protocol 
number:
V201_01
Generic name of study vaccines:
Investigational MF59-adjuvanted Quadrivalent 
Subunit Inactivated Cell-derived Influenza 
Vaccine (aQIVc) 
Quadrivalent Subunit Inactivated Cell-derived 
Influenza Vaccine (QIVc)
Blinding: Observer-blind study.
Blood sample schedule: A blood sample will be collected from all subjects on Day 1, Day 
29 and Day 181.
Data collection: electronic Case Reporting Form (eCRF) and eDiary.
Study periods: The study has a treatment period (Day 1 to Day 29) and a follow-up period 
(Day 30 to Day 181).
Study clinic visits: Three clinic visits for each subject: at Day 1, Day 29 and Day 181.
Safety phone call: Two safety phone calls (Day 7 and Day 91) will be conducted: on Day 7 
to review the eDiary data as well as to collect any unsolicited AEs, and their related 
medications and any vaccinations, and on Day 91 to collect only SAEs, MAAEs, AEs 
leading to withdrawal, and AESIs, and their related medications and any vaccinations.
Reactogenicity data collection: Solicited AEs, occurring at the day of study vaccination 
and the following 6 days (Day 1 through Day 7, or longer if the events are not resolved), 
will be recorded daily using an eDiary as completed by the subject. The qualified 
healthcare professional will separately evaluate the solicited systemic events and injection 
site pain during the Day 7 safety phone call.
Safety data collection: All unsolicted AEs will be collected for 28 days after vaccination. 
During the follow-up period (from 29 days after vaccination onwards), only unsolicited 
AEs will be collected that are SAEs, MAAEs, AEs leading to study withdrawal, and/or 
AESIs. These data will be captured by interviewing the subject during the clinic visits and 
safety phone calls and by review of available medical records. Subjects will be instructed 
to call the site in the event of any AE which they perceive as being of concern during the 
entire study period.


Protocol Number: V201_01
Product Name: aQIVc
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Name of Sponsor:
Seqirus Ltd UK
Protocol 
number:
V201_01
Generic name of study vaccines:
Investigational MF59-adjuvanted Quadrivalent 
Subunit Inactivated Cell-derived Influenza 
Vaccine (aQIVc) 
Quadrivalent Subunit Inactivated Cell-derived 
Influenza Vaccine (QIVc)
Number of Subjects planned:
Approximately 800 subjects will be enrolled into this study, approximately 100 subjects in 
each group. Subjects will be approximately equally split between two age groups; ≥50-64 
and ≥65 years of age.
Study Population and Subject Characteristics:
This study will enroll male and female adults aged 50 years and older who are healthy or 
have co-morbidities which increase their risk of complications from influenza infection. 
The list of inclusion and exclusion criteria are included in protocol Section 4, Selection of 
Study Population.
Study Procedures:
Written informed consent must be obtained prior to any study-related procedures. 
On Day 1, screening evaluations will be performed and will include a review of relevant 
medical history, physical examination, including body temperature and an eligibility 
assessment. 
All eligible subjects will then be randomized and subsequently receive a single dose of 
study vaccine to which they were assigned. Blood samples for serology testing will be 
collected on Day 1 (prior to vaccination), Day 29 and Day 181. Subjects will be followed 
up for safety for 180 days after vaccination.
Further details on the study procedures are presented at the end of the synopsis in Table
0-1 and in Section 5, Study Procedures.


Protocol Number: V201_01
Product Name: aQIVc
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Name of Sponsor:
Seqirus Ltd UK
Protocol 
number:
V201_01
Generic name of study vaccines:
Investigational MF59-adjuvanted Quadrivalent 
Subunit Inactivated Cell-derived Influenza 
Vaccine (aQIVc) 
Quadrivalent Subunit Inactivated Cell-derived 
Influenza Vaccine (QIVc)
Study Vaccines:
Regardless of the type of vaccine assigned in the trial, subjects will receive a single dose, 
administered intramuscular into the deltoid muscle of the upper arm, preferably in the 
non-dominant arm.
The study will include 8 treatment arms. An overview of the study vaccines per arm can be 
found in Table 3-1.
Investigational Vaccines:

aQIVc: Quadrivalent Subunit Inactivated Cell-derived Influenza Vaccine, which 
includes different amounts of MF59 adjuvant and HA antigens. HA antigens are 
derived from the four influenza virus strains (A/H1N1, A/H3N2, B/Yamagata and 
Victoria lineage) recommended by the WHO (World Health Organization) for 
quadrivalent vaccines for the respective season. 
Comparator vaccine:

QIVc: Non-adjuvanted Quadrivalent Subunit Inactivated Cell-derived Influenza 
Vaccine. HA antigens are derived from the four influenza virus strains (A/H1N1, 
A/H3N2, B/Yamagata and Victoria lineage) recommended by the WHO for 
quadrivalent vaccines for the respective season.
Statistical Analyses:
The primary analysis will be conducted on the immunogenicity and safety data collected 
up to Day 29.
All HI and MN titers below the lower limit of detection (i.e., < LLOQ) will be set to half 
that limit (i.e., ½ * LLOQ). A general linear model (GLM) will be fitted with 
logarithmically transformed (base 10) antibody titer as the response variable, with antigen 
levels and MF59 levels as factors, and age subgroup, previous vaccination history and 
log10 pre-vaccination titer as covariates. Geometric mean titers and the associated CIs will 


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Name of Sponsor:
Seqirus Ltd UK
Protocol 
number:
V201_01
Generic name of study vaccines:
Investigational MF59-adjuvanted Quadrivalent 
Subunit Inactivated Cell-derived Influenza 
Vaccine (aQIVc) 
Quadrivalent Subunit Inactivated Cell-derived 
Influenza Vaccine (QIVc)
be calculated by exponentiation (base 10) of the least square means of the logarithmically 
transformed (base 10) antibody titer. From the model, an adjusted difference in the least 
square means (on the log scale) will be produced with 95% confidence limits. The 
estimated difference and the confidence limits will be back-transformed to obtain an 
adjusted GMT ratio (each aQIVc formulation versus QIVc) with 95% confidence limits. 
Each of the four strains will be analyzed separately.
The statistical analysis of the Geometric Mean Fold Increase (GMFI) in (HI or MN) GMTs 
post-vaccination (Day 29) compared to pre-vaccination (Day 1) will be conducted using 
the same model described above excluding the pre-vaccination titer as a co-variate.
The difference in seroconversion rates (SCRs) or percentage of subjects with HI titer ≥1:40 
at Day 29 from each aQIVc formulation versus QIVc will be presented with 95% CIs 
using the Miettinen and Nurminen method without adjustment for the age stratum. Each of 
the four strains will be analyzed separately. Additional exploratory analysis will be done 
including antigen and MF59 levels and adjustment for age and previous vaccination 
history using a generalized model for binary data.
The statistical analyses of all safety endpoints will be descriptive in nature. Percentage of 
subjects will be presented by vaccine group for the overall age group as well as by age 
subgroup.  
Additional details on the statistical analysis can be found in Section 8 Statistical 
Considerations.
Sample Size: 
A total of 800 subjects will be enrolled in this study and are randomized according to the 
schedule as presented in Table 3-1. Anticipating 5% of the subjects will be non-evaluable, 
this will result in approximately 95 evaluable subjects for each of the aQIVc and QIVc 
comparator vaccine groups. More details on the sample size calculation are provided in 
Section 8.5, Sample Size and Power Considerations of Primary Objectives. 


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Name of Sponsor:
Seqirus Ltd UK
Protocol 
number:
V201_01
Generic name of study vaccines:
Investigational MF59-adjuvanted Quadrivalent 
Subunit Inactivated Cell-derived Influenza 
Vaccine (aQIVc) 
Quadrivalent Subunit Inactivated Cell-derived 
Influenza Vaccine (QIVc)
Interim Analysis:
An interim analysis is not planned for this study.
Internal Safety Team and Data Monitoring Committee:
Because the higher dose formulations of the investigational vaccine (aQIVc) have never 
been administered to humans, additional safety precautions are implemented for this study. 
An IST, independent from the clinical study, will review the safety information on a 
regular basis during the study conduct against pre-defined pausing rules. In addition, a 
Data Monitoring Committee (DMC) will be used in this study. Further details can be found 
in Section 3.8 Stopping/Pausing Guidelines and Internal Safety Team and Section 3.9 Data 
Monitoring Committee.


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Table 0-1: Time and Event Schedule
Visit Type
Clinic Visit
Safety Phone 
Call
Clinic Visit*
Safety Phone 
Call
Clinic Visit*
Study Day
1
7
29
91
181
Visit Window (Days)
n/a
+3 day
-7/+3 days
+/-7 days
+/-14 days
Visit Number
1
2
3
4
5
Study Event
References
Screening and Randomization
Informed Consenta  
Section 5.1.1
X
Medical Historyb
Section 5.1.2
X
Physical Exam
Section 5.1.2
X
Targeted Physical Examc
Section 5.4.1
X
X
Measuring body temperature
Section 5.1.2
X
Pregnancy testd
Section 5.1.2
X
Exclusion/Inclusion Criteria 
Section 4
X
Randomization
Section 5.1.4
X
Study Treatment
Vaccination 
Section 5.2
X
Safety
30 Minutes Post Injection 
Assessment
Section 5.3
X
Train and Dispense Subject eDiary 
Section 5.3
X
Review of eDiary data and 
compliance
Section 3.6.2
Ongoing during eDiary use
Assess unsolicited AEs
Section 7.1.2
X
X
X
Assess SAEs
Section 7.1.4
X
X
X
X
X


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Visit Type
Clinic Visit
Safety Phone 
Call
Clinic Visit*
Safety Phone 
Call
Clinic Visit*
Study Day
1
7
29
91
181
Visit Window (Days)
n/a
+3 day
-7/+3 days
+/-7 days
+/-14 days
Visit Number
1
2
3
4
5
Study Event
References
Assess for AEs leading to 
withdrawal, medically attended AEs 
and AESIs
Section 7.1.4.1
X
X
X
X
X
Assess relevant medications and 
vaccinations
Section 6.5
X
X
X
X
X
Immunogenicity
Serology blood draw
Section 5.1.5
Xe
X
X
Study End
Study Completion/termination
Section 5.6
Xf
* In the exceptional situation in which a clinic visit is not possible, such as in the case when a site is not able to see study subjects at the clinic due to local regulations related to the 
coronavirus pandemic, a home visit may be considered.
Notes: a Consent form(s) should be signed prior to performing any procedures. The informed consent process may be conducted earlier, but within 10 days prior to Day 1. b 
Medical history includes existing comorbidities. c Based on specific complaints as indicated by the subject. dA pregnancy test should be done for females of childbearing potential 
in order to rule out any pregnancy. e Blood draw to be done prior to vaccination. f Subjects who terminate the study early will be requested to complete all safety-related Study 
Completion procedures. Abbreviations: AE = adverse event; AESI = adverse event of special interest; n/a = not applicable; SAE = serious adverse event.


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Definition of Terms 
Term
Definition
Clinic Visit
A clinic visit is a visit to the study site. 
Females of non-
childbearing potential
A female is considered to be of non-childbearing potential prior 
to menarche and after natural or induced menopause. Natural 
menopause is recognized to have occurred after 12 consecutive 
months of amenorrhea for which there is no other obvious 
pathological or physiological cause. Induced menopause is 
recognized to have occurred after hysterectomy, after bilateral 
oophorectomy, or iatrogenic ablation of ovarian function.
Follow-up period
The follow-up period starts for subjects 28 days after 
vaccination and continues for up to 180 days post-vaccination.
Qualified healthcare 
professional
Any licensed healthcare professional who is permitted by 
institutional policy to perform clinical interventions and 
assessments such as physical examinations is trained on the 
study procedure(s) and who is identified within the site signature 
and delegation log.
Trained healthcare 
professionals
Any healthcare professional who is permitted by institutional 
policy, trained to perform delegated tasks, is trained on the study 
procedure(s) and who is identified within the site signature and 
delegation log.
Treatment period
The treatment period begins at the time of vaccination and ends 
28 days after vaccination.


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List of Abbreviations
AE
Adverse event
AESI
Adverse events of special interest
aQIV
MF59-adjuvanted Quadrivalent Subunit Inactivated egg-derived Influenza 
Vaccine 
aQIVc
MF59-adjuvanted Quadrivalent Subunit Inactivated cell-derived Influenza 
Vaccine
CDC
Center for Disease Control and Prevention
CHMP
Committee on Human Medicinal Products
CI
confidence interval
CRF
case report form
CRO
Contract Research Organization
CSR
Clinical Study Report
DMC
Data Monitoring Committee
DNA
deoxyribonucleic acid
eCRF
electronic Case Report Form
eDiaries
electronic Diaries
EC
Ethics Committee (paired with Institutional Review Board)
EDC
Electronic Data Capture
EMA
European Medicines Agency
FAS
Full Analysis Set
FDA
Food and Drug Administration
GCP
Good Clinical Practices
GDPR
General Data Protection Regulation
GMFI
geometric mean fold increase
GMT
geometric mean titer
HA
hemagglutinin
HI
hemagglutination inhibition
HIPAA 
Health Insurance Portability and Accountability Act 
ICF
Informed Consent Form
ICH
International Committee on Harmonization
IEC
Independent Ethics Committee
IM
intramuscular
IRB
Institutional Review Board
IRT
Interactive Response Technology
IST
Internal Safety Team
IV
Intravenous
LL
lower limit
LLOQ
Lower Limit of Quantitation
MAAEs
Medically attended Adverse Events


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MCAR
Missing Completely at Random
MDCK
Madin Darby Canine Kidney 
MF59
MF59C.1 adjuvant
MN
microneutralization
NA
neuraminidase 
NH
Northern Hemisphere
PO
per os, i.e., by mouth
PPS
Per Protocol Set
PT
preferred term
PVRM
Pharmacovigilance and Risk Management
QIVc 
Quadrivalent subunit inactivated cell-derived influenza vaccine 
RTSM
Randomization and Trial Supply Management
SAE
serious adverse event
SAP
Statistical analysis plan 
SD
standard deviation 
SCR
seroconversion rate
SDA
Source Data Agreement
SH
Southern Hemisphere
SUSAR
Suspected Unexpected Serious Adverse Reaction
US
United States
WHO
World Health Organization


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1
BACKGROUND AND RATIONALE
1.1
Background
Influenza is an infectious acute respiratory disease characterized by the abrupt onset of 
respiratory and systemic symptoms, such as fever, cough, sore throat and rhinitis (Temte and 
Prunuske 2010). Influenza occurs in annual epidemics in the northern hemisphere (NH) and 
southern hemisphere (SH) generally affecting individuals during the winter months in 
temperate climates. In tropical climates, it can occur all year round.
Influenza in humans can be caused by the influenza virus type A, B, and C, of which type A 
and B viruses are most clinically relevant. Type A viruses are associated with both annual 
epidemics and pandemics, and B viruses contribute to annual epidemics (WHO 2018). The 
type A viruses are further divided into different subtypes, of which the A/H3N2 and A/H1N1 
viruses are the most clinically relevant for annual influenza disease burden. For influenza B, 
only a single type is known to exist, but 2 distinct genetic lineages are identified: Yamagata 
and Victoria (CDC 2019a). All influenza viruses experience some form of antigenic drift, but 
it is most pronounced in the influenza A virus for which new circulating variants are 
frequently observed over the influenza season. Such evolution of the influenza virus 
influences the host specificity and pathogenicity of these viruses which can differ 
geographically, from season to season and within a season.
Influenza is an acute and highly contagious viral infection with global circulation. Every year 
across the globe, there are an estimated 1 billion cases of influenza disease, of which 3 to 5 
million are severe cases, resulting in 290 000 to 650 000 influenza-related respiratory deaths 
(WHO 2018; Iuliano et al. 2018). In the United States (US), the influenza-related 
hospitalization rate over 15 seasons was estimated to be 309 per 100 000 in individuals 65 
years and older (Zhou et al. 2012). Although the rate of hospitalization associated with 
influenza is lower in subjects 50 to less than 65 years of age compared to 65 years and older, 
the highest number of influenza illnesses and medical visits is reported for the 50 to 64 years 
of age cohort, which indicates also a high burden of disease in this age group (CDC 2019b).
Vaccination is the primary method for preventing influenza and its potentially severe 
complications. Efficacy of the conventional influenza vaccines in the healthy adult 
population (18-50 years of age) is demonstrated to be high (Samuel and Miller 2019). In 


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contrast, the efficacy in elderly individuals is significantly lower due to the aging of the 
immune system (ie immunosenescence) and underlying medical conditions that can both 
increase the risk of influenza complications as well as interfere with immune responses 
(Sasaki and Sullivan 2011). 
In view of the limitations of conventional influenza vaccines in older adults, there continues 
to be an unmet need for a new generation of influenza vaccines that provides more consistent 
and broader coverage against all seasonal virus subtypes and variants (Wong and Webby 
2013; Reber et al. 2012).
One approach to overcoming immunosenescence is to enhance the immune response by the 
addition of an adjuvant, such as the squalene and water emulsion adjuvant, MF59. The 
addition of MF59 increases antigen uptake, macrophage recruitment, macrophage migration, 
and the spectrum of antibody recognition of HA epitopes (Calabro et al. 2011; O’Hagan et al.
2012). Thus, when added to influenza vaccines, MF59 increases vaccine immunogenicity 
against both homologous and heterologous strains. Fluad®, Seqirus’s trivalent seasonal 
influenza egg-based vaccine adjuvanted with MF59, has been licensed for use in Europe 
since 1997 and in the US since 2015. It has been shown to generate significantly higher 
geometric mean hemagglutination inhibition (HI) titers and rates of seroconversion than a 
nonadjuvanted trivalent influenza vaccine comparator in elderly subjects (Frey et al. 2014).
The investigational vaccine in this study, MF59-Adjuvanted Quadrivalent Subunit Inactivated 
Cell-derived Influenza Vaccine (aQIVc), combines benefits of both cell-based and adjuvanted 
technologies to meet the unmet need of influenza prevention in older adults. The cell-derived 
antigen provides a better match to circulating wild-type strains, therefore addressing 
mismatch due to egg adaptation (Hedge 2015; Lambert et al. 2010), which could improve 
vaccine effectiveness. The effect of inclusion of MF59 adjuvant to the vaccine is expected to 
enable increased magnitude, breadth, and duration of immune response. Results of a dose-
finding study with different dosages of antigen and adjuvant for the egg-based adjuvanted 
trivalent influenza vaccine (aTIV) indicate that the increase of the antigen and MF59 dose 
may be associated with a higher antigen-specific immune response (Otten et al. 2020). As 
such, optimization of the aQIVc vaccine composition may provide additional benefits 
compared to the currently licensed egg-based standard-dose adjuvanted influenza vaccine 
(Fluad Quadrivalent).


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1.2
Rationale
The aim of this study is to assess immunogenicity and safety of different formulations of the 
adjuvanted quadrivalent cell-based (aQIVc) vaccine versus the non-adjuvanted quadrivalent 
cell based influenza (QIVc) vaccine.
Immunogenicity and safety will be assessed in the overall study population (adults ≥50 years 
and above) and in the age subgroups ≥50-64 years and ≥65 years to determine the appropriate
age population for this vaccine. Data from this study will be used to select the optimal dose to 
be tested in the pivotal Phase 3 immunogenicity and safety study in older adults.
1.3
Potential Risks and Benefits
Subjects will be exposed to aQIVc or QIVc vaccines.
As with all injectable vaccines, immediate systemic allergic reactions to vaccination can 
occur. The reactions for anaphylaxis are sporadic and estimated to be 1.3 per million vaccine 
doses administered for all vaccines (McNeil 2019). As a precautionary measure, all subjects 
will remain under observation at the study site for at least 30 minutes after vaccination. 
Vasovagal syncope (fainting) can occur before or after any vaccination, is usually triggered 
by the pain or anxiety caused by the injection and is not related to the substance injected. 
Therefore, it is important that standard precautions and procedures be followed to avoid 
injury from fainting. Intramuscular vaccination commonly precipitates a transient and self-
limiting local inflammatory reaction. This typically includes injection site pain, erythema 
(redness), or induration (hardness).
aQIVc is expected to provide benefit over QIVc influenza vaccines in older adults by the 
inclusion of the MF59 adjuvant as well as the use of antigens produced in cell culture that 
more closely resemble the wild-type strains (Hedge 2015; Lambert et al. 2010). In addition, 
results of a dose-finding study with different dosages of antigen and adjuvant for the egg-
based adjuvanted trivalent influenza vaccine (aTIV) indicate that the increase of the antigen 
and MF59 dose may be associated with a higher antigen-specific immune response (Otten et 
al. 2020). As such, optimization of the aQIVc vaccine composition may provide additional 
benefits for the older adult population.


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Overall, the clinical safety experience with aQIV, different formulations of aTIV, and QIVc 
in older adults, in conjunction with data from the repeat dose aQIVc toxicology study are 
sufficient to support the initiation of this Phase 2 study, looking at the safety and 
immunogenicity of various aQIVc formulations in adults 50 years and older. In the repeat-
dose toxicity study, three doses of aQIVc, containing 
 HA/dose (
 HA/strain), were 
administered by intramuscular injection to male and female rabbits at 3-week intervals. The 
vaccine was immunogenic and there was no evidence of local or systemic toxicity. The most 
common local and systemic reactions observed in the egg-based formulation of the
adjuvanted vaccine (aQIV) in older adults were injection site pain, headache, and fatigue. 
Most reactions were reported as mild or moderate in intensity. The most common local and 
systemic reactions associated with administration of the QIVc comparator vaccine in older 
adults included injection site pain, erythema, induration, headache, fatigue, and myalgia. 
Most reactions were reported as mild or moderate in intensity. In the dose-finding study with 
aTIV, the different formulations were all generally well tolerated. The incidence of solicited
adverse events (AEs) was higher in subjects who received the highest dosages of MF59, but 
there were no apparent trends observed in terms of the incidence of unsolicited AEs. The 
solicited adverse reactions associated with the higher dosages of MF59 were mostly mild to 
moderate in severity. MF59 typically increases local reactogenicity in older individuals, 
which may reflect the enhanced immune response due to the adjuvant. Increasing dosages of 
MF59 did not appear to result in an increase in systemic solicited AEs, with the possible 
exception of fatigue (Otten et al. 2020).
Because most of the various formulations of the investigational vaccine (aQIVc) have never 
been administered to humans, additional safety precautions have been incorporated in this 
study (see Section 3.8, Stopping/Pausing Guidelines and Internal Safety Team).
Please refer to the current Investigator’s Brochure (IB) for aQIVc for a summary of potential 
risks and benefits of aQIVc and to the Product Information for QIVc for a summary of 
potential risks and benefits of QIVc.


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2
STUDY OBJECTIVES 
2.1
Primary Objective 
2.1.1 Primary Immunogenicity Objective
1. To assess the immunogenicity of the different aQIVc formulations in comparison with 
the non-adjuvanted QIVc vaccine as measured by hemagglutination inhibition (HI) 
assay* at 28 days after vaccination.
* In case of lack of agglutination for a specific strain using HI assay, immunogenicity for that strain will be 
assessed as measured by microneutralization (MN) assay as an acceptable alternative.
2.1.2 Primary Safety Objective:
1. To assess the reactogenicity of all aQIVc formulations and the comparator, QIVc, for 
7 days after vaccination.
2. To assess the safety of all aQIVc formulations and the comparator, QIVc, for 28 days 
after vaccination.
For primary endpoints, refer to Section 8.1.1, Primary Endpoints.
2.2
Secondary Objectives 
2.2.1 Secondary Immunogenicity Objective(s)
1. To assess the immunogenicity of the different aQIVc formulations in comparison with 
the QIVc vaccine as measured by HI assay* at 180 days after vaccination.
2. To assess the immunogenicity of the different aQIVc formulations in comparison with 
the QIVc vaccine as measured by MN assay at 28 and 180 days after vaccination.
* In case of lack of agglutination for a specific strain, immunogenicity for that strain will be assessed as 
measured by MN assay as an acceptable alternative.
For secondary immunogenicity endpoints, refer to Section 8.1.2.1, Secondary 
Immunogenicity Endpoints.


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2.2.2 Secondary Safety Objectives
1. To assess the safety of all aQIVc formulations and the comparator, QIVc for 180 days 
after vaccination.
For secondary safety endpoints, refer to 8.1.2.2.
2.3
Exploratory Objectives
2.3.1 Exploratory Immunogenicity Objectives
1.
To assess the immunogenicity of the different aQIVc formulations in comparison with 
the QIVc vaccine as measured by MN assay using heterologous target viruses at 28 
days after vaccination in a subset of collected samples.
2.
Additional exploratory immunogenicity analyses may be conducted to further 
characterize the immune response of the study vaccines.
For exploratory endpoints, refer to Section 8.1.3, Exploratory Endpoints.
The results of these exploratory analyses may be presented in an addendum to the Clinical 
Study Report (CSR).
3
STUDY DESIGN
3.1
Overview of Study Design
Experimental design: This is a Phase 2, randomized, stratified, observer-blind, antigen and 
adjuvant dose-ranging clinical study in approximately 800 male and female adults aged 50 
years and older (approximately equally split between two age groups; ≥50-64 and ≥65 years 
of age) who are healthy or have co-morbidities which increase their risk of complications 
from influenza infection.
Duration of the study: The study duration is approximately 6 months for each subject. The 
study will be conducted in one single influenza season.
Vaccination schedule: Single vaccination (Day 1).


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Investigational Vaccine: The study will use 7 investigational formulations of aQIVc (see 
Table 3-1)
Comparator Vaccine: The study will use 1 comparator vaccine (see Table 3-1)
Treatment groups: All treatment groups will include approximately 100 subjects aged ≥50 
years of age (see Table 3-1). All subjects will receive one dose of study vaccine at Day 1. The 
aQIVc formulation numbers correspond to the design space as presented in Figure 3-1. Not 
all formulations within the design space will be directly evaluated in this study, but the study 
results will make it possible to model the effect of the non-tested formulations within the 
design space.
Table 3-1: Overview of study vaccines
Group
Vaccine
Type of 
vaccine
HA antigen 
content/
strain (µg)
MF59 
squalene 
content (mg)
Total 
injected 
volume (mL)
Number of 
randomized 
subjects
A
aQIVc 1
Investigational
100
B
aQIVc 3
Investigational
100
C
aQIVc 6
Investigational
100
D
aQIVc 7
Investigational
100
E
aQIVc 9
Investigational
100
F
aQIVc 10
Investigational
100
G
aQIVc 11
Investigational
100
H
QIVc
Comparator
15
0
0.5
100
The treatments for Groups B, C, D, E, F and G will be obtained by ‘bed-side’ mixing of QIVc, standard dose 
of aQIVc (formulation aQIVc 1) and MF59 solution. Mixing is not required for Groups Aand H.


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Figure 3-1: Design Space
Randomization: an Interactive Response Technology (IRT) will be used in the study with 
stratification factors for age (≥50-64 and ≥65 years of age) and history of any influenza 
vaccination within 3 previous years (yes/no) to randomly assign each subject to one of the 8
treatments.
Blinding: Observer-blind study.
Blood sample schedule: A blood sample will be collected from all subjects on Day 1 (prior to 
vaccination), Day 29 and Day 181. 
Data collection: electronic Case Reporting Form (eCRF) and electronic Diary (eDiary).
Study periods: The study has a treatment period (Day 1 to Day 29) and a follow-up period 
(Day 30 to Day 181).
Study clinic visits: Three clinic visits for each subject: at Day 1, Day 29 and Day 181. 
Safety phone call: Two safety phone calls (Day 7 and Day 91) will be conducted: on Day 7 to 
review the eDiary data as well as collect any unsolicited AEs, and their related medications 
and any vaccinations, and on Day 91 to collect only SAEs, medically attended adverse events


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(MAAEs), AEs leading to withdrawal, and AESIs, and their related medications and any 
vaccinations.
Reactogenicity data collection: Solicited AEs, occurring at the day of study vaccination and 
the following 6 days (Day 1 through Day 7, or longer if the events are not resolved), will be 
recorded daily using an eDiary as completed by the subject. The qualified healthcare 
professional will separately evaluate the solicited systemic events and injection site pain 
during the Day 7 safety phone call.
Safety data collection: All unsolicted AEs will be collected for 28 days after vaccination. 
During the follow-up period (from 29 days after vaccination onwards), only unsolicited AEs 
will be collected that are SAEs, MAAEs, AEs leading to study withdrawal, and/or AESIs. 
These data will be captured by interviewing the subject during the clinic visits and safety 
phone calls and by review of available medical records. Subjects will be instructed to call the 
site in the event of any AE which they perceive as being of concern during the entire study 
period.
Serological assays: 

HI assay for homologous vaccine strains, using cell-derived target virus, will be 
performed in all subjects on serum samples collected on Days 1, 29 and 181.

MN assay for homologous vaccine strains, using cell-derived target virus, will be 
performed in all subjects on serum samples collected on Days 1, 29 and 181.

HI assay for heterologous vaccine strains, using cell-derived target virus, will be 
performed in a subset of collected samples from serum samples collected on Days 1 
and 29.
3.2
Scientific Rationale for Study Design
The design, conduct, and analysis of this study comply with international and regional 
standards for clinical research in humans, and for investigating the immunogenicity and 
safety of seasonal influenza virus vaccines.
Since the manufacturing process of aQIVc and QIVc is the same, with the exception of the 
addition of the MF59 adjuvant in aQIVc, QIVc is chosen as a comparator to assess the effect 


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of different combinations of adjuvant (MF59) and antigen (concentration of HA) in aQIVc
formulations in terms of immunogenicity and safety.
3.3
Justification for Dose
In an earlier dose-finding study with different aTIV formulations, up to 
 times the
standard (9.8 mg) MF59 dose and up to 
 times the standard (15 µg) HA dose per strain
have been tested. The results from this study showed a dose response effect of MF59 as well 
as of antigen and which continued to increase without evidence of plateau (Otten et al. 2020).
Therefore, in the current study with aQIVc, higher dosages than the standard dose will be 
evaluated; formulations with up to 
 times the standard MF59 dose and up to 
 times 
the standard HA dose. Increasing the amounts of MF59 and HA is expected to offer 
immunogenic benefit for subjects, but on the other hand it might also increase the 
reactogenicity profile of the compound. Therefore, the safety and immunogenicity of several 
aQIVc formulations will be evaluated in this study. The optimal formulation, demonstrating a 
positive benefit risk profile, will be taken forward for testing in Phase 3. 
3.4
Study Period
Each subject should expect to participate in the study for approximately 6 months, from the 
time of enrolment through the last study visit.
3.5
Blinding Procedures
The study is designed as an observer-blind study. Observer-blind means that during the 
course of the study, designated and trained unblinded nurse(s), physician(s), or other 
qualified healthcare professional(s) will be responsible for administering the study vaccines 
to the subjects. They will be instructed not to reveal the identity of the study vaccines either 
to the subject or to the investigative site personnel (i.e., investigator and study nurse) 
involved in the conduct of the trial, except in an emergency if unblinding in IRT is not 
possible. Vaccine administration should be shielded from the subject and blinded study 
personnel. The unblinded personnel should not be involved in data collection, data entry or 
data review such as safety assessments and/or collect study data after the vaccinations. Study 
vaccines will be assigned through an IRT system. 


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Details of the IRT unblinding procedures will be described in the IRT Manual. If unblinding 
occurs, either accidental or out of necessity, the Sponsor should be notified promptly.
Investigators, subjects, the Sponsor study team, all laboratory personnel involved in 
processing samples and performing laboratory assays and others who are directly involved in 
the conduct of the study or in the analysis of the primary study results, or have contact with 
study centers, will remain blinded to the treatment codes until the database has been locked 
for primary analysis.
3.6
Data Collection
3.6.1 Data Collected from Subjects
The following data will be collected from each subject over the duration of their study 
participation:

Demographic Information.

Medical History, including existing comorbidities.

Influenza vaccination history.

Physical examination including temperature, height, and weight.

Reactogenicity: Solicited AEs for 7 consecutive days (or longer if the events are not 
resolved) following vaccination (Day 1 through Day 7).

Unsolicited AEs for 28 days following vaccination (Day 1 to Day 29).

SAEs, MAAEs, AEs leading to withdrawal from the study, AESIs as collected from 
Day 1 to Day 181.

Concomitant Medications/vaccinations (as defined in Section 6.5).

Reason for early study termination.
All data collected must only be identified using the Sponsor Subject ID, as described in 
Section 5.1.4, Randomization.
3.6.2 Tools Used for Data Collection
Data will be recorded in the Subject eDiary and collected on electronic Case Report Forms 
(eCRFs).


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Subject eDiary
Electronic Diaries (eDiaries), hereafter referred to as Subject eDiaries, will be the primary 
source document for solicited local and systemic AEs (including body temperature 
measurements). These AEs will be recorded in the eDiary by the subject, starting after the 
initial 30-minute post-vaccination period at the clinic, and continuing for 7 consecutive days 
(or longer if the events are not resolved). Refer to the user’s manual for further details. 
The Investigator or delegated, qualified healthcare staff should review the Subject’s eDiary 
data and monitor the Subject’s eDiary status for compliance on an ongoing basis during 
eDiary use (Day 1 through Day 7) or longer if events are not resolved.
1. The Subject eDiary will be designed in such a way as to prevent any blank, 
incomplete or biologically implausible entries during eDiary completion. Subjects 
will be instructed to fully complete the Subject eDiary each day, as per the 
instructions provided.
2. Before or just after vaccination, site staff must ensure that each subject’s eDiary is 
prepared for data capture in the ensuing post-vaccination period.
3. Any new safety information reported during the site visit cannot be entered into the 
Subject eDiary. Such information must be described in the source notes as a verbally-
reported event. Any unsolicited adverse reaction reported in this fashion must be 
described as an unsolicited reaction and therefore entered on the AE page of the 
eCRF.
4. For any solicited systemic reactions or injection site pain where the investigator 
evaluation in the Day 7 Safety Follow-up call differs from the eDiary data (eg.
presence/absence or severity), the maximum severity and start- and stop dates will be 
reported in the ‘Safety Phone Call Day 7 eCRF’ page.
Electronic Case Report Forms
This study utilizes electronic Case Report Forms (eCRFs) to collect study-related data from 
each subject. A qualified site staff member(s) is required to enter subject data in the eCRFs in 
English based on the medical information available in each subject’s source record.


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Data should be entered into the eCRF in a timely fashion following each subject’s clinic visit, 
study procedure, or phone call. Each subject’s eCRF casebook will be compared with the
subject’s source records by a Sponsor-approved study monitor (or designee) over the duration 
of the study in order to ensure data collection accuracy.
3.7
Collection of Clinical Specimens
The following clinical specimens are required to be collected from subjects in this study:

Blood at Days 1, 29, and 181.

Urine at Day 1 for females of childbearing potential in order to rule out any pregnancy. 
The processing of each specimen should be completed by a qualified site member and in 
accordance with the study-specific manual. The testing of clinical specimens (with the 
exception of the pregnancy test) will be performed by the Sponsor or designated laboratory. 
Refer to the study-specific Clinical Specimen Laboratory Manual for additional details.
Blood Specimens
Approximately 10 mL sample of blood will be drawn from all subjects on Day 1 before 
vaccination and at Days 29 and 181.
The blood will be used for immunological assays. See Section 7, Assessments for additional 
details.
The total amount of blood collected over the study period per subject will be approximately 
30 mL.
Urine Specimens
Urine will be collected for pregnancy testing in females of childbearing potential. Urine will 
be collected on Day 1 before vaccination, results will be recorded in the source document.
Testing will be done at the study site.


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3.8
Stopping/Pausing Guidelines and Internal Safety Team
Most formulations of aQIVc used in this study have never been administered to humans. 
Therefore, additional safety precautions have been included. The Internal Safety Team (IST), 
which is independent of the clinical study team involved in the execution of the study, will be 
alerted when the rules as listed in Table 3-2 are met. An ad-hoc review of unblinded data will 
be scheduled during which the IST will assess the data and judge if the data constitutes a 
potential safety concern. In addition, the IST will review the safety information at two pre-
defined timepoints during the study conduct, using the pause rules as provided in Table 3-3. 
These reviews by the IST will be performed as soon as 7-day safety follow-up data are 
available for the first 80 and 240 enrolled subjects. Additional ad-hoc safety reviews can be 
scheduled if needed. 
If a pause rule is met or a safety concern is identified by the IST, the enrolment into the study 
and study vaccination will be paused pending the independent Data Monitoring Committee 
(DMC) review. It is the Sponsor’s responsibility to put the enrolment (or the study 
vaccination) on hold at all sites via the IRT. The DMC will review all available safety data 
and make a recommendation to the Sponsor whether the study should be permanently 
stopped, modified or continue unchanged. The final decision will be made by the Sponsor in 
consultation with the DMC (and, if needed, the Investigators). The decision will be 
documented and provided in writing to the investigators. 
Independent of the DMC, the Sponsor can halt the study at any time. If the study is halted, 
the Sponsor or delegate will promptly notify the health authorities and investigators, who will 
promptly inform the study subjects and local Ethics Committee/ Institutional Review Board 
(EC/IRB) as per local regulations. Study vaccinations and further enrolment will only occur 
after written authorization is provided by the Sponsor in conjunction with a recommendation 
to proceed by the DMC and in consultation with the health authorities and EC/IRB, as 
appropriate.
The responsibilities, roles, and procedures (including blinding) of the IST are described in the 
IST Charter.


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Table 3-2: Alert Rules for Ad-hoc IST Review
No.
Criteria
No. of subjects
1
Any SAE that cannot be reasonably attributed to a cause 
other than vaccinationa, according to the Investigator’s 
assessment, within 7 days post-vaccination. 
≥1 subject 
2
Severe systemic hypersensitivity such as anaphylaxis, 
within 24 hours after study vaccination. 
≥1 subject
a at least probably related. 
Abbreviations:  IST = internal safety team; No. = number; SAE = serious adverse event. 
Table 3-3: Pause Rules for Planned IST Review
No
Criteria
No. of subjects
1
Any Grade 3 (severe) local solicited AEa lasting ≥2 
consecutive days, within 7 days postvaccination
≥25% (and ≥2 subjects) in one of the
aQIVc groups
2
Any Grade 3 (severe) systemic solicited AEa lasting ≥2 
consecutive days, within 7 days postvaccination
≥25% (and ≥2 subjects) in one of the
aQIVc groups
3
Any severe unsolicited AE that cannot be reasonably 
attributed to a cause other than vaccinationb, within 7 days 
postvaccination
≥10% (and ≥2 subjects) in one of the
aQIVc groups
a based on eDiary data as entered by the subject
b at least probably related. 
Abbreviations: AE = adverse event; aQIVc = MF59-adjuvanted Quadrivalent Subunit Inactivated Cell-derived Influenza 
Vaccine; IST = internal safety team; No. = number. 
3.9
Data Monitoring Committee
A DMC will be established to evaluate subject safety throughout the study at a group and 
subject level, as appropriate. The responsibilities, roles, and procedures (including blinding) 
of the DMC are described in the DMC Charter.
3.10 End of Study
Most clinical trials intended to support the immunogenicity and safety of an Investigational 
Product proceed to full completion of planned sample size accrual.


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Evaluation of the primary and/or secondary immunogenicity objectives requires the testing of 
biological samples from the study subjects, which can only be completed after all samples are 
collected. The last samples for the analysis of the primary and/or secondary objectives will be 
taken on Day 181. For the purpose of this protocol, the end of study is defined as the 
completion of the testing of such biological samples, to be achieved no later than 8 months 
after the collection of the last biological sample Day 181.
4
SELECTION OF STUDY POPULATION
4.1
Inclusion Criteria
In order to participate in this study, all subjects must meet ALL of the inclusion criteria 
described.
1. Individuals ≥50 years of age on the day of informed consent.
2. Individuals who have voluntarily given written consent after the nature of the study has 
been explained according to local regulatory requirements, prior to study entry.
3. Individuals who can comply with study procedures including follow-up1.
4. Males, females of non-childbearing potential or females of childbearing potential2 who are 
using an effective birth control method, at least 30 days prior to informed consent,3 which 
they intend to use for at least 2 months after the study vaccination.
                                               
1A Subject is considered to be compliant if the Investigator judges that the subject will complete the Subject 
eDiary, return for all the follow-up visits and will be available for the telephone calls as scheduled in the study. 
In some situations support from a caregiver is allowed for the completion of the eDiary; this is up to the 
judgement of the Investigator.
2 A female is considered to be of non-childbearing potential prior to menarche and after natural or induced 
menopause. Natural menopause is recognized to have occurred after 12 consecutive months of amenorrhea for 
which there is no other obvious pathological or physiological cause. Induced menopause is recognized to have 
occurred after hysterectomy, after bilateral oophorectomy, or iatrogenic ablation of ovarian function.
3 The following birth control methods are considered effective: abstinence, hormonal contraception (such as 
oral, injection, transdermal patch, implant) if used for at least 30 days prior to informed consent, diaphragm with 
spermicide, tubal occlusion device, intrauterine device, tubal ligation, male partner using condom with 
spermicide, male partner having been vasectomized at least six months prior to informed consent.


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4.2
Exclusion Criteria
In order to participate in this study, all subjects must not meet ANY of the exclusion criteria 
described below:
1. Females of childbearing potential who are pregnant, lactating, or who have not adhered to 
a specified set of contraceptive methods from at least 30 days prior to informed consent
and who do not plan to do so for at least 2 months after the study vaccination.
2. Progressive, unstable or uncontrolled clinical conditions.
3. Hypersensitivity, including allergy, to any component of vaccines whose use is foreseen in 
this study.
4. History of any medical condition considered an adverse event of special interest (AESI)
(see Appendix 2 – List of Adverse Events of Special Interest).
5. Known history of Guillain-Barré syndrome or another demyelinating disease such as 
encephalomyelitis and transverse myelitis.
6. Clinical conditions representing a contraindication to intramuscular administration of 
vaccines or blood draw.
7. Abnormal function of the immune system resulting from:
a) Clinical conditions.
b) Systemic administration of corticosteroids (PO/IV/IM)4 at a dose of ≥20 mg/day of 
prednisone or equivalent for more than 14 consecutive days within 90 days prior to 
informed consent5. 
c) Administration of antineoplastic and immunomodulating agents or radiotherapy 
within 90 days prior to informed consent.
8. Receipt of immunoglobulins or any blood products within 180 days prior to informed 
consent.
                                               
4 PO= by mouth; IV=intravenous; IM= intramuscular
5  Topical, inhaled and intranasal corticosteroids are permitted. Intermitted use (one dose in 30 days) of intra-
articular corticosteroids are also permitted.


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9. Receipt of an investigational or non-registered medicinal product within 30 days prior to 
vaccination.
10. Individuals who received any other vaccines within 14 days (for inactivated vaccines) or 
28 days (for live vaccines) prior to enrolment in this study or who are planning to receive 
any vaccine within 28 days from the study vaccines.
11. Study personnel or immediate family or household member of study personnel.
12. Receipt of any influenza vaccine within 6 months prior to vaccination in this study, or 
plan to receive an influenza vaccine during the study period.
13.
Acute (severe) febrile illness (see Section 4.3, Criteria for Delay of Vaccination).
14. Any other clinical condition that, in the opinion of the investigator, might interfere with 
the results of the study or pose additional risk to the subject due to participation in the 
study.
4.3
Criteria for Delay of Vaccination
There may be instances when individuals meet all eligibility criteria for vaccination yet have 
a transient clinical circumstance, such as an infection, which may warrant delay of 
vaccination: body temperature elevation [≥38.0°C] within 3 days prior to intended study 
vaccination, or acute use of any systemic antipyretics and/or analgesic medications within 24 
hours prior to vaccination. Under such circumstances, a subject may be considered eligible 
for study enrolment after the described window for the delay has passed and 
inclusion/exclusion criteria have been rechecked, and if the subject is confirmed to be 
eligible.
4.4
Criteria for Repeat Vaccination in the Study
Not applicable.
4.5
Premature Withdrawal from Study
Subjects may decide to withdraw at any time, or be withdrawn from the study at the 
discretion of the investigator should any untoward effects occur and/or for safety reasons. In 
addition, a subject may be withdrawn by the investigator or the Sponsor if he/she violates the 


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study plan or for administrative reasons. The investigator or study coordinator must notify the 
Sponsor immediately when a subject has been withdrawn due to an AE.
The circumstances above are referred to as premature withdrawal from the study, and the 
reason for premature withdrawal should be clearly documented and detailed in the source 
documentation and eCRF. The investigator should make every attempt to evaluate the 
subject’s safety, including the resolution of ongoing AEs, at the time of premature 
withdrawal. If a subject wants to withdraw from the study prior to the last planned study visit, 
the subject will be asked to be followed for safety for the duration of the study. When a 
subject withdraws or is withdrawn, from the study, the procedures described in Section 5.6.1, 
Early Termination Visit should be completed if possible. Subjects who have been prematurely 
withdrawn from the study will not be replaced.
Any subject who becomes pregnant during the study, despite the protocol requirement for 
adequate contraception, should be encouraged to continue participating in the study for safety 
follow-up. The site must record a confirmation of pregnancy in the eCRF and must complete 
a Pregnancy Report Form as soon as possible after learning of pregnancy occurrence (see 
Section 7.1.6, Pregnancies for further details). If the pregnant subject withdraws from the 
study for any of the below categories except death, the site will obtain permission from the 
subject to continue to remain in contact with her until the outcome of the pregnancy is 
known, even if the outcome is not known until after the subject reaches the end of the follow-
up period.
The reasons for premature withdrawal from the study include; AE, death, withdrawal of 
consent, lost to follow-up, and protocol deviation. These reasons are described in greater 
detail below.
Adverse Event
For any subject withdrawn from study participation prior to the planned End of Study Visit, it 
is important to determine if an AE was associated with the reason for discontinuing the study. 
This AE must be identified on the AE eCRF page by indicating “Withdrawn from the study 
due to AE”. Any ongoing AEs at the time of study withdrawal must be followed until 
resolution or stabilization.


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Death
For any subject withdrawn from study participation due to death, this should be noted on the 
Study Completion eCRF page and the associated SAE that led to the death must be reported.
Withdrawal of consent
The subject can withdraw consent for participation in the study at any time without penalty or 
loss of benefit to which the subject is otherwise entitled. The reason for early termination 
should be deemed as “withdrawal of consent” if the subject withdraws from participation due 
to a non-medical reason (i.e., a reason other than AE). If the subject intends to withdraw 
consent from the study, the investigator should clarify and document if the subject will 
withdraw completely from the study or if the subject will continue study participation for 
safety, or a subset of other study procedures. If the subject requests complete withdrawal 
from the study, no further study interventions will be performed with the subject.
If a subject withdraws consent, the Sponsor will have full access to all of the subject’s 
medical records prior to the date and time of written revocation.
Lost to Follow-Up
For subjects who fail to show up for the Study Completion visit, or for three consecutive 
visits (clinic or telephone contacts), study staff are encouraged to make at least three 
documented attempts to contact the subject by telephone and at least one documented written 
attempt to contact the subject to encourage the completion of study early termination 
procedures. These efforts to contact the subject should be recorded in the source document. 
The termination date for the subject to be captured on the End of Study eCRF page is the date 
of the last successful contact (clinic visit or telephone) with the subject.
Other Reasons
Examples for subjects withdrawn from the study due to an “other” reason can include: the 
Sponsor’s decision to terminate the study, subject meeting a pre-specified withdrawal 
criterion, subject discontinuation for insurance issues, moving, no time, etc. This reason 
should be noted at the End of Study eCRF page and any ongoing AEs at the time of study 
withdrawal must be followed until resolution/stabilization. Note: If the clinical study is 


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prematurely terminated by the Sponsor, the investigator is to promptly inform the study 
subjects and local EC/IRB and should assure appropriate therapy and follow up for the 
subjects. All procedures and requirements pertaining to the archiving of study documents 
should be followed. All other study materials (study medication/vaccines, etc.) must be 
returned to the Sponsor.
Protocol Deviation
A protocol deviation is any change, divergence, or departure from the study design or 
procedures of a study protocol. In general, subjects associated with protocol deviations may 
remain in the study unless continuation in the study jeopardizes the subject’s health, safety, or 
rights.
Investigators will apply due diligence to avoid protocol deviations. Under no circumstances 
should the investigator contact the Sponsor or its agents, if any, monitoring the study to 
request approval of a protocol deviation, as no authorized deviations are permitted. If the 
investigator feels a change to the protocol would improve the conduct of the study this must 
be considered a protocol amendment, and unless such an amendment is agreed upon by the 
Sponsor and approved by the IRB/EC and health authorities it cannot be implemented. 
5
STUDY PROCEDURES
The sections that follow provide an overview of the procedures that are to be followed in 
enrolling, evaluating, and following subjects who participate in this clinical study. Visits can 
be either clinic visits or safety follow-up telephone calls, as specified in both Table 0-1 and 
below in Table 5-1.


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Table 5-1: Study Procedures
Visit Category
Procedures
Pre-vaccination Procedures and Vaccination 
Clinic Visit 
Section 5.1 describes procedures to be 
followed 
prior 
to 
study 
vaccination: 
informed consent, screening, enrolment, and 
randomization, and blood draw. Section 5.2
and Section 5.3 describe procedures to be 
followed 
during 
the 
clinic 
visit 
for 
vaccination: vaccination and immediate 
post-vaccination procedures.
Post-vaccination Visits
Section 5.4 describes follow-up clinic visits 
and safety follow-up calls.
Study Completion Visit
Section 5.6 describes procedures to be 
followed at the last study visit for a subject 
(may include early termination visit).
5.1
Pre-vaccination Procedures: Screening/Randomization
This section describes the procedures that must be performed for each potential subject prior 
to vaccination, including obtaining informed consent, screening, enrolment, and 
randomization.
5.1.1 Informed Consent
Informed consent is the voluntary agreement of an individual to participate in research. 
Consent must be given with free will of choice and without undue inducement. The 
individual must have sufficient knowledge and understanding of the nature of the proposed 
research, the anticipated risks, and potential benefits, and the requirements of the research to 
be able to make an informed decision.
Informed consent following local IRB/EC guidance must be obtained before conducting any 
study-specific procedure (i.e., all of the procedures described in the protocol). The informed 
consent process may be conducted earlier, but within 10 days prior to Day 1. The process of 


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obtaining informed consent should be documented in the subject source document in addition 
to maintaining a copy of the signed and dated informed consent. Additional specifics 
regarding the informed consent procedures are described in Section 13.2, Informed Consent 
Procedures.
If a subject is unable to read, an impartial witness should be present during the entire 
informed consent discussion. An impartial witness is defined as a person who is independent 
from study conduct, who cannot be unfairly influenced by those involved with the study, who 
attends the informed consent process if the subject cannot read, and who reads the informed 
consent form (ICF) and any other written information supplied to the subject. After the 
written ICF and any other written information to be provided to subjects, is read and 
explained to the subject, and after the subject has verbally consented to the subject’s 
participation in the study and, if capable of doing so, has signed and personally dated the ICF, 
the witness should sign and personally date the consent form. By signing the consent form, 
the witness attests that the information in the consent form and any other written information 
was accurately explained to, and apparently understood by, the subject and that informed 
consent was freely given by the subject. 
5.1.2 Screening
After an individual has consented to participate in the study and informed consent is signed, 
that individual will be given a unique Screening Number, manually created by the 
investigator. The subject’s unique Screening Number will be documented in the Screening 
and Enrolment log. The eligibility of the subject will be determined based on the inclusion 
and exclusion criteria listed in Section 4, Selection of Study Population and evaluated during 
this screening procedure.
The medical history will also be collected, including but not limited to any medical history 
that may be relevant to subject eligibility for study participation such as prior influenza 
vaccination in the 3 years prior to subject enrolment, concomitant medications, and previous 
and ongoing illnesses or injuries. Relevant medical history can also include any medical 
history that contributes to the understanding of an AE that occurs during study participation 
if it represents an exacerbation of an underlying disease/pre-existing problem. In case a 
subject is recruited from outside the clinic, verbal recall of medical history and associated 
concomitant medication is acceptable if medical records are not available.


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If applicable, prior and concomitant medications or vaccinations should be collected (refer to 
Section 6.5, Prior and Concomitant Medications and Vaccines for further details). The use of 
any systemic analgesics and/or antipyretics within 24 hours prior to vaccination is a criterion 
for delay of vaccination (see Section 4.3, Criteria for Delay of Vaccination).
Also, data to quantify the risk of complications from influenza will be collected. The 
‘Prediction Rule for Estimating the Probability of Hospitalization Due to Pneumonia or 
Influenza and Death Due to Any Cause (Hak et al. 2004) found in Appendix 1, will be used. 
The risk assessment, which incorporates comorbidity among other baseline characteristics 
such as outpatient visits during the previous year and previous hospitalizations due to 
pneumonia or influenza, is a validated predictor of the risk of complications from influenza 
in elderly subjects. Using this model, a score of <50 is considered low risk and a score of ≥50 
is considered high risk. Determination of pre-existing medical conditions should be made by 
the investigator based on his or her clinical judgment.  
Pre-vaccination body temperature (preferably oral) will be collected. If body temperature is 
≥38.0°C at the time of screening, vaccination must be postponed until 3 days after the fever 
has resolved (see Section 4.3, Criteria for Delay of Vaccination). Height and weight will also 
be measured. Measurements must be conducted by a trained healthcare professional. For 
women of childbearing potential, a pregnancy test should be performed (see Section 3.7,
Collection of Clinical Specimens). 
A general physical examination is to be performed by a qualified healthcare professional. 
These data will be written in the source document (see Section 9.1, Source Documentation). 
Should the physical assessment reveal any abnormal values or events, these must be 
documented in the eCRF.
In the event that the individual is determined ineligible for study participation, he/she is 
considered a screen failure. Re-screening with a new screening number, or further attempts to 
enroll or randomize the subject are not permitted. The reason for screen failure must be 
documented in the Screening and Enrolment log. If the individual is determined to be eligible 
for the study, he/she will be enrolled in the study.


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5.1.3 Enrolment
After signing the ICF, if an individual is determined to be eligible for study participation, the 
investigator will enroll the subject in the EDC system. Stratification information should be 
provided before randomization.
5.1.4 Randomization
Enrolled subjects will be randomized to one of the eight treatment groups in equal ratios
(Groups A-H, see Table 3-1). Subjects will be stratified based on age (≥50 to 64 years and 
≥65 years) before being randomized. History of any influenza vaccination within 3 previous 
influenza seasons (yes/no) is also a stratification factor that will be applied to all subjects. 
The subject will be randomized in the IRT/ Randomization and Trial Supply Management
(RTSM) system. The subject will receive a unique Subject ID that will be used for all eCRFs 
and associated study documentation for the duration of the study. After randomization, the 
Screening Number ceases to be used and remains in the Screening and Enrolment Log only. 
The list of randomization assignments is produced by the IRT service provider and approved 
by the Sponsor according to the applicable Sponsor’s Standard Operating Procedure (SOP).
If for any reason, after signing the ICF, the subject who is eligible and enrolled fails to be 
randomized, this is called a randomization failure and the early termination study procedures 
must be applied. The reason for all randomization failures should be recorded in the 
Screening and Enrolment Log and in the source document. The information on subjects who 
are randomization failures should be kept distinct from subjects who are screen failures, as 
described in Section 5.1.2, Screening.
If for any reason, after randomization the subject fails to undergo treatment, the subject has to 
be discontinued, and the reason should be recorded on the End of Study eCRF. The 
information on discontinued subjects should be kept distinct in the source documentation 
from randomization failures.
5.1.5 Blood Draw 
Prior to vaccination, approximately 10 mL of blood will be drawn from subjects for the 
immunogenicity testing (see Section 3.7, Collection of Clinical Specimens).


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5.2
Vaccination Clinic Visit
Vaccination will be performed on Day 1.
Ensure the blood sample is taken prior to vaccination.
After completing the pre-vaccination procedures on Day 1, the vaccine will be administered 
according to the procedures described in Section 6.3, Vaccine Preparation and 
Administration. The blinding procedures as described in Section 3.5, Blinding Procedures
should be taken into account.
Prior to the administration of the vaccine, it should be confirmed that the subject is eligible 
and does not meet any criteria for delaying study vaccination as described in Section 4.3, 
Criteria for Delay of Vaccination.
5.3
Post-Vaccination Procedures
The following post-vaccination procedures will be performed on Day 1 
Observation Period
After vaccination, the subject will be observed for at least 30 minutes including observation 
for immediate post-vaccination AEs (unsolicited). All safety data collected during this time 
will be recorded in the subject’s source document and in the Adverse Event eCRF.
Subject eDiary Training
A Subject eDiary will be dispensed (provisioned device or as an app on their own device) in 
this study to document solicited AEs. The Subject eDiary is the primary source for the 
collection of these data; therefore, it is critical that the subject completes the Subject eDiary 
correctly. The subject should be trained on how and when to complete each field of the 
Subject eDiary.
The subject should be trained on how to self-measure local solicited adverse events and body 
temperature (preferably oral). The measurement of solicited local AEs is to be performed 
using the ruler provided by the site.


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The subject should be instructed on how to perform body temperature measurement using the 
thermometer provided by the site. If the subject feels unusually hot or cold during the day, the 
subject should check body temperature. If the subject has a fever, the highest body 
temperature observed that day should be recorded in the Subject eDiary.
Subject eDiary training should be directed at the individual(s) who will perform the 
measurements of AEs and who will enter the information into the Subject eDiary. In some 
situations, the Subject eDiary may be completed by somebody other than the subject, e.g. 
caregiver, spouse, partner (except study site personnel). If a person other than the subject 
enters information into the Subject eDiary, the reason that the subject cannot complete their 
own eDiary and the person’s identity and relationship to the subject must be documented in 
the subject’s source. Any individual who makes entries into the eDiary must receive training 
on completion of the eDiary at the time of the visit. This training must be documented in the 
subject’s source. Ideally, the same individual should complete the eDiary throughout the 
course of the study. Refer to the study-specific eDiary Manual for additional details on the 
assignment and use. 
Subject Compliance: Subjects should start to complete the eDiary on the day of vaccination 
for 7 consecutive days (or longer if the events are not resolved). Diary data should be 
submitted every day. Note: The subject must be contacted by phone if they fail to submit data 
on 2 or more consecutive days during the first 7 days after vaccination. 
Schedule the next study activity
The site should schedule the next study activity with the subject. It is recommended for the 
site to schedule in advance the remaining upcoming study activities. Activities include clinic 
visits or safety phone calls. 
The subject should be reminded of the next planned study activity, a safety phone call on Day 
7. The subject will be reminded to complete the Subject eDiary and to contact the site if there 
are any questions, and to contact the site immediately (or as soon as the subject is medically 
stable) if the subject has a medical condition that leads to a hospitalization or an emergency 
room visit or to a visit to/by a doctor or is otherwise of concern. 


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5.3.1 eDiary Reminder Alerts
The subject will receive daily reminders via the Subject eDiary device’s built-in audio-visual 
alarms to alert the user to complete the diary during the post-vaccination period. From Day 1 
through Day 7, the users will receive daily alerts through the Subject eDiary to record the 
presence (yes/no) of any unsolicited AE of concern (this includes, but is not limited to 
medically attended [defined as symptoms or illnesses requiring hospitalization, or emergency 
room visit, or visit to/by a qualified healthcare professional] unsolicited AEs). In case of such 
events, the subject will be instructed to contact the site as soon as possible to report the 
event(s). 
Reports from the Subject eDiary system should be regularly (eg. daily) generated and
subjects may need to be contacted between Day 1 and Day 7 due to:

Non-compliance (i.e. failing to enter or transmit diary data),

Reporting of any severe solicited reactions (one or more) that may suggest the need for 
medical support (as per medical judgement of the qualified healthcare professional).
Contact with the subject must be written down in the source document. Please refer to 
Section 4.5, Premature Withdrawal from Study and 7.1.3, Evaluation of Adverse Events for 
guidance on necessary action in the event of one of these alerts.
5.4
Post -Vaccination Visits 
Post-vaccination clinic visits or safety calls will be performed on: Day 7 (Safety Phone Call), 
Day 29 (Clinic Visit), Day 91 (Safety Phone Call) and Day 181 (Clinic Visit).
5.4.1 Follow-up Clinic Visit
Post-vaccination clinic visits will be performed on Day 29 (visit 3), and at Day 181 (visit 5) 
(see Section 5.6, Study Completion Visit for assessments to be performed at Study 
Completion Visit). In the exceptional situation in which a clinic visit is not possible, such as 
in the case when a site is not able to see study subjects at the clinic due to local regulations 
related to the coronavirus pandemic, a home visit may be considered.
During the follow-up clinic visit on Day 29, the subject will be interviewed to determine if 
any unsolicited AEs occurred including SAEs, AESIs, MAAEs, and if any concomitant 


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medications or vaccines were taken/received in the time since the last contact. The qualified 
healthcare professional reviewing these data will discuss the symptoms (if any) reported by 
the subject and will determine if any additional diagnoses and/or AEs are present. Adverse 
events reported by the subject at this follow-up clinic visit must be recorded in the subject's 
source document and on an AE eCRF, as specified in Section 7.1, Safety Assessments.
If applicable, a qualified healthcare professional will perform a targeted physical examination 
if necessary based on specific complaints indicated by the subject.
The subject will be asked to return any electronic device that was provisioned for the 
collection of the solicited AEs, if applicable. If the eDiary application was installed on a 
personal phone, it will be removed. During the visit, approximately 10 mL of blood will be 
drawn from all subjects (see Section 3.7, Collection of Clinical Specimens). 
In addition, the site should schedule the next study activity, a safety phone call on Day 91, 
with the subject. The subject will be reminded to contact the site if there are any questions 
and to contact the site immediately (or as soon as the subject is medically stable) if the 
subject has a medical condition that leads to hospitalization or an emergency room visit or 
has any other concern.
5.4.2 Safety Follow-up Calls
Safety follow-up calls will be performed on Days 7 and 91. 
Safety follow-up calls are calls made to the subject by a qualified healthcare professional 
designated on the site’s roles and responsibilities log. These calls will follow a script as 
guidance, which will facilitate the collection of relevant safety information. The subject will 
be interviewed, and information relating to the following AEs will be collected:
Day 7 Safety Follow-up Call

After review of the eDiary entries with the subject, the qualified healthcare professional
will assess if any discrepancies exist (eg. presence/absence or severity) and if yes, make 
his/her own evaluation of the solicited systemic events and injection site pain in the 
relevant sections of the eCRF.

All unsolicited AEs (including, but not limited to: SAEs, AESIs, MAAEs, and AEs 
leading to withdrawal)


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
Concomitant medication associated with the events above or vaccinations.
Day 91 Safety Follow-up Call

SAEs, AESIs, MAAEs, and AEs leading to withdrawal

Concomitant medication associated with the events above or vaccinations.
Contact details including all safety information described by the subject must be written 
down in a designated location within the source document.
In addition, the site should schedule the next study activity with the subject. The subject will 
be reminded to contact the site if there are any questions and to contact the site immediately 
(or as soon as the subject is medically stable) if the subject has a medical condition that leads 
to hospitalization or an emergency room visit or has any other concern.
5.5
Unscheduled Visits
Not applicable to this study.
5.6
Study Completion Visit
The study completion visit will occur on Day 181 and will be a clinic visit. In the exceptional 
situation in which a clinic visit is not possible, such as in the case when a site is not able to 
see study subjects at the clinic due to local regulations related to the coronavirus pandemic, a 
home visit may be considered. The date of study completion is the date of the last contact 
(clinic visit or telephone call) in which the subject’s health status was assessed or, in cases 
where the subject does not agree to any further safety follow-up, it is the date consent is 
withdrawn. This date should be recorded at the end of Study eCRF page. For visit procedures 
to be performed for a subject whose planned study participation ends prematurely, please see
Section 5.6.1, Early Termination Visit.
During the study completion clinic visit, the subject will be interviewed to determine if any 
SAEs, AESIs or MAAEs occurred and if any associated concomitant medications or vaccines 
were taken/received in the time since the last safety assessment. The qualified healthcare 
professional reviewing these data will discuss the symptoms (if any) reported by the subject 
and will determine if any additional diagnoses and/or AEs s are present. Adverse events 
reported by the subject at this follow-up clinic visit must be recorded in the subject's source 
document and on an Adverse Events eCRF, as specified in Section 7.1, Safety Assessments.


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If applicable, a qualified healthcare professional will perform a targeted physical examination
based on specific complaints indicated by the subject. 
During the visit, approximately 10 mL of blood will be drawn from all subjects (see Section 
3.7, Collection of Clinical Specimens).
The site will review with the subject the plan of when information relating to the subject’s 
participation in the study may be available (e.g., study results, treatment assignments). It will 
also be discussed how information relating to the subject’s participation in the study will be 
shared with the subject’s healthcare provider if the subject chooses to share this information.
The site will complete the end of Study eCRF page and this will mark the completion of the 
subject’s participation in the study.
5.6.1 Early Termination Visit
When a subject discontinues the study, the investigator will perform the safety assessment 
procedures during study completion (see Section 5.6, Study Completion Visit). The reason(s) 
for the early termination will be included in the subject’s source documentation. If the Early 
Termination Visit is a telephone call, as much information as possible should be collected. 
Early Termination Visits include subjects who were randomized but not treated.
The site will review with the subject the plan of when information relating to the subject’s 
participation in the study may be available (e.g., study results, treatment assignments). It will 
also be discussed how information relating to the subject’s participation in the study will be 
shared with the subject’s healthcare provider if the subject chooses to share this information.
The site will complete the end of Study eCRF page and this will mark the completion of the 
subject’s participation in the study.
6
TREATMENT OF SUBJECTS
All vaccines associated with this study are to be stored separately from other vaccines and 
medications in a secure location under appropriate storage conditions with temperature 
monitoring. All vaccines associated with this study must be checked for their expiration date
and if the vaccine has experienced a temperature deviation prior to use. Expired vaccines or 
those that have experienced a temperature deviation must not be administered to 


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subjects, as specified in Section 6.6, Vaccine Supply, Labeling, Storage and Tracking and in 
the Investigator’s Manual.
6.1
Study Vaccine(s)
The term ‘study vaccine’ refers to those vaccines provided by the Sponsor, which will be 
evaluated as part of the study objectives. The study vaccines specific to this study are 
described below.
Investigational Vaccine: different formulations of aQIVc
Quadrivalent Subunit Inactivated Cell-derived Influenza Vaccine, which includes MF59 
adjuvant. HA antigens are derived from the four influenza virus strains (A/H1N1, A/H3N2, 
B/Yamagata and Victoria lineage) recommended by the WHO (World Health Organization) 
for quadrivalent vaccines for the respective season. aQIVc formulations B-G (see Table 3-1)
will be obtained through bed-side mixing of aQIVc, QIVc and MF95. The full composition 
of these products is reported in Table 6-1, Table 6-2, and Table 6-3. A detailed instruction on 
how to perform the bed-side mixing can be found in the Investigator’s Manual.


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Table 6-1: aQIVc Vaccine Composition 
Name of Ingredients
Final Quantity per 
0.5mL dose
Function
Active Ingredients
HA and NA antigens from the influenza virus strains 
recommended by the WHO for the respective season

A/ (H1N1)

A/ (H3N2)

B/ (Yamagata lineage)

B/ (Victoria lineage)
 HA
(per strain)
HA Antigen
(active ingredient)
Adjuvant
Squalene
Oil phase
Polysorbate 80
Surfactant
Sorbitan trioleate
Surfactant
Sodium citrate
Buffer
Citric acid
Buffer
Other Ingredients
Phosphate-buffered saline
Sodium chloride
Isotonic acid
Potassium chloride
Isotonic acid
Magnesium chloride hexahydrate
Stabilizer
Disodium phosphate dihydrate
Buffer
Potassium dihydrogen phosphate
Buffer
Water for injection
Up to 0.50 mL
Diluent
Notes: MDCK cell protein (≤25.2 μg), protein other than HA (≤240 μg), MDCK cell deoxyribonucleic acid (DNA) (≤10 
ng), cetyltrimethlyammonium bromide (≤18 μg), beta-propiolactone (<0.5 μg), polysorbate 80 (≤2.675 μg), and sorbitan 
trioleate (≤1.175 μg).
Abbreviations: aQIVc = MF59-adjuvanted Quadrivalent Subunit inactivated cell-derived Influenza Vaccine; DNA = 
deoxyribonucleic acid; HA = hemagglutinin; MDCK = Madin-Darby Canine Kidney; NA = neuraminidase; WHO = 
World Health Organization.


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Table 6-2: QIVc Vaccine Composition
Names of Ingredients
Quantity per 0.5 mL 
dose
Function
Active Ingredients
HA and NA antigens from the influenza virus strains 
recommended by the WHO for the respective season

A/ (H1N1)

A/ (H3N2)

B/ (Yamagata lineage)

B/ (Victoria lineage)
15 µg HA
(per strain)
Active ingredient
Other Ingredients
Buffer M (PBS) pH 7.2
Sodium chloride
Isotonic aid
Potassium chloride
Buffer
Magnesium chloride hexahydrate
Stabilizer
Disodium hydrogen phosphate dihydrate
Buffer
Potassium dihydrogen phosphate
Buffer
Water for injection
Up to 0.5 mL
Diluent
Notes:  residues of special relevance are cetyltrimethlyammonium bromide, polysorbate 80, and β-propiolactone. 
Abbreviations:  HA = hemagglutinin; NA = neuraminidase; PBS = phosphate buffered saline; QIVc = Quadrivalent 
Subunit Inactivated Cell-derived Influenza Vaccine; WHO = World Health Organization.
Table 6-3: MF59 Composition
Names of Ingredients
Quantity per 1 mL 
Function
Squalene
Oil phase
Polysorbate 80
Surfactant
Sorbitan Trioleate
Surfactant
Sodium Citrate, dihyrate
Buffer
Citric Acid, monohydrate
Buffer
Water for Injection
q.s. 1 mL
Aqueous phase
Nitrogen
overlay
Inert gas
Abbreviation: q.s. = quantum satis (to fill up to 1 mL)


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Comparator vaccine: QIVc 
Non-adjuvanted Quadrivalent Subunit Inactivated Cell-derived Influenza Vaccine. HA 
antigens are derived from the four influenza virus strains (A/H1N1, A/H3N2, B/Yamagata 
and Victoria lineage) recommended by the WHO for quadrivalent vaccines for the respective 
season. Fifteen (15) μg of each HA antigen is present in 0.5 mL of the vaccine, which is 
formulated in a prefilled syringe (PFS). The full composition of the vaccine is reported in
Table 6-2.
6.2
Non-Study Vaccines
Not applicable.
6.3
Vaccine Preparation and Administration
The investigator or designee will be responsible for oversight of the administration of the 
vaccine to subjects enrolled in the study according to the procedures stipulated in this study 
protocol. Refer to the Investigator’s Manual for additional details. All vaccines will be 
administered only by unblinded personnel who are qualified to perform that function under 
applicable local laws and regulations for the specific study site.
Regardless of the type of vaccine assigned in the trial, subjects will receive a single dose, 
administered intramuscularly into the deltoid muscle of the upper arm, preferably in the 
non-dominant arm. Detailed administration instructions will be provided to investigators 
prior to the study start.
The study will include 8 treatment arms as described below in Table 6-4.


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Table 6-4: Treatment Arms
Group
Treatment
Type of vaccine
HA antigen 
content/strain 
(µg)
MF59 
squalene 
content (mg)
Total injected 
volume (mL)
A
aQIVc 1
Investigational
B
aQIVc 3
Investigational
C
aQIVc 6
Investigational
D
aQIVc 7
Investigational
E
aQIVc 9
Investigational
F
aQIVc 10
Investigational
G
aQIVc 11
Investigational
H 
QIVc
Comparator
15
0
0.5
The treatments for Groups B, C, D, E, F and G will be obtained by ‘bed-side’ mixing of QIVc, standard dose of 
aQIVc (formulation 1) and MF59 solution. Mixing is not required for Groups A and, H. Mixing instructions will 
be provided in the Investigator’s Manual.
PRECAUTIONS TO BE OBSERVED IN ADMINISTERING STUDY VACCINE:
Prior to vaccination, subjects must be determined to be eligible for study vaccination and it 
must be clinically appropriate in the judgment of the investigator to vaccinate. Eligibility for 
vaccination prior to first study vaccine administration is determined by evaluating the entry 
criteria outlined in protocol Sections 4.1, Inclusion Criteria and 4.2, Exclusion Criteria.
Delay of study vaccination is determined by following the criteria outlined in Section 4.3, 
Criteria for Delay of Vaccination.
Study vaccines should not be administered to individuals with known hypersensitivity to any 
component of the vaccines.
Standard immunization practices are to be observed and care should be taken to administer 
the injection intramuscularly. Before administering the vaccine, the vaccination site is to be 
disinfected with a skin disinfectant (e.g., 70% alcohol). Allow the skin to dry. DO NOT 
inject intravascularly.


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As with all injectable vaccines, trained medical personnel and appropriate medical treatment 
should be readily available in case of anaphylactic reactions following vaccine 
administration. For example, epinephrine 1:1000, diphenhydramine, and/or other medications 
for treating anaphylaxis should be available.
6.4
Vaccine Administration Error or Overdose of Vaccine
A vaccine administration error is defined as receiving a dose of study vaccine that was 
administered by a different route from the intended route of administration. An overdose of 
study vaccine (whether accidental or intentional) is defined when a dosage higher than the 
recommended dosage is administered as per the dosing regimen described in Section 6.1, 
Study Vaccines.
Any vaccine administration error or overdose of study vaccine detailed in this protocol must 
be reported as an AE and as a Protocol Deviation. If the vaccine administration error or 
overdose is associated with an SAE, it must be reported as such within 24 hours to the 
Sponsor. 
6.5
Prior and Concomitant Medications and Vaccines
Prior medications and vaccines
All influenza vaccination history from the 3 years prior to subject enrolment into the study is 
to be obtained from the subject and recorded in the subject’s source records. For each season 
it will be recorded in the eCRF whether the subject was vaccinated (yes/no), including the type 
of vaccination (if available). In addition, the date of the last influenza vaccination and the 
source of the influenza history (recall or medical source) will be captured in the eCRF. All 
subjects who received at least one influenza vaccination within 3 previous influenza seasons
will be categorized as subjects with previous influenza vaccination.
All medications and vaccines taken or received by the subject within 30 days prior to the start 
of the study and related to the subject’s medical history are to be recorded on the Concomitant 
Medications and Non-Study Vaccinations eCRF.
Note that this includes all medications and vaccines described in the inclusion and exclusion 
criteria, including:


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
Immunoglobulins or any blood products within 180 days prior to informed consent.

Administration of antineoplastic and immunomodulating agents or radiotherapy 
within 90 days prior to informed consent. 

An investigational or non-registered medicinal product within 30 days prior to 
informed consent.

Systemic administration of corticosteroids (PO/IV/IM) at a dose of ≥20 mg/day of 
prednisone or equivalent for more than 14 consecutive days within 90 days prior to 
informed consent. 

Any influenza vaccine within 6 months prior to vaccination in this study.
Please note as a reminder, that when the subject is using any of these medications or 
vaccines, the subject should not be enrolled in the study as per the exclusion criteria.
The use of antipyretics and/or analgesic medications within 24 hours prior to vaccination 
must be identified and the reason for their use (prophylaxis versus treatment) must be 
described in the source document and Concomitant Medications eCRF. The use of 
antipyretics/analgesics within 24 hours prior to vaccine administration is a reason to delay 
study vaccination (see Section 4.3, Criteria for Delay of Vaccination).
Medications taken for prophylaxis are those intended to prevent the onset of symptoms. 
Medications taken for treatment are intended to reduce or eliminate the presence of 
symptoms that are present.
Concomitant medications and vaccines
Concomitant medications include all medications (including vaccines) taken by/administered 
to the subject at and after enrolment and must be documented on the Concomitant 
Medications eCRF.
The following concomitant medications will be recorded in the Concomitant Medications 
CRF:

All concomitant medications from Day 1 to Day 29.

All medications associated with SAEs, MAAEs, AESIs, and AEs that lead to 
premature withdrawal from the study, from Day 1 to study completion.


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
All vaccines, including any seasonal or pandemic influenza vaccines, from Day 1 to 
study completion.

Any investigational and non-registered medicinal product (other than the study 
vaccines) during the entire study period (from Visit 1 to study completion).
In addition, the use of the following concomitant medications, for conditions reported in the 
medical history, from enrolment until end of the study should be documented on the 
Concomitant Medication eCRF page as they may have an effect on the interpretation of the 
study objectives and therefore if used, may be determined to be a reason for exclusion from 
one of the analysis sets.

Blood, blood products or a parenteral immunoglobulin preparation. 

Oral or systemic corticosteroids.

Other immunomodulating agents.
6.6
Vaccine Supply, Labeling, Storage and Tracking
The Sponsor will ensure the following:

Supply the study vaccines.

Appropriate labeling of all study vaccines provided, that complies with the legal 
requirements of each country where the study is to be performed.

Appropriate storage and distribution of study vaccines.
The investigator must ensure the following:

Acknowledge receipt of the study vaccines by a designated staff member at the site, 
including:
–
Confirmation that the vaccines were received in good condition and in the right 
amount
–
Confirmation to the Sponsor that the temperature range during shipment from the 
Sponsor to the investigator’s designated storage location is in the correct range (2°C 
to 8°C) 
–
Report any temperature deviation and do not use vaccines until further confirmation 
by the Sponsor or delegate that the vaccines are authorized for use.


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
Proper storage of the study vaccines, including:
–
Storage in a secure, locked, temperature-controlled location.
–
Proper storage according to the instructions specified on the labels and in the 
Investigator’s Manual.
–
Appropriate record-keeping and inventory of the study vaccines, including regular 
documentation of adequate storage temperature.

Appropriate use of the study vaccines, including:
–
No use of vaccines prior to receipt of authorization for use from the Sponsor.
–
Use only in accordance with the approved protocol.
–
Proper handling, including confirmation that the vaccine has not expired prior to 
administration.
–
Appropriate documentation of the administration of vaccines to study subjects 
including:
o
Date, dosage, batch/lot numbers, expiration dates, unique identifying numbers 
assigned to subjects and study vaccines, and time of vaccine administration. 
This information will be maintained in an accountability log that will be 
reviewed by the site monitor.
o
Reconciliation of all vaccines received from the Sponsor. Reconciliation is
defined as maintaining records of which and how many vaccines were 
received, which vaccines were administered to subjects, which vaccines were 
destroyed at the site, and which vaccines were returned to the Sponsor, as 
applicable.

Proper adherence to the local institutional policy with respect to the destruction of study 
vaccines.

Complete record keeping of vaccine use, wastage, return or destruction, including 
documentation of:
–
Copy of the site’s procedure for the destruction of hazardous material.
–
The number of doses destroyed, date of destruction, destruction code (if available), 
method of destruction, and name of individual performing destruction.


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Vaccines that have been stored differently from the manufacturer’s indications must not be 
used unless the Sponsor provides written authorization for use. In the event that their use 
cannot be authorized, the Sponsor will make every effort to replace the vaccine supply. All 
vaccines used in conjunction with this protocol must be stored separately from normal 
hospital/practice stocks to prevent unintentional use of study vaccines outside of the clinical 
study setting.
Monitoring of vaccine accountability will be performed by the study monitor during site 
visits and at the completion of the study.
At the conclusion of the study, and as appropriate during the course of the study, the 
investigator must ensure that all unused study vaccines, packaging, and supplementary labels 
are destroyed locally (upon approval from the Sponsor) or returned to the Sponsor.
7
ASSESSMENTS
7.1
Safety Assessments
The measures of safety used in this study are routine clinical procedures. They include a 
close vigilance for and stringent reporting of selected local and systemic AEs routinely 
monitored in vaccine studies as indicators of reactogenicity. 
An AE is defined as any untoward medical occurrence in a subject or clinical investigation 
subject administered a pharmaceutical product at any dose that does not necessarily have to 
have a causal relationship with this treatment. Therefore, an AE can be any unfavorable and 
unintended sign (including an abnormal laboratory finding, for example), symptom, or 
disease temporally associated with the use of an investigational product, whether or not 
considered related to the investigational product. This definition includes intercurrent 
illnesses or injuries and exacerbation of pre-existing conditions.
The period of observation for AEs extends from the time immediately after the subject has 
received the study vaccine until he or she completes the specified safety follow-up period at 
the end of the influenza season. When a subject terminates the study early, the period of 
observation will stop as well. Events occurring prior to receiving study vaccine/product will 
be documented as medical history and recorded within the source document. Adverse events 


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are collected as either solicited or unsolicited; solicited events are derived from organized 
data collection systems, such as Subject eDiaries or interviews.
7.1.1 Solicited Adverse Events
The term “reactogenicity” refers to solicited signs and symptoms (“solicited adverse events”) 
occurring in the hours and days following vaccination, to be collected by the subject for 7 
consecutive days (Day 1 through Day 7), using a Subject eDiary with pre-defined events. If a 
solicited local or systemic adverse event continues beyond day 7 after vaccination, it will 
continue to be recorded in the Subject eDiary.
During the Day 7 Safety Phone Call, the investigator or qualified healthcare professional
will review the eDiary data with the subject. In case the investigator evaluation differs from 
the eDiary data (eg. presence/absence or severity), the investigator or qualified healthcare 
professional will make his/her own evaluation of the solicited systemic events and injection 
site pain for maximum severity and start and stop date (induration, erythema and fever are 
recorded in the eDiary through exact measurements and therefore not included in the 
investigator evaluation). This evaluation will be entered on the ‘Safety Phone Call Day 7 
eCRF’. This evaluation is separate from the subject reported eDiary data.
Each solicited AE is to be assessed according to a defined severity grading scale; see 
specifics of the solicited event and grading system below in Table 7-1.
Table 7-1: Severity Grading for Solicited Local and Systemic Adverse Events
Solicited Event
Grade 1/Mild
Grade 2/Moderate
Grade 3/Severe
Injection site pain
No interference with 
daily activity
Interferes with daily 
activity
Prevents daily activity
Erythema
25-50 mm
51-100 mm
> 100 mm
Induration
25-50 mm
51-100 mm
> 100 mm
Loss of appetite
Eating less than usual 
with no effect on normal 
activity
Eating less than usual 
/interfered with normal 
activity
Not eating at all
Nausea
No interference with 
daily activity
Interferes with daily 
activity
Prevents daily activity


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Solicited Event
Grade 1/Mild
Grade 2/Moderate
Grade 3/Severe
Fatigue
No interference with 
daily activity
Interferes with daily 
activity
Prevents daily activity
Myalgia
No interference with 
daily activity
Interferes with daily 
activity
Prevents daily activity
Arthralgia
No interference with 
daily activity
Interferes with daily 
activity
Prevents daily activity
Headache
No interference with 
daily activity
Interferes with daily 
activity
Prevents daily activity
Chills
No interference with 
daily activity
Interferes with daily 
activity
Prevents daily activity
Fever
38.0 - 38.4°C
100.4 – 101.1°F
38.5 – 38.9°C
101.2 – 102.0°F
≥39.0°C
≥102.1°F
Other Indicators of Reactogenicity
The use of analgesics/antipyretics will be captured as “absent” or “present” separately by 
reason “for treatment” or “for prevention”.
The study staff must review the data entered into the Subject eDiary as described in Section 
3.6.2, Tools Used for Data Collection and Section 5.4, Post-vaccination Visits.
Note: Any solicited AE that meets any of the following criteria must be entered into subjects’ 
source document (see Section 9.1, Source Documentation) and also as an AE on the AE 
eCRF:

Solicited local or systemic AE that leads to an unscheduled visit to a healthcare provider 
(MAAE, see Section 7.1.3, Evaluation of Adverse Events).

Solicited local or systemic AE leading to the subject withdrawing from the study or the 
subject being withdrawn from the study by the investigator (AE leading to withdrawal, 
see Section 7.1.3, Evaluation of Adverse Events).

Solicited local or systemic AE that otherwise meets the definition of a serious AE (see 
Section 7.1.4, Serious Adverse Events).


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7.1.2 Unsolicited Adverse Events
An unsolicited AE is an AE that was not solicited using a Subject eDiary and that was 
spontaneously communicated by a subject who was vaccinated with one of the study 
vaccines.
Potential unsolicited AEs may be medically attended (defined as symptoms or illnesses 
requiring hospitalization, or emergency room visit, or visit to/by a qualified healthcare
professional), or were of concern to the subject. In case of such events, subjects will be 
instructed to contact the site as soon as possible to report the event(s). The detailed 
information about the reported unsolicited AEs will be collected by the qualified site 
personnel during the interview and will be documented in the subject’s records.
The period of observation for AEs extends from the time the subject has received the study 
vaccine until the subject either completes the specified safety follow-up period (Day 181) or 
at early study termination. 
All unsolicited AEs that start during the treatment period (Day 1 to Day 29) will be recorded 
in the eCRF. During the follow-up (Day 30 to Day 181), unsolicited AEs that meet any 
reporting criterion (an SAE, AESI, AE leading to withdrawal or a MAAE) are to be recorded 
in the eCRF. 
7.1.3 Evaluation of Adverse Events
Every effort should be made by the investigator to evaluate safety information reported by a 
subject for an underlying diagnosis and to capture this diagnosis as the event on the AE page. 
In other words, the practice of reporting only symptoms (e.g., “cough” or “ear pain”) are 
better reported according to the underlying cause (e.g., “asthma exacerbation” or “otitis 
media”).
The severity of events reported on the AE eCRF will be determined by the investigator as:
Mild:
transient with no limitation in normal daily activity.
Moderate:
some limitations in normal daily activity.
Severe:
unable to perform normal daily activity.


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The relationship of the study treatment to an unsolicited AE will be determined by the 
investigator based on the following definitions:
1. Not Related
The AE is not related to an investigational vaccine if there is evidence that clearly indicates 
an alternative explanation. If the timing of the exposure to the vaccine and the onset of the 
AE is not reasonably related in time, or other facts, evidence or arguments exist that 
reasonably suggest an alternative explanation, then the AE is not related.
2. Possibly Related
The administration of the investigational vaccine and AE are considered reasonably related in 
time and the AE could be explained by exposure to the investigational vaccine or by other 
causes.
3. Probably Related
Exposure to the investigational vaccine and AE are reasonably related in time and no 
alternative explanation has been identified.
The relationship of the study treatment to an unsolicited AE will be determined by the 
investigator. 
Adverse events will also be evaluated by the investigator for the co-existence of any of the 
other following conditions:

Medically attended adverse event: an AE that leads to a visit to a healthcare provider.

AESI see Section 7.1.4.1, Adverse Events of Special Interest.

AEs leading to withdrawal: adverse events leading to study withdrawal. 
If solicited or unsolicited AEs have been reported and the subject indicated that the symptoms 
required medical attendance or were of concern, the subject must be contacted for further 
information.
When the subject is contacted for any of these reasons, the contact and all reported safety 
information must be documented in the subject’s source documentation.


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All AEs, regardless of severity, will be monitored until resolution or until the investigator 
assesses them as chronic or stable. All subjects experiencing AEs - whether considered 
associated with the use of the study vaccine or not - must be monitored until symptoms 
subside and any abnormal laboratory values have returned to baseline, or until there is a 
satisfactory explanation for the changes observed, or until death, in which case a full 
pathologist’s report should be supplied, if possible. The investigator’s assessment of ongoing 
AEs at the time of each subject’s last visit should be documented in the subject’s medical 
chart.
7.1.4 Serious Adverse Events
An SAE is defined as any untoward medical occurrence that at any dose results in one or more 
of the following:

Death.

Is life-threatening (i.e., the subject was, in the opinion of the investigator, at immediate 
risk of death from the event as it occurred); it does not refer to an event which 
hypothetically might have caused death if it were more severe.

Required or prolonged hospitalization.

Persistent or significant disability/incapacity (i.e., the event causes a substantial 
disruption of a person’s ability to conduct normal life functions).

Congenital anomaly/or birth defect.

An important and significant medical event that may not be immediately life-threatening 
or resulting in death or hospitalization but, based upon appropriate medical judgment, 
may jeopardize the subject or may require intervention to prevent one of the other 
outcomes listed above.
Adverse events that do not fall into these categories are defined as non-serious.
It should be noted that severe AEs need not be serious in nature and that an SAE need not, by 
definition, be severe.


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All SAEs will be evaluated by the investigator for the relationship of the event to the study 
vaccine. SAEs that are judged to be possibly or probably related to the study vaccine should 
be reported to the Sponsor as related/suspected events.
The relationship of the study treatment to an SAE will be determined by the investigator 
based on the following definitions:
1. Related/suspected
The SAE is judged by the investigator to be possibly or probably related to the study vaccine 
on the AE eCRF page (see Section 7.1.3, Evaluation of Adverse Events).
2. Not Related
The SAE is not related if the occurrence of the SAE is not reasonably related in time, or the 
SAE is considered unlikely to be related to using of the study vaccine, i.e., there are no facts 
(evidence) or arguments to suggest a causal relationship.
The relationship of the study vaccine to an SAE will be determined by the investigator.
In addition, SAEs will be evaluated by the Sponsor or designee for “expectedness.” An 
unexpected AE is one that is not listed in the current Product Information or the Reference 
Safety Information in the Investigator’s Brochure or an event that is by nature more specific 
or more severe than a listed event.
A pre-existing event or condition should be recorded on the Medical History eCRF. The 
worsening of a pre-existing event or condition should be reported as an AE as described in 
Sections 5.1.2, Screening and 7.1.2, Unsolicited Adverse Events. Worsening of a pre-existing 
event or condition should be reported as SAE if meets any of SAE criteria. If the subject was 
hospitalized and the onset of an event leading to hospitalization occurred before the subject 
entered the study (e.g., any pre-planned hospitalization for conditions like cosmetic 
treatments or for non-emergency routine visits for a pre-existing condition), the 
hospitalization would not lead to an AE being classified as serious unless, in the view of the 
investigator, hospitalization was prolonged as a result of participation in the clinical study or 
was necessary due to a worsening of the pre-existing condition.


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7.1.4.1
Adverse Events of Special Interest
Adverse events of Special Interest (AESI)s are a pre-defined list of adverse events that are of 
potential immune-mediated medical conditions; the list can be found in Appendix 2. 
Subjects will be assessed at each clinic visit and safety phone call for any new medical events 
or signs or symptoms that could possibly indicate an AESI. The subject will be asked whether 
any new diagnosis has been given to the subject through a review of recent medical history. 
Should a qualified healthcare professional who is not the investigator suspect a potential 
AESI, she/he should promptly inform the investigator. 
A diagnosis of an AESI is to be reported in the same manner and time frame as an SAE and 
will be captured on the AE eCRF. If the eCRF is not available, then the study site must 
complete the paper SAE Report Form and send it to the Sponsor’s Pharmacovigilance and 
Risk Management (PVRM) (or delegate) at AE.reporting@Seqirus.com. The investigator 
must notify the Sponsor within 24 hours. The AESI diagnosis, as well as if any medication is 
taken to treat the condition, will be recorded in the subject’s source documents and on the AE 
eCRF and, as applicable, the Concomitant Medication eCRF.
7.1.5 Methods for Recording Adverse Events and Serious Adverse Events
Findings regarding AEs must be reported on an AE eCRF, as specified in Section 7.1.2, 
Unsolicited Adverse Events. All findings in subjects experiencing AEs must be reported also 
in the subject's source document.
All SAEs which occur during the course of the study, whether considered to be associated 
with the study vaccination or not, must be documented on the AE eCRF within 24 hours of 
the site becoming aware of the event.
If the eCRF is not available, then the study site must complete the paper SAE Report Form 
and send it to the Sponsor's PVRM (or delegate) at AE.reporting@Seqirus.com within 24 
hours of becoming aware. Once the eCRF is available, the SAE should be documented on 
the AE eCRF as soon as possible.
Any medication or other therapeutic measures used to treat the AE will be recorded on the 
appropriate eCRF(s) in addition to the outcome of the AE.


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After receipt of the initial report, representatives of the Sponsor or its designee will contact 
the investigator if it is necessary to obtain further information for an assessment of the event.
All SAEs must be reported by the investigator to his/her corresponding EC/IRB or applicable 
regulatory authorities in accordance with institutional policy/regulatory requirements and 
adequate documentation of this notification must be provided to the Sponsor.
Sponsor or its designee must also comply with the applicable regulatory requirement(s) 
related to the reporting of suspected unexpected serious adverse vaccine reactions (SUSARs) 
to the regulatory authority(ies) and the IRB/EC. If a SUSAR or other safety signal relating to 
the use of one of the study vaccines is reported to the Sponsor or its designee, the Sponsor 
will communicate the information to the investigator and the investigator will be responsible 
for submitting this information to the EC/IRB and other relevant authorities.
7.1.5.1
Post-Study Events
Any SAE that occurs outside of the protocol-specified observation period or after the end of 
the study but is considered to be caused by the study vaccine will be processed by the 
Sponsor PVRM (or delegate) and must be reported using the email address: 
AE.reporting@Seqirus.com.
These SAEs will be considered as part of the spontaneous reporting towards the 
investigational study vaccine in order to ensure the safety of all subjects.
7.1.6 Pregnancies
If a subject becomes pregnant after vaccination, confirmation of pregnancy should be 
recorded in the eCRF. To ensure subjects’ safety, each pregnancy in a subject after study 
vaccination must be reported to the Sponsor’s PVRM (or delegate) within 72 hours of the site 
learning of its occurrence. The study site must complete the paper Pregnancy 
Reporting/Outcome Form and send it to AE.reporting@Seqirus.com. If the subject agrees to 
submit this information, the pregnancy must be followed to determine the outcome, including 
spontaneous or voluntary termination, details of the birth, and the presence or absence of any 
birth defects, congenital abnormalities, or maternal and/or newborn complications. This 
follow-up should occur even if the intended duration of safety follow-up for the study has 
ended.


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Any pregnancy outcome meeting the definition of a SAE (see Section 7.1.4, Serious Adverse 
Events) must also be reported on the AE eCRF.
7.1.7 Safety Laboratory Measurements
No scheduled safety laboratory measurements are planned for this study.
7.2
Efficacy Assessment
There is no assessment of efficacy in this study. 
7.3
Immunogenicity Assessment
The measures of immunogenicity used in this study are standard, i.e., widely used and 
generally recognized as reliable, accurate, and relevant (able to describe the quality and 
extent of the immune response).
The immunogenicity analysis will evaluate the immunogenicity of the study vaccine, which 
will be measured by the HI and MN assay by titrating antibodies against the homologous 
influenza strains. 
For the primary immunogenicity objectives, HI antibody responses using a cell-derived target 
virus will be evaluated for all strains. In case of a lack of agglutination for a specific strain 
using HI assay, immunogenicity for that strain will be assessed as measured by the MN assay. 
For the secondary immunogenicity objectives, HI and MN antibody responses using cell-
derived target virus will be evaluated for all strains. In case of a lack of agglutination for a 
specific strain using HI assay, immunogenicity for that strain will be assessed as measured by 
the MN assay only.
For the exploratory immunogenicity evaluation, HI antibody responses may be evaluated for 
all strains using egg-derived and cell-derived target viruses. For the exploratory objective 
related to immunogenicity against heterologous strains MN assays and cell-derived target 
viruses will be used. 
Testing will be conducted by the Sponsor or designated laboratory in a blinded manner 
towards the treatment arm.


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8
STATISTICAL CONSIDERATIONS
A complete description of the statistical analyses and methods will be available in the 
Statistical Analysis Plan (SAP), which will be drafted before the first subject is enrolled and 
finalized before the database is locked for the primary analysis. The primary analysis will be 
conducted on the immunogenicity and safety data collected up to Day 29. 
8.1
Endpoints
8.1.1 Primary Endpoint(s)
The primary objective of the study is to assess the immunogenicity of aQIVc formulations in 
comparison with QIVc vaccine, as measured by HI assay using cell-derived target viruses, 
and to assess the reactogenicity of different aQIVc formulations in comparison to QIVc for 7 
days after vaccination and the safety of all vaccines for 28 days after vaccination.    
8.1.1.1
Primary Immunogenicity Endpoints
Humoral immune response of the aQIVc formulations in comparison with the QIVc vaccine 
in terms of HI antibody response against homologous vaccine strains:

Geometric Mean Titer (GMT): Geometric mean of HI antibodies at Day 1 and Day
29.

Geometric Mean Fold Increase (GMFI): The Geometric mean of the fold increase in 
serum HI titer post-vaccination (Day 29) compared to pre-vaccination (Day 1).

Percentages of subjects with HI titers ≥ 1:40 at Day 1 and Day 29.

Percentage of subjects with seroconversion (defined as a ≥4-fold increase in titer post-
vaccination in those with pre-vaccination titer above the LLOQ (1:10), or a post-
vaccination titer ≥1:40 for subjects with baseline titer below the LLOQ (1:10) for HI 
antibodies at Day 29).
Note that the HI assay could be replaced by the MN assay due to a lack of agglutination.
Derived variables at Day 29:

The GMT ratio (aQIVc formulation/QIVc) for each strain.

The inter-group difference in the SCRs (aQIVc formulation minus QIVc) for each 
strain.


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
The inter-group difference in the percentage of subjects with HI titer ≥1:40 (aQIVc 
formulation minus QIVc) for each strain.
8.1.1.2
Primary Safety Endpoints

Solicited local or systemic AEs for 7 days following vaccination (Day 1 through 
Day 7), based on the subject reported data (eDiary).

Severe solicited local or systemic AEs for 7 days following vaccination (Day 1 
through Day 7), based on the subject reported data (eDiary).

Unsolicited AEs for 28 days following vaccination (Day 1 to Day 29).

SAEs, MAAE, AEs leading to withdrawal from the study, AESIs for 28 days after 
vaccination (Day 1 to Day 29).
8.1.2
Secondary Endpoints
Secondary endpoints evaluated in the study include a safety assessment of all vaccines for 180 
days after vaccination and the immunogenicity endpoints measured by the HI assay assessed 
at Day 181 and the immunogenicity endpoints measured by the MN assay both at Day 29 and 
Day 181. Also, the solicited systemic events and injection site pain (both any and severe) will 
be assessed using the investigator evaluation (see section 7.1.1 Solicited Adverse Events).
8.1.2.1
Secondary Immunogenicity Endpoints
Humoral immune response of the aQIVc formulations in comparison with the QIVc vaccine in 
terms of HI antibody response against homologous vaccine strains:

GMT: Geometric mean of HI antibodies at Day 181.

GMFI: The Geometric mean of the fold increase in serum HI titer post-vaccination 
(Day 181) compared to pre-vaccination (Day 1).

Percentages of subjects with HI titers ≥1:40 on Day 181.
Derived variables:

The GMT ratio (aQIVc formulation/QIVc) for each strain.

The inter-group difference in the percentage of subjects with HI titer ≥1:40 (aQIVc 
formulation minus QIVc) for each strain.


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In addition, the humoral immune response of the aQIVc formulations in comparison with the 
QIVc vaccine in terms of MN antibody response against homologous vaccine strains:

GMT: Geometric mean of MN antibodies at Days 1, 29 and 181.

GMFI: The Geometric mean of the fold increase in serum MN titer post-vaccination 
(Day 29 and Day 181) compared to pre-vaccination (Day 1).

Percentages of subjects with seroconversion (defined as ≥4-fold increase for subjects 
with pre-vaccination MN titers ≥ Lower Limit of Quantitation (LLOQ) or as ≥ 
4LLOQ for subjects with pre-vaccination MN titer <LLOQ) at Day 29.
Derived variables:

The GMT ratio (aQIVc formulation/QIVc) for each strain

The inter-group difference in the SCRs (aQIVc formulation minus QIVc) for each 
strain.
8.1.2.2
Secondary Safety Endpoints

SAEs, MAAEs, AEs leading to withdrawal or AESIs as collected during the study 
(Day 1 to End of Study). 

Solicited adverse events as evaluated by the investigator.
8.1.3 Exploratory Endpoints
Exploratory endpoints that may be assessed in the study include the immunogenicity 
endpoints as measured on Day 29 by HI or MN assay using cell-derived target viruses or cell-
derived heterologous target viruses. Details will be described in the Statistical Analysis Plan. 
The results of these exploratory analyses may be presented in an addendum to the Clinical 
Study Report (CSR).
8.2
Success Criteria
This study is a Phase 2 study to explore the immunogenicity of different formulations of 
aQIVc versus QIVc. Although no success criteria are pre-specified, 95% confidence interval 
bounds will be used for statistical comparisons. 


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8.3
Analysis Sets
8.3.1 All Enrolled Set
All screened subjects who provide informed consent, receive a subject ID and provide 
demographic and/or baseline screening assessments, regardless of the subject’s treatment 
status in the study. 
8.3.2 All Exposed Set
All subjects in the All Enrolled Set who are randomized and received study vaccination.
8.3.3 Safety Set
Subjects will be analyzed as “treated” (i.e., according to the vaccine a subject received, rather 
than the vaccine to which the subject may have been randomized).
Solicited Safety Set
All subjects in the Exposed Set with any solicited adverse event data including body 
temperature measurements or use of analgesics/antipyretics.  
Unsolicited Safety Set (unsolicited adverse events)
All subjects in the Exposed Set for whom safety has been assessed after vaccination
(including 30-minute post-vaccination assessment).
Overall Safety Set
All subjects who are in the Solicited Safety Set and/or in the Unsolicited Safety Set.
8.3.4 Full Analysis Set (FAS) Immunogenicity 
Full Analysis Set Immunogenicity
All subjects in the All Enrolled Set who are randomized, received study vaccination and 
provided immunogenicity data on Day 1 and Day 29 assessment. 
In case of vaccination error, subjects in the FAS sets will be analyzed “as-treated” (i.e., 
according to the vaccine the subject actually received).


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8.3.5 Per Protocol Set (PPS) Immunogenicity
All subjects in the FAS Immunogenicity who:

Correctly receive the vaccine (i.e., receive the vaccine to which the subject is randomized 
within the defined window).

Have no protocol deviations leading to exclusion (see Section 8.3.8, Protocol Deviations) 
as defined prior to unblinding / analysis.

Are not excluded due to other reasons defined prior to unblinding or analysis, including 
subjects who withdrew informed consent prior to Day 28.  
8.3.6 Other Analysis Sets
Other applicable analysis sets will be described in the SAP.
8.3.7 Subgroups
Primary and secondary analyses of immunogenicity and safety endpoints will be done for the 
total population and by age subgroup (50 – 64 years and ≥ 65 years). 
Additional subgroup analysis will be conducted for previous vaccination history, sex and 
comorbidity risk score (below 50 and above 50). Refer to the SAP for further details. 
8.3.8 Protocol Deviations
A protocol deviation is any change, divergence, or departure from the study design or 
procedures of a study protocol. A protocol deviation may be a reason to remove data from an 
analysis set at the time of analysis. CSR-reportable protocol deviations will be defined as 
exclusionary from the analysis according to protocol objectives and endpoints, which will be 
specified in the statistical analysis plan. In some cases, exclusion of data may be due to a 
reason other than a protocol deviation, e.g. early termination.


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8.4
Statistical Analysis Plan
8.4.1 Analysis of Demographic and Baseline Characteristics
Descriptive statistics (mean, standard deviation, median, minimum and maximum) for age, 
height, weight, BMI and comorbidity risk score at enrolment will be calculated overall and 
by vaccine group.
Distributions of subjects by sex, race and ethnic origin, prior vaccination status and country
will be summarized overall and by vaccine group.
Demographic and baseline characteristics will also be presented by the age subgroup.
8.4.2 Analysis of Primary Objectives
8.4.2.1
Analysis of Primary Safety Objectives
The primary objective is to assess the safety using the solicited local and systemic adverse 
events (AEs) for 7 days following vaccination (Day 1 through Day 7), reported severe 
solicited local or systemic AEs for 7 days following vaccination (Day 1 through Day 7), 
reported unsolicited adverse events for 28 days following vaccination ((Day 1 to Day 29), 
and reported SAEs, MAAEs and AESIs for 28 days after vaccination (Day 1 to Day 29).
8.4.2.1.1
Statistical Hypothesis
No statistical hypothesis testing will be performed for the safety objectives. 
8.4.2.1.2
Analysis Sets
All safety objectives will be evaluated based on the Solicited Safety Set and Unsolicited 
Safety Set. 
8.4.2.1.3
Statistical Methods
The statistical analyses of all safety endpoints will be descriptive in nature. Percentage of 
subjects will be presented by group for the overall age group as well as by age subgroup.  


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A logistic regression analysis will be applied with the binary response (presence of solicited 
adverse reaction or no reaction as reported by the subject), to explore the relation with 
antigen and MF59 levels as factors as well as with age and previous vaccination history. This 
model will be applied for both reported solicited local or systemic adverse events (AEs) for 7 
days following vaccination and reported severe solicited local or systemic AEs for 7 days 
following vaccination (Day 1 through Day 7).
8.4.2.2
Analysis of Primary Immunogenicity Objectives
The primary objective is to assess the immunogenicity assessed using the HI or MN assay at
Day 29.
8.4.2.2.1
Statistical Hypothesis
No formal statistical hypothesis testing will be performed for the immunogenicity objectives. 
8.4.2.2.2
Analysis Sets
All immunogenicity objectives will be evaluated based on the PPS Immunogenicity. In the
case that there is >5% difference in the total number of subjects between the PPS and the 
FAS Immunogenicity, additional analysis based on the FAS Immunogenicity will be 
conducted. 
8.4.2.2.3
Statistical Methods
All statistical analyses for HI (and MN) titers will be performed on the logarithmically (base 
10) transformed values. Individual HI titers below the detection limit (<10) will be set to half 
of that limit (5); values above the upper limit of quantification will be set to the upper limit.
Reverse cumulative distribution curves of HI and MN titers will be presented graphically by 
time-point and strain. 
Unadjusted estimates for GMTs, GMFIs and pertaining 2-sided 95% CIs will be calculated 
assuming a log-normal distribution of the titers and will be completed by providing 
minimum, maximum and median titers for each vaccine group. Binary data (i.e., percentages 
of subjects with seroconversion and with titer ≥ 1:40) will be summarized for each group and 


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will be reported together with 2-sided 95% CIs calculated according to Clopper Pearson’s 
method. 
For immunogenicity data, it may be reasonable to consider missing immunogenicity values 
as missing completely at random (MCAR), i.e., not informative. Therefore, the 
immunogenicity analysis will comprise a complete case analysis only, without introducing 
any bias. 
The analysis model for the HI GMT will be done using a general linear model on log-
transformed (base 10) Day 29 titers as the outcome variable, with antigen levels and MF59 
levels as factors, and age subgroup, previous vaccination history and log10 pre-vaccination 
titer as covariates. From this model, adjusted differences in the least square means (on the log 
scale) between the adjuvanted vaccine arms and the non-adjuvanted arm will be produced
with 95% confidence limits. The estimated difference and the confidence limits will be back-
transformed to obtain an adjusted GMT ratio (each aQIVc formulation versus QIVc) with 
95% confidence limits. Each of the four strains will be analyzed separately. In addition, 
unadjusted GMT ratios for each aQIVc formulation versus QIVc will be presented.
Interaction effects between the antigen and MF59 factors will be explored. Other models may 
be investigated. In addition, adjusted GMT ratios will be calculated using formulation as 
categorical factor in this general linear model. 
A similar model as described above will be used for the GMFI on Day 29 excluding the pre-
vaccination titer as covariate.
The binary endpoints (i.e. seroconversion or titer ≥1:40) at Day 29 from each aQIVc 
formulation versus QIVc will be compared using the Miettinen and Nurminen method
without adjustment for the age stratum. Each of the four strains will be analyzed separately. 
Additional exploratory analysis will be done including antigen and MF59 levels and 
adjustment for age and previous vaccination history using a generalized linear model for 
binary data.


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8.4.3 Analysis of Secondary Objectives
8.4.3.1
Analysis of Secondary Safety Objectives
8.4.3.1.1
Analysis of Extent of Exposure
The number of subjects vaccinated will be presented by vaccine group.
8.4.3.1.2
Analysis of Solicited Local, Systemic and Other Adverse Events
Solicited adverse events are reported daily from Day 1 up to and including Day 7 post-
vaccination directly by the subject and as evaluated by the investigator. A solicited adverse 
event will be defined as “present” or at least mild on any of the recorded days to be counted. 
This will be summarized as total and for local, systemic and the other category separately. 
The same tables will be created at each timepoint.  
Frequencies and percentages of subjects experiencing each adverse event will be presented 
overall and for each maximum symptom severity and by vaccine group. Post-vaccination 
solicited adverse events reported from Day 1 through Day 7 will also be summarized for the 
interval’s Day 1-3 and Day 4-7 overall and for each maximal severity and by vaccine group.
The severity of solicited local adverse events will be summarized according to the grading. 
For the categories based on linear measurement for local AEs erythema and induration: 25-50 
mm is mild, 51-100 mm is moderate and >100 mm is severe. Solicited local adverse events 
measuring less than 25 mm will not be considered as an adverse event. For temperature: any 
fever is ≥ 38°C, mild is ≥38°C-38.4°C, moderate is ≥38.5°C-38.9°C and severe is ≥39°C. 
The use of antipyretics and analgesics will be summarized by type of use (prophylactic 
versus treatment) as the number and percentage of subjects reporting use. 
Body temperature will be summarized by 0.5°C increments from 36.0°C up to ≥40°C. 
8.4.3.1.3
Analysis of Unsolicited Adverse Events
This analysis applies to all AEs occurring during the study, judged either as probably related, 
possibly related, or not related to vaccination by the investigator, recorded in the AE eCRF, 
with a start date on or after the date of vaccination. The original verbatim terms used by 


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investigators to identify adverse events in the eCRFs will be mapped to preferred terms using 
the MedDRA dictionary. 
The adverse events will then be grouped by MedDRA preferred terms into frequency tables 
according to system organ class. All reported AEs, as well as AEs judged by the investigator 
as at least possibly related to study vaccine, will be summarized according to system organ 
class and the preferred term within system organ class. These summaries will be presented by 
the vaccination group and by the interval of study observation (Day 1 to Day 29, Day 30 to 
end of study). When an AE occurs more than once for a subject, the maximal severity and 
strongest relationship to the vaccine group will be counted.
Separate summaries will be produced for the following categories

AEs

SAEs

AEs that are possibly or probably related to the vaccine

SAEs that are possibly or probably related to the vaccine

AESIs

MAAEs

AEs leading to withdrawal
Data listings of all adverse events will be provided by subject. 
8.4.3.1.4
Statistical Hypotheses
No statistical hypotheses will be tested.
8.4.3.1.5
Analysis Sets
The Solicited Safety Set for the solicited AEs and the Unsolicited Safety Set for all 
unsolicited AEs.
8.4.3.1.6
Statistical Methods
Only descriptive statistics will be calculated and presented.


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8.4.3.2
Analysis of Secondary Immunogenicity Objective(s)
The analysis of the secondary immunogenicity endpoints will be conducted in the same way 
as described for the primary immunogenicity endpoints.
8.4.3.2.1
Statistical Hypotheses
No statistical hypotheses will be tested.
8.4.3.2.2
Analysis Sets
All secondary immunogenicity objectives will be evaluated based on the PPS 
Immunogenicity. Note that the PPS for the Day 181 analysis may exclude additional subjects 
in case of major protocol deviations related to the use of concomitant medication interfering 
with the immunogenicity assessment. In the case that there is >5% difference in the total 
number of subjects between the PPS and the FAS Immunogenicity, additional analysis based 
on the FAS Immunogenicity will be conducted. 
8.4.3.2.3
Statistical Methods
The statistical methods will follow the same methods as described for the primary endpoints 
in Section 8.4.2.2.3, Statistical Methods.
8.4.4 Analysis of Other Objectives
The exploratory analysis may be done based on the immunogenicity endpoints by the HI 
assay using the egg-derived target viruses. Results from these analyses will be reported 
separately.
8.4.4.1
Analysis of Other Safety Objectives
No exploratory safety analysis was defined.
8.4.4.2
Analysis of Other Immunogenicity Objectives
The exploratory endpoints will be evaluated using the same methods as described above in
Section 8.4.2.2.3, Statistical Methods.


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8.5
Sample Size and Power Considerations of Primary Objectives
The total sample size of 800 subjects for this exploratory Phase 2 study with different 
formulations has been based on feasibility and to provide information for further 
development. The power calculations are provided to illustrate what differences can be 
detected.
Anticipating 5% of the subjects will be non-evaluable over the course of the study (due to 
being lost to follow-up, having insufficient samples, incomplete laboratory results, or 
protocol violations, etc.), approximately 95 evaluable older adult subjects for each vaccine 
group will be included in the statistical analysis. Based on data from Seqirus study V130_01
(Otten et al. 2020), it is assumed that the within group standard deviations for the log10 HI 
titers for all strains are about 0.7 or lower. 
Assuming a standard deviation (SD) of 0.7 for the log 10 HI titer at Day 29 , a comparison 
between dose groups with n=95 per group has at least 80% power with a two-sided α=0.05 if 
the GMT ratio between the two groups is at least 1.93, and at least 90% power if the ratio is 
at least 2.14.
The table below describes the statistical power to detect that the GMT ratio of aQIVc 
formulation versus QIVc is significantly higher than 1 or higher than 1.5 at a two-sided 5% 
level of significance assuming 95 evaluable subjects per formulation and a standard deviation 
of 0.7 for the log10 titers.
Power Calculations Comparing the aQIVc formulations with QIVc is given below in Table 
8-1.


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Table 8-1: Power Calculations
Power % to detect
Actual GMT ratio
Lower limit of GMT ratio above 1
Lower limit of GMT 
ratio above 1.5
2.00
83.86
23.08
2.50
97.37
58.44
3.00
99.67
83.86
3.50
99.96
95.00
4.00
100
98.65 
N=95 per group and two-sided alpha = 0.05
8.6
Interim Analysis
No interim analysis of data from this study is planned.
Two final analyses will be performed stepwise as follows:
1. A final analysis including all immunogenicity and safety data collected from Visit 1 to Visit 
3 (Day 29) and the primary and applicable secondary objectives will be conducted on cleaned 
and locked data. This early read-out is done to inform phase 3 clinical development. No 
individual listings and unblinded data will be generated at this stage. Access to information 
about treatment allocation will be limited by a biostatistician in charge of statistical analysis.
Other study team members will be either group-unblinded or remain blinded until the full 
study database is locked.
2. The analysis of immunogenicity and safety data collected between Visit 3 and Visit 5 will 
be performed after all data is available. The results of this analysis will cover the entire study 
duration. All results will be presented in the clinical study report and will include individual 
data listings and unblinded information.
9
SOURCE DOCUMENTATION, STUDY MONITORING, AND 
AUDITING
In order to ensure consistency across sites, monitoring and auditing will be standardized and 
performed in accordance with the Sponsor’s or delegated contract research organization’s 
(CRO) standard operating procedures and applicable regulatory requirements (e.g., FDA, 
EMA, and ICH guidelines).


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Prior to the enrolment of the first study subject, the Sponsor or delegate will train 
investigators and/or their study staff on the study protocol, all applicable study procedures, 
documentation practices, and all electronic systems. eCRFs supplied by the Sponsor must be 
completed for each enrolled subject (see Section 8.3.1, All Enrolled Set for the definition of 
the enrolled subject). Documentation of screened but not enrolled subjects must be 
maintained at the site and made available for review by the site monitor. Data and documents 
will be checked by the Sponsor and/or monitor.
9.1
Source Documentation
Prior to the start of the study, the site staff participating in the study conduct will be 
instructed on what documents will be required for review as source documents. The kinds of 
documents that will serve as source documents will be agreed between the Sponsor or 
delegate and investigator and designees and specified in the Source Data Agreement (SDA)
prior to subject enrolment.
In addition, source documentation must include all of the following: subject identification 
(on each page), eligibility and participation, proper informed consent procedures, dates of 
visits, adherence to protocol procedures, adequate reporting and follow-up of adverse events, 
documentation of prior/concomitant medication/vaccines, study vaccine 
receipt/dispensing/return records, study vaccine administration information, any data 
collected by a telephone conversation with the subject and date of completion and reason.
The subject must also allow access to the subject’s medical records. Each subject must be 
informed of this prior to the start of the study and consent for access to medical records may 
be required in accordance with local regulations.
All safety data reported by subjects must be written down in source documents prior to entry 
of the data into eCRFs. If there are multiple sources of information (e.g., Subject eDiary, 
verbal report of the subject, telephone contact details, medical chart) supporting the diagnosis 
of an adverse event, these sources must be identified in the source documents, discrepancies 
between sources clarified, the ultimate diagnosis must be justified and written in the source 
documents, and this diagnosis must be captured in the Adverse Event eCRF (AE eCRF). 


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The Subject eDiary source data is hosted by a vendor engaged for this study, on behalf of the 
study investigators. Each investigator will be provided with a certified archive copy of all 
diary data relating to subjects at that site and must confirm it is readable. 
9.2
Study Monitoring, Auditing and Source Data Verification
Prior to the enrolment of the first study subject, the Sponsor or its designee (e.g., a CRO) will 
develop a Clinical Monitoring Plan to specify how centralized and/or on-site monitoring, 
including clinical specimens reconciliation, will be performed for the study. Study progress 
will be monitored by the Sponsor or its designee as frequently as necessary to ensure:

that the rights and well-being of human subjects are protected,

the reported study data are accurate, complete, and verifiable from the source documents 
and

the conduct of the study is in compliance with the current approved 
protocol/amendment(s), GCP and applicable regulatory requirements.
Contact details for the Sponsor’s team or its designee involved in study monitoring will be 
provided to the investigator. Study data recorded on eCRFs will be verified by checking the 
eCRF entries against source documents in order to ensure data completeness and accuracy as 
required by study protocol except for those parameters which are specifically described in
Section 7, Assessments being entered directly into the EDC system.
Data verification may also be performed through a centralized review of data (e.g., checking 
for outliers or other anomalies). Additional documents such as the investigator site file, 
pharmacy records, and informed consent documentation must also be available for review if 
requested. Arrangements for monitoring visits will be made in advance in accordance with 
the monitoring plan, except in case of an emergency.
The investigator and/or site staff must make source documents of subjects enrolled in this 
study available for inspection by the Sponsor or its representative at the time of each 
monitoring visit and Sponsor’s audits, when applicable. These documents must also be 
available for inspection, verification, and copying, as required by regulations, by officials of 
the regulatory health authorities (e.g., FDA, EMA, and others) and/or ECs/IRBs. The 
investigator and study site staff must comply with applicable privacy, data protection and 


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medical confidentiality laws for use and disclosure of information related to the study and 
enrolled subjects.
10 DATA MANAGEMENT
10.1
Data Entry and Management
In this study, all clinical data (including, but not limited to, AE/SAEs, concomitant 
medications, medical history, and physical assessments) and blood sampling will be entered 
onto eCRFs in a timely fashion by the investigator and/or the investigator’s dedicated site 
staff. Data entered onto eCRFs are stored on a secure website. The data collected on this 
secure website are assimilated into an electronic data capture (EDC) system, which is 
compliant with Title 21 Part 11 policies of the Code of Federal Regulations (FDA 1997). The 
data system includes password protection and internal quality checks. The EDC system will 
be designed and validated by the Sponsor prior to activation for data entry by sites. The 
investigator or designated delegate must review data entered and electronically sign the 
eCRFs to verify their accuracy.
Access to the EDC system for data entry or review will require training and distinct 
individual access code assignments to those site staff members who will be entering study 
data and those involved in study oversight who may review study data. Data are collected 
within the EDC system, to which the Sponsor and site monitors have exclusively “read-only” 
access.
10.2
Data Clarification
As part of the conduct of the trial, the Sponsor may have questions about the data entered by 
the site, referred to as queries. The monitors and the Sponsor or its designated CRO are the 
only parties that can generate a query. All corrections and clarifications will be entered into 
the EDC system and will be identified by the person entering the information, the reason for 
the change, as well as the time of the changes made. If changes are made to a previously and 
electronically signed eCRF, the investigator must confirm and endorse the changes.


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10.3
Data Protection
The sponsor respects the subjects’ rights to privacy and will ensure the confidentiality of their 
medical information in accordance with all applicable laws and regulations.
The Sponsor as Data Controller according to the General Data Protection Regulation 
(“GDPR”) on the protection of individuals with regard to the processing of personal data and 
on the free movement of such data confirms herewith compliance to GDPR in all stages of 
Data Management.
11 RECORD RETENTION
Investigators must retain all study records required by the Sponsor and by the applicable 
regulations in a secure and safe facility. The investigator must consult the Sponsor’s 
representative before disposal of any study records and must notify the Sponsor of any 
change in the location, disposition, or custody of the study files. 
The sponsor-specific essential documents should be retained until at least 2-years after the 
last approval of a marketing application in an ICH region and until there are no pending or 
contemplated marketing applications in an ICH region or at least 2-years have elapsed since 
the formal discontinuation of clinical development of the investigational product. These 
documents should be retained for a longer period however if required by the applicable 
regulatory requirement(s) or if needed by the sponsor (ICH E6 (R2)). 
“Essential documents” are defined as documents that individually and collectively permit 
evaluation of the conduct of a study and the quality of the data produced. These documents 
should be retained for a longer period, however, if required by the applicable national 
regulatory or institutional requirements (ICH E6 (R2)).
The sponsor should inform the investigator(s)/institution(s) in writing of the need for record 
retention and should notify the investigator(s)/institution(s) in writing when the trial-related 
records are no longer needed (ICH E6 (R2)).
The principles of record retention will also be applied to the storage of laboratory samples, 
provided that the integrity of the stored sample permits testing. These laboratory samples will 
be securely stored for future testing at a global Sponsor’s or Sponsor’s controlled/contracted 
facility for up to 15 years, for purposes to conduct additional analyses needed related to the 


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study, or ultimately for future analysis to further understand the immune response to the 
vaccine or to influenza disease. After this period, the samples will be destroyed. Only 
laboratory staff performing the testing will have access to these samples. By signing the ICF, 
the subject agrees that samples will be retained for use limited to additional analyses related 
to this study. If the subject also agrees to have the subject’s samples stored for future testing -
not related to this study- after the study is completed, this can be indicated on the ICF.
12 USE OF INFORMATION AND PUBLICATION
The Sponsor assures that the key design elements of this protocol will be posted in a publicly 
accessible database such as clinicaltrials.gov, and in compliance with current regulations.
The Sponsor also assures that key results of this clinical study will be posted in a publicly 
accessible database within the required time-frame from the end of the study as defined in 
Section 3.10, End of Study.
In accordance with standard editorial, ethical practices and current guidelines of Good 
Publication Practice (Graf 2009), the Sponsor will generally support publication of 
multicenter studies only in their entirety and not as individual center data. In this case, a 
coordinating investigator will be designated by mutual agreement prior to the start of the 
study. The coordinating investigator will also sign the clinical study report on behalf of the 
principal investigators (CPMP/EWP/2747/00). Authorship will be determined by mutual 
agreement and in line with the International Committee of Medical Journal Editors' 
authorship requirements. Any formal publication of the study in which the contribution of the 
Sponsor’s personnel exceeded that of conventional monitoring will be considered as a joint 
publication by the investigator and the appropriate Sponsor’s personnel.
The Sponsor must be notified of any intent to publish data collected from the study and prior 
approval from the Sponsor must be obtained prior to submission for publication. 
13 ETHICAL CONSIDERATIONS
13.1
Regulatory and Ethical Compliance
The study will be conducted in compliance with the protocol, GCP and applicable regulatory 
requirement(s).


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This clinical study was designed and shall be implemented and reported in accordance with 
the ICH Harmonized Tripartite Guidelines for Good Clinical Practice (ICH E6(R2), with 
applicable local regulations including European Directive 2001/20/EC; US Code of Federal 
Regulations Title 21, Sponsor’s codes on protection of human rights, and with the ethical 
principles laid down in the Declaration of Helsinki (European Council 2001; US Code of 
Federal Regulations; ICH 2016).
13.2
Informed Consent Procedures
Eligible subjects may only be included in the study after providing written informed consent, 
as described in Section 5.1.1, Informed Consent. Before the start of the study, the investigator 
will have informed consent and any other materials that will be provided to the subjects 
reviewed and approved by the IRB/EC. This review and approval will be documented and 
stored with other study documents. The investigator or designee must fully inform the subject 
of all pertinent aspects of the study. A copy of the written informed consent will be given to 
the subject. The subject must be allowed ample time to ask about the details of the study and 
to make a decision as to whether or not to participate in the study. The subject must sign the 
consent form indicating their agreement to participate in the study before any study-related 
procedures are conducted. The informed consent process may be conducted up to 10 days 
prior to vaccination on Day 1. If the subject is unable to read and write, a witness must be 
present during the informed consent discussion and at the time of informed consent signature.
Prior to the start of the study, the Sponsor will provide investigators a proposed informed 
consent form that complies with the ICH GCP guideline and regulatory requirements and is 
considered appropriate for this study. Any changes to the proposed consent form suggested 
by the investigator must be agreed to by the Sponsor before submission to the IRB/EC and a 
copy of the approved version must be provided to the Sponsor monitor after IRB/EC 
approval. 
Women of childbearing potential should be informed that taking the study medication may 
involve unknown risks to the fetus if the pregnancy were to occur during the study and agree 
that in order to participate in the study they must adhere to the contraception requirements 
indicated in the protocol for the duration of the study. If the case of doubts on the ability of a 
subject to adhering to these requirements, that subject should not be allowed in the study.


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13.3
Responsibilities of the Investigator and IRB/IEC
The protocol and the proposed informed consent form must be reviewed and approved by a 
properly constituted IRB/EC before the study start. Properly constituted IRB/EC is defined in 
the integrated addendum to ICH E6: ICH Guideline for Good Clinical Practice E6 (R2). A 
signed and dated statement that the protocol and informed consent have been approved by the 
IRB/EC must be given to the Sponsor before study initiation. Prior to study start and at any 
time the protocol is amended during study conduct, the investigator is required to sign a 
protocol signature page confirming his/her agreement to conduct the study in accordance 
with these documents and all of the instructions and procedures found in this protocol and to 
give access to all relevant data and records to the Sponsor’s monitors, auditors, Sponsor’s 
Clinical Quality Assurance representatives, designated agents of the Sponsor, IRBs/ECs, and 
regulatory authorities as required. If an inspection of the clinical site is requested by a 
regulatory authority, the investigator must inform the Sponsor immediately that this request 
has been made.
The investigator also responsible for the following:

Maintaining a list of appropriately qualified persons to whom the investigator has delegated 
significant study-related duties.

Demonstrating the capability of recruiting the required number of suitable subjects within 
the recruitment period.

Demonstrating sufficient time and staffing to properly conduct and complete the study 
within the agreed study period.

Ensuring that all persons assisting with the study are adequately informed about the 
protocol, the investigational product(s), and their study-related duties and functions

Ensuring that appropriately qualified healthcare professionals are responsible for all study-
related medical decisions and for ensuring appropriate medical care of subjects 
experiencing any adverse event related to the study.

If permission to do so is given by the subject, ensuring that the subject’s primary healthcare 
provider is informed of the subject’s participation in the study.


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The investigator should not implement any deviation from, or changes of the protocol 
without agreement by the Sponsor and prior review and documented approval/favorable 
opinion from the IRB/IEC of an amendment, except where necessary to eliminate an 
immediate hazard(s) to study subjects, or when the change(s) involves only logistical or 
administrative aspects of the study (e.g., change in monitor(s), change of telephone 
number(s). In addition, the investigator, or person designated by the investigator, should 
document and explain any deviation from the approved protocol.
The investigator may implement a deviation from, or a change of, the protocol to eliminate 
an immediate hazard(s) to study subjects without prior IRB/IEC approval/favorable opinion. 
As soon as possible, the implemented deviation or change, the reasons for it, and, if 
appropriate, the proposed protocol amendment(s) should be submitted:
(a) to the IRB/IEC for review and approval/favorable opinion,
(b) to the Sponsor for agreement and, if required,
(c) to the regulatory authority(ies).
13.4
Protocol Amendments
An amendment is a written description of change(s) to or formal clarification of a study 
protocol which may impact on the conduct of the clinical study, potential benefit of the 
clinical study, or may affect subject safety, including changes of study objectives, study 
design, subject population, sample sizes, study procedures, or significant administrative 
aspects. An administrative change of a study protocol is a minor correction or clarification 
that has no significant impact on the way the clinical study is to be conducted and no effect 
on subject safety (e.g., change of telephone number(s), logistical changes). Protocol 
amendments must be approved by the Sponsor, health authorities where required, and the 
IRB/EC. In cases when the amendment is required in order to protect the subject's safety 
urgently, the amendment can be implemented prior to IRB/EC approval. Notwithstanding, the 
need for formal approval of a protocol amendment, the investigator is expected to take any 
immediate action required for the safety of any subject included in this study, even if this 
action represents a deviation from the protocol. In such cases, the Sponsor should be notified 
of this action, the IRB/EC at the study site, and, if required by local regulations, the relevant 
health authority) should be informed within 10 working days.


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14 REFERENCE LIST
Calabro S, Totroli M, Baudner BC, Paccito A et al. Vaccine adjuvants alum and MF59 induce 
rapid recruitment of neutrophils and monocytes that participate in antigen transport to 
draining lymph nodes. Vaccine. 2011;29(9):1812-1823.
Centers for Disease Control and Prevention, 2019a. Seasonal Influenza (Flu), Types of 
Influenza Vaccines. November 18, 2019 [Accessed December 23, 2019] Available from 
https://www.cdc.gov/flu/about/viruses/types.htm.
Centers for Disease Control and Prevention, 2019b. Past seasons estimated influenza disease 
burden. November 22, 2019 [Assessed December 23, 2019]. Available from: 
https://www.cdc.gov/flu/about/burden/2017-2018.htm.
Code of Federal Regulations, 1997: Food and Drug Administration, U.S. Department of 
Health and Human Services: Title 21, Part 11: Electronic Records Electronic Signatures. 
Federal Register 62:13464.
Committee for Medicinal Products for Human Use. Note for guidance on coordinating 
investigator signature of clinical study reports (CPMP/EWP/2747/00).
European Parliament, 2001. Directive 2001/20/EC of the European Parliament and of the 
Council of 4 April 2001. Official Journal of the European Communities. L 121/34-44.
Frey SE, Reyes MR, Reynales H, Bermal NN, et al. Comparison of the safety and 
immunogenicity of an MF59®-adjuvanted with a non-adjuvanted seasonal influenza vaccine 
in elderly subjects. Vaccine. 2014;32(39):5027-5034.
Graf C, Battisti WP, Bridges D. Good publication practice for communicating company 
Sponsored medical research: the GPP2 guidelines. BMJ. 2009;339:b4330.
Hak E, Wei F, Nordin J, Mullooly J et al. Development and validation of a clinical prediction 
rule for hospitalization due to pneumonia or influenza or death during influenza epidemics 
among community-dwelling elderly persons. J Infect Dis. 2004;189:450-458.
Hedge N. Cell culture-based influenza vaccines: A necessary and indispensable investment 
for the future. Hum Vacc Immunother. 2015;11(5):1223-1234
ICH E6 (R2), 2016. ICH Harmonized Tripartite ICH Guideline for Good Clinical Practices 
E6 (R2), EMA/CHMP/ICH/135/1995.


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Iuliano A, Roguski K, Chang HH, Muscatello D et al. Estimates of global seasonal influenza-
associated respiratory mortality: A modeling study. Lancet. 2018;391(10127):1285-1300.
Lambert LC, Fauci AS. Influenza vaccines for the future. N Engl J Med. 2010;363:2036-
2044.
McNeil, M. Vaccine-associated anaphylaxis. Curr Treat Options Allergy. 2019;6:297–308
O'Hagan DT, Ott GS, De Gregorio E, Seubert A. The mechanism of action of MF59 - an 
innately attractive adjuvant formulation. Vaccine. 2012;30(29):4341-8. Review Erratum in 
Vaccine. 2013;31(14):1877.
Otten G, Matassa V, Ciarlet M, Leav B. A phase 1, randomized, observer blind, antigen and 
adjuvant dosage finding clinical trial to evaluate the safety and immunogenicity of an 
adjuvanted, trivalent subunit influenza vaccine in adults ≥65 years of age. Vaccine. 
2020;38:578-587.
Reber AJ, Chirkova T, Kim JH, Cao W et al. Immunosenescence and challenges of 
vaccination against influenza in the aging population. Aging and Disease. 2012;3:68-90.
Samuel R and Miller J. Is the influenza vaccine effective in decreasing infection, 
hospitalization, pneumonia, and mortality in healthy adults? OSMAJ. 2019;112(3):86–87.
Sasaki S and Sullivan M. Limited efficacy of inactivated influenza vaccine in elderly 
individual is associated with decreased production of vaccine-specific antibodies. J Clin 
Invest. 2011;121(8):3109-3119.
Temte J and Prunuske J. Seasonal influenza in primary care settings: Review for primary care 
physicians. WMJ. 2010;109(4):193-200.
World Health Organization, 2018. Influenza (seasonal) Factsheet, November 2018 [accessed 
6 December 2018]; Available from: https://www.who.int/news-room/fact-
sheets/detail/influenza-(seasonal)
Wong SS and Webby RJ. Traditional and new influenza vaccines. Clin Microbiol Rev.
2013;26:476-492.
Zhou H, Thompson WW, Viboud CG, Ringholz CM, et al. Hospitalizations associated with 
influenza and respiratory syncytial virus in the United States, 1993-2008. Clin Infect Dis. 
2012;54(10):1427-1436.


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APPENDIX 1 – COMORBIDITY RISK SCORE
Prediction Rule for Estimating the Probability of Hospitalization Due to Pneumonia or 
Influenza and Death Due to Any Cause (Hak et al. 2004)
Characteristic
Scorea
Age, years
<70
0
70-74
14
75-79
28
80-89
42
≥90
56
Sex
Female 
0
Male 
9
Outpatient visits during the previous year
0
0
1-6
11
7-12
22
>13
33
Previous hospitalization due to pneumonia or influenza
No
0
Yes
63
Comorbidityb
Pulmonary disease
18
Heart disease
6
Renal disease or renal transplant
12
Dementia or stroke
22
Non-hematological and hematological cancer
48
Subject total score
Notes:
a The prognostic score for a given subject can be obtained by adding the scores for each applicable 
characteristic.
b Pre-existing medical conditions of eligible subjects will be scored following a judgment by the investigator. 


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APPENDIX 2 – LIST OF ADVERSE EVENTS OF SPECIAL INTEREST
Gastrointestinal disorders
•
Celiac disease
•
Crohn’s disease
•
Ulcerative colitis
•
Ulcerative proctitis
Liver disorders
•
Autoimmune cholangitis
•
Autoimmune hepatitis
•
Primary biliary cirrhosis
•
Primary sclerosing cholangitis
Metabolic diseases
•
Addison’s disease
•
Autoimmune thyroiditis (including Hashimoto thyroiditis)
•
Diabetes mellitus type I
•
Grave's or Basedow’s disease
Musculoskeletal disorders
•
Antisynthetase syndrome
•
Dermatomyositis
•
Juvenile chronic arthritis (including Still’s disease)
•
Mixed connective tissue disorder
•
Polymyalgia rheumatic
•
Polymyositis
•
Psoriatic arthropathy
•
Relapsing polychondritis
•
Rheumatoid arthritis
•
Scleroderma, including diffuse systemic form and CREST syndrome
•
Spondyloarthritis, including ankylosing spondylitis, reactive arthritis (Reiter's Syndrome) 
and undifferentiated spondyloarthritis
•
Systemic lupus erythematosus
•
Systemic sclerosis
Neuroinflammatory disorders
•
Acute disseminated encephalomyelitis, including site-specific variants (e.g., non-
infectious encephalitis, encephalomyelitis, myelitis, radiculomyelitis)
•
Cranial nerve disorders, including paralyses/paresis (e.g., Bell’s palsy)
•
Guillain-Barré syndrome, including Miller Fisher syndrome and other variants


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•
Immune-mediated peripheral neuropathies and plexopathies, including chronic 
inflammatory demyelinating polyneuropathy, multifocal motor neuropathy and 
polyneuropathies associated with monoclonal gammopathy
•
Multiple sclerosis
•
Narcolepsy
•
Optic neuritis
•
Transverse myelitis
•
Myasthenia gravis, including Eaton-Lambert syndrome
Skin disorders
•
Alopecia areata
•
Autoimmune bullous skin diseases, including pemphigus, pemphigoid and dermatitis 
herpetiformis
•
Cutaneous lupus erythematosus
•
Erythema nodosum
•
Morphoea
•
Lichen planus
•
Psoriasis
•
Sweet’s syndrome
•
Vitiligo
Vasculitides
•
Large vessels vasculitis including: giant cell arteritis such as Takayasu's arteritis and 
temporal arteritis
•
Medium sized and/or small vessels vasculitis including: polyarteritis nodosa, Kawasaki's 
disease, microscopic polyangiitis, Wegener's granulomatosis, Churg–Strauss syndrome 
(allergic granulomatous angiitis), Buerger’s disease thromboangiitis obliterans, 
necrotizing vasculitis and anti-neutrophil cytoplasmic antibody (ANCA) positive 
vasculitis (type unspecified), Henoch- Schonlein purpura, Behcet's syndrome, 
leukocytoclastic vasculitis
Others
•
Antiphospholipid syndrome
•
Autoimmune hemolytic anemia
•
Autoimmune glomerulonephritis (including IgA nephropathy, glomerulonephritis rapidly 
progressive, membranous glomerulonephritis, membranoproliferative glomerulonephritis, 
and mesangioproliferative glomerulonephritis)
•
Autoimmune myocarditis/cardiomyopathy
•
Autoimmune thrombocytopenia
•
Goodpasture syndrome
•
Idiopathic pulmonary fibrosis
•
Pernicious anemia


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•
Raynaud’s phenomenon
•
Sarcoidosis
•
Sjögren’s syndrome
•
Stevens-Johnson syndrome
•
Uveitis


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APPENDIX 3 – SPONSOR AND INVESTIGATOR SIGNATURE PAGES


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SIGNATURE OF INVESTIGATOR
I have read the protocol entitled “A Phase 2, Randomized, Observer-Blind, antigen and 
Adjuvant dose-ranging Clinical Study to Evaluate Safety and Immunogenicity of Different 
Formulations of MF59-Adjuvanted Quadrivalent Subunit Inactivated Cell-derived Influenza 
Vaccine (aQIVc) in Older Adults ≥50 years of age”, and I agree to conduct the clinical study, 
after approval by an Institutional Review Board or Independent Ethics Committee (as 
appropriate), in accordance with the protocol, the principles of the Declaration of Helsinki, 
the standards of Good Clinical Practice (as defined by the International Conference on 
Harmonisation) and applicable regulatory requirements.
Changes to the protocol will only be implemented after written approval is received from the 
sponsor and the Institutional Review Board or Independent Ethics Committee (as 
appropriate), with the exception of medical emergencies.
I will ensure that the study staff fully understand and follow the protocol.
Signature: 
……………………...……………………… Date:
…………………………
(DD MMM YYYY)
Name, qualifications and affiliation:          ……………………………………………………
Address of Investigator:
…………………………………………………………………


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APPENDIX 4 – GENERAL INFORMATION
Name
Title
Address (including telephone 
number and email address)
Sponsor’s Responsible Representatives
Person(s) Authorized to sign the protocol and the protocol amendment(s)
 
 
Paasheuvelweg 28
1105 BJ Amsterdam
The Netherlands
Sponsor’s Medical Expert
Paasheuvelweg 28
1105 BJ Amsterdam
The Netherlands
/ 
External Service Provider Responsible for Monitoring Activities
IQVIA
Full service provider
201 Broadway, 5th Floor
Cambridge, MA 02139
+1 919-998-2000/ +1 617- 621-1600


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Investigator(s)
-
Name(s) and title(s) of investigator(s) who is/are responsible for conduct of the trial 
and the address(es) and telephone number(s) of the trial site(s) are provided in a 
separate document.
-
Qualified physician who is responsible for a trial-site medical decisions (if other 
than investigator) provided in a separate document.
Central Clinical Laboratories involved in the trial.
VisMederi
Str. del Petriccio e Belriguardo 35
53100 Siena SI
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SAP_001.pdf: 
 
Statistical Analysis Plan, Protocol Number V201_01 
26 Augustus 2021 Final Version 2.0  
 
 
 
 
Document #: FORM2-000092701, Ed: 3.0  
Source Document #: SOP-00092497 
 
 
 
 
 
 
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STATISTICAL ANALYSIS PLAN 
 
 
 
Study Title: 
A Phase 2, Randomized, Observer-Blind, Antigen and 
Adjuvant Dose-Ranging Clinical Study to Evaluate Safety 
and Immunogenicity of Different Formulations of MF59-
Adjuvanted Quadrivalent Subunit Inactivated Cell-derived 
Influenza Vaccine (aQIVc) in Older Adults ≥50 Years of 
Age 
 
Study Number: 
 
 
V201_01 
 
Protocol Version and Date:  
2, 22 JAN 2021 
 
Phase of Development: 
 
Phase 2 
 
Plan Prepared by: 
 
 
, 
 
 
 
 
 
 
 
 
Version and Date: 
 
 
Version 2.0: 26 AUG 2021 
 
 
 
 
 
 
Approvers: 
 
 
 
, 
 
 
 
 
 
 
, 
 
, 
 
 
 
 
Property of Seqirus, Confidential 
May not be used, divulged, published or otherwise disclosed without written consent of 
Seqirus 
 
 


 
Statistical Analysis Plan, Protocol Number V201_01 
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TABLE OF CONTENTS 
 
LIST OF ABBREVIATIONS .......................................................................................................... 6 
1. 
Background and Rationale ....................................................................................................... 7 
2. 
Objectives ................................................................................................................................. 8 
2.1 
Primary Objective .............................................................................................................. 8 
2.1.1 
Primary Immunogenicity Objective ........................................................................... 8 
2.1.2 
Primary Safety Objective ........................................................................................... 8 
2.2 
Secondary Objectives ........................................................................................................ 8 
2.2.1 
Secondary Immunogenicity Objective(s) ................................................................... 8 
2.2.2 
Secondary Safety Objectives ...................................................................................... 8 
2.3 
Exploratory Objectives ...................................................................................................... 9 
2.3.1 
Exploratory Immunogenicity Objectives ................................................................... 9 
3. 
Study design ........................................................................................................................... 10 
3.1 
Overview of Study Design .............................................................................................. 10 
4. 
Randomization and Blinding .................................................................................................. 16 
4.1 
Method of Group Assignment and Randomization ......................................................... 16 
4.1.1 
Definition of Randomization/Vaccination Errors .................................................... 16 
4.1.2 
Forced Randomization ............................................................................................. 17 
4.2 
Blinding and Unblinding ................................................................................................. 17 
5. 
Sample Size and Power Considerations ................................................................................. 18 
6. 
Determination of Protocol Deviations .................................................................................... 19 
6.1 
Definition of Protocol Deviations .................................................................................... 19 
6.2 
Determination of Protocol Deviations ............................................................................. 19 
7. 
Analysis Set ............................................................................................................................ 20 
7.1 
All Enrolled Set ............................................................................................................... 20 
7.2 
Exposed Set ..................................................................................................................... 20 
7.3 
Full Analysis Set (FAS) Immunogenicity Set ................................................................. 20 
7.4 
Per Protocol Set (PPS) Immunogenicity ......................................................................... 20 
7.5 
Safety Set ......................................................................................................................... 21 
7.5.1 
Restricted Safety Set ................................................................................................ 21 


 
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7.6 
Other Analysis Set ........................................................................................................... 21 
8. 
General Issues for Statistical Analyses .................................................................................. 23 
8.1 
Adjustment for Covariates ............................................................................................... 23 
8.2 
Handling of Dropouts, Missing Data ............................................................................... 23 
8.3 
Multicenter Studies .......................................................................................................... 24 
8.4 
Multiple Comparisons and Multiplicity .......................................................................... 24 
8.5 
Immunogenicity/Safety/Other Subsets ............................................................................ 24 
8.6 
Subgroups ........................................................................................................................ 24 
8.7 
Data Transformation ........................................................................................................ 25 
8.8 
Derived and Computed Variables .................................................................................... 25 
8.9 
Treatment groups ............................................................................................................. 27 
8.10 
Analysis Software ........................................................................................................ 28 
9. 
Study Subjects ........................................................................................................................ 29 
9.1 
Disposition of Subjects and Withdrawals ........................................................................ 29 
10. 
Demographics and Other Baseline Characteristics ............................................................. 29 
10.1 
Demographics .............................................................................................................. 29 
10.2 
Medical History ........................................................................................................... 30 
11. 
Immunogenicity Analysis ................................................................................................... 30 
11.1 
Blood samples .............................................................................................................. 30 
11.2 
Primary Objectives Analysis ........................................................................................ 30 
11.3 
Secondary Objectives Analysis .................................................................................... 32 
11.4 
Exploratory Objectives Analysis ................................................................................. 33 
12. 
Efficacy Analysis ................................................................................................................ 33 
13. 
Safety Analysis .................................................................................................................... 33 
13.1 
Analysis of Extent of Exposure ................................................................................... 33 
13.1.1 Safety Completeness ................................................................................................ 34 
13.2 
Solicited Local and Systemic Adverse Events ............................................................. 34 
13.3 
Investigator assessment of Solicited Adverse Events .................................................. 38 
13.4 
Unsolicited Adverse Events ......................................................................................... 38 
13.5 
Combined Solicited and Unsolicited Adverse Events ................................................. 40 
13.6 
Clinical Safety Laboratory Investigations ................................................................... 40 


 
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13.7 
Concomitant Medication .............................................................................................. 40 
14. 
Interim Analysis .................................................................................................................. 40 
15. 
Data Monitoring Committees .............................................................................................. 41 
16. 
Changes to planned analysis ............................................................................................... 41 
17. 
List of Final Report Tables, Listings and Figures ............................................................... 42 
18. 
References ........................................................................................................................... 42 
 
 


 
Statistical Analysis Plan, Protocol Number V201_01 
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Approvals 
 
                                                           
 
 
, 
 
 
 
 
 
 
 
 
, 
 
 
 
 
 
 
, 
 
 
 
 
 
 
 
 
 
 


 
Statistical Analysis Plan, Protocol Number V201_01 
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LIST OF ABBREVIATIONS 
 
AE 
 
 
Adverse Event 
AESI  
 
Adverse Events of Special Interest 
ANCOVA 
 
Analysis of Covariance 
ANOVA 
 
Analysis of Variance 
CSR 
 
 
Clinical Study Report 
DMC  
 
Data Monitoring Committee 
FAS 
 
 
Full Analysis Set 
GMR  
 
Geometric Mean Ratios 
GMT  
 
Geometric Mean Titers 
HI 
 
 
Hemagglutination Inhibition 
ICH 
 
 
International Council for Harmonisation 
IRT 
 
 
Interactive Response Technology 
ITT 
 
 
Intention-to-Treat 
MedDRA 
 
Medical Dictionary for Regulatory Activities 
MITT  
 
Modified Intention-to-Treat 
PD 
 
 
Protocol Deviation 
PPS 
 
 
Per Protocol Set 
SAE 
 
 
Serious Adverse Event  
SAP 
 
 
Statistical Analysis Plan  
SP 
 
 
Statistical Programmer 
SUSAR 
 
Suspected Unexpected Serious Adverse Reactions 
TFL 
 
 
Tables, Figures and Listings 
TOC  
 
Table of Content 
 


 
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1. BACKGROUND AND RATIONALE 
 
Vaccination is the primary method for preventing influenza and its potentially severe 
complications. Efficacy of the conventional influenza vaccines in the adult population is 
demonstrated to be high. In contrast, the efficacy in elderly individuals is significantly lower due 
to the aging of the immune system (i.e. immunosenescence) and underlying medical conditions 
that can both increase the risk of influenza complications as well as interfere with immune 
responses (Sasaki and Sullivan 2011). In view of the limitations of conventional influenza vaccines 
in older adults, there continues to be an unmet need for a new generation of influenza vaccines that 
provides more consistent and broader coverage against all seasonal virus subtypes and variants 
(Wong and Webby 2013; Reber et al. 2012). The investigational vaccines in this study are seven 
different formulations of the adjuvanted quadrivalent cell-based (aQIVc) vaccine, including 
different amount of antigen and different dose of the adjuvant MF-59. The primary aim of this 
study is to assess immunogenicity and safety of the seven different formulations of the aQIVc 
vaccine versus the non-adjuvanted quadrivalent cell-based influenza (QIVc) vaccine. The results 
are to be used to select the optimal formulation to study in Phase 3 study. 
 
For further details please refer to Section 1.0 of the protocol. 
 
This plan describes all details related to the statistical analysis of the data collected in the study 
V201_01 and is based on protocol version 2, 22 Jan 2021.  
This analysis plan is compliant with ICH Harmonized Tripartite Guideline, 5 February 1998, 
Statistical Principles for Clinical Trials, E9; World Health Organization, WHO Technical Report, 
Series No. 924. 2004, Annex 1: Guidelines on Clinical Evaluation of Vaccines: Regulatory 
Expectations; and FDA Center for Biologics Evaluation and Research (CBER) Guidance for 
Industry, May 2007, Clinical Data Needed to Support the Licensure of Seasonal Inactivated 
Influenza Vaccines. 
 
 


 
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2. OBJECTIVES 
 
2.1 
Primary Objective  
2.1.1 Primary Immunogenicity Objective 
1. To assess the immunogenicity of the different aQIVc formulations in comparison with the 
non-adjuvanted QIVc vaccine as measured by hemagglutination inhibition (HI) assay* at 
28 days after vaccination. 
* In case of lack of agglutination for a specific strain using HI assay, immunogenicity for that strain will be assessed 
as measured by microneutralization (MN) assay as an acceptable alternative. 
2.1.2 Primary Safety Objective 
1. To assess the reactogenicity of all aQIVc formulations and the comparator, QIVc, for 7 
days after vaccination. 
2. To assess the safety of all aQIVc formulations and the comparator, QIVc, for 28 days after 
vaccination. 
 
2.2 
Secondary Objectives  
2.2.1 Secondary Immunogenicity Objective(s) 
1. To assess the immunogenicity of the different aQIVc formulations in comparison with the 
QIVc vaccine as measured by HI assay* at 180 days after vaccination. 
2. To assess the immunogenicity of the different aQIVc formulations in comparison with the 
QIVc vaccine as measured by MN assay at 28 and 180 days after vaccination. 
* In case of lack of agglutination for a specific strain, immunogenicity for that strain will be assessed as measured by 
MN assay as an acceptable alternative. 
2.2.2 Secondary Safety Objectives 
1. To assess the safety of all aQIVc formulations and the comparator, QIVc for 180 days 
after vaccination. 
 


 
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2.3 
Exploratory Objectives 
2.3.1 Exploratory Immunogenicity Objectives 
1. 
To assess the immunogenicity of the different aQIVc formulations in comparison with the 
QIVc vaccine as measured by MN assay using heterologous target viruses at 28 days after 
vaccination in a subset of collected samples. 
2. 
Additional exploratory immunogenicity analyses may be conducted to further characterize 
the immune response of the study vaccines. 
 
The results of these exploratory analyses may be presented in an addendum to the Clinical Study 
Report (CSR). 
 
 


 
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3. STUDY DESIGN  
3.1 
Overview of Study Design 
Experimental design: This is a Phase 2, randomized, stratified, observer-blind, antigen and adjuvant 
dose-ranging clinical study in approximately 800 male and female adults aged 50 years and older 
(approximately equally split between two age groups; ≥50-64 and ≥65 years of age) who are 
healthy or have co-morbidities which increase their risk of complications from influenza infection.  
Duration of the study: The study duration is approximately 6 months for each subject. The study 
will be conducted in one single influenza season. 
 
Vaccination schedule: Single vaccination (Day 1). 
Investigational Vaccine: The study will use 7 investigational formulations of aQIVc (see Table 1) 
Comparator Vaccine: The study will use 1 comparator vaccine (see Table 1) 
Treatment groups: All treatment groups will include approximately 100 subjects aged ≥50 years of 
age (see Table 1). All subjects will receive one dose of study vaccine at Day 1. The aQIVc 
formulation numbers correspond to the design space as presented in Figure 3-1. Not all 
formulations within the design space will be directly evaluated in this study, but the study results 
will make it possible to model the effect of the non-tested formulations within the design space. 
 
 
 
 
 
 


 
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Table 1: Overview of study vaccines 
Group 
Vaccine 
Type of vaccine 
HA antigen 
content/ strain 
(µg) 
MF59 
squalene 
content (mg) 
Total 
injected 
volume (mL) 
Number of 
randomized 
subjects 
A 
aQIVc 1 
Investigational 
 
 
 
100 
B 
aQIVc 3 
Investigational 
 
 
 
100 
C 
aQIVc 6 
Investigational 
 
 
 
100 
D 
aQIVc 7 
Investigational 
 
 
 
100 
E 
aQIVc 9  
Investigational 
 
 
 
100 
F 
aQIVc 10 
Investigational 
 
 
 
100 
G 
aQIVc 11 
Investigational 
 
 
 
100 
H 
QIVc 
Comparator 
15 
0 
0.5 
100 
The treatments for Groups B, C, D, E, F and G will be obtained by ‘bed-side’ mixing of QIVc, standard dose of 
aQIVc (formulation aQIVc 1) and MF59 solution. Mixing is not required for Groups A and H. 
Figure 3-1: Design Space 
 
Randomization: An Interactive Response Technology (IRT) will be used in the study with 
stratification factors for age (≥50-64 and ≥65 years of age) and history of any influenza 


 
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vaccination within 3 previous years (yes/no) to randomly assign each subject to one of the 8 
treatments. 
Blinding: Observer-blind study. 
Blood sample schedule: A blood sample will be collected from all subjects on Day 1 (prior to 
vaccination), Day 29 and Day 181.  
Data collection: electronic Case Reporting Form (eCRF) and electronic Diary (eDiary). 
Study periods: The study has a treatment period (Day 1 to Day 29) and a follow-up period (Day 
30 to Day 181). 
Study clinic visits: Three clinic visits for each subject: at Day 1, Day 29 and Day 181.  
Safety phone call: Two safety phone calls (Day 7 and Day 91) will be conducted: on Day 7 to 
review the eDiary as well as to collect any unsolicited AEs, and their related medications and any 
vaccinations, and on Day 91 to collect only SAEs, medically attended adverse events (MAAEs), 
AEs leading to withdrawal, and AESIs, and their related medications and any vaccinations. 
Reactogenicity data collection: Solicited AEs, occurring at the day of study vaccination and the 
following 6 days (Day 1 through Day 7, or longer if the events are not resolved), will be recorded 
daily using an eDiary as completed by the subject. The qualified healthcare professional will 
separately evaluate the solicited systemic events and injection site pain during the Day 7 safety 
phone call. 
Safety data collection: All unsolicited AEs will be collected for 28 days after vaccination. During 
the follow-up period (from 29 days after vaccination onwards), only unsolicited AEs will be 
collected that are SAEs, MAAEs, AEs leading to study withdrawal, and/or AESIs. These data 
will be captured by interviewing the subject during the clinic visits and safety phone calls and by 
review of available medical records. Subjects will be instructed to call the site in the event of any 
AE which they perceive as being of concern during the entire study period. 
Serological assays:  
• HI assay for homologous vaccine strains, using cell-derived target virus, will be 
performed in all subjects on serum samples collected on Days 1, 29 and 181. 


 
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• MN assay for homologous vaccine strains, using cell-derived target virus, will be 
performed in all subjects on serum samples collected on Days 1, 29 and 181. 
• HI assay for heterologous vaccine strains, using cell-derived target virus, will be 
performed in a subset of collected samples from serum samples collected on Days 1 and 
29. 
For further details please refer to Section 3.0 of the protocol. 
 
 
 


 
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Table 2 Time and Event Schedule 
 
 
Visit Type 
Clinic Visit 
Safety Phone 
Call 
Clinic Visit* 
Safety Phone 
Call 
Clinic Visit* 
Study Day 
1 
7 
29 
91 
181 
Visit Window (Days) 
n/a 
+3 day 
-7/+3 days 
+/-7 days 
+/-14 days 
 
Visit Number 
1 
2 
3 
4 
5 
Study Event 
References 
 
 
 
 
 
Screening and Randomization 
 
 
 
 
 
 
Informed Consenta   
Section 5.1.1 
X 
 
 
 
 
Medical Historyb 
Section 5.1.2 
X 
 
 
 
 
Physical Exam 
Section 5.1.2  
X 
 
 
 
 
Targeted Physical Examc 
Section 5.4.1 
 
 
X 
 
X 
Measuring body temperature 
Section 5.1.2 
X 
 
 
 
 
Pregnancy testd 
Section 5.1.2  
X 
 
 
 
 
Exclusion/Inclusion Criteria  
Section 4  
X 
 
 
 
 
Randomization 
Section 5.1.4  
X 
 
 
 
 
Study Treatment 
Vaccination  
Section 5.2 
X 
 
 
 
 
Safety 
30 Minutes Post Injection 
Assessment 
Section 5.3  
X 
 
 
 
 
Train and Dispense Subject eDiary  
Section 5.3  
X 
 
 
 
 
Review of eDiary data and 
compliance 
Section 3.6.2 
Ongoing during eDiary use 
 
 
Assess unsolicited AEs 
Section 7.1.2 
X 
X 
X 
 
 
Assess SAEs 
Section 7.1.4 
X 
X 
X 
X 
X 


 
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Visit Type 
Clinic Visit 
Safety Phone 
Call 
Clinic Visit* 
Safety Phone 
Call 
Clinic Visit* 
Study Day 
1 
7 
29 
91 
181 
Visit Window (Days) 
n/a 
+3 day 
-7/+3 days 
+/-7 days 
+/-14 days 
 
Visit Number 
1 
2 
3 
4 
5 
Study Event 
References 
 
 
 
 
 
Assess for AEs leading to 
withdrawal, medically attended 
AEs and AESIs 
Section 7.1.4.1 
X 
X 
X 
X 
X 
Assess relevant medications and 
vaccinations 
Section 6.5  
X 
X 
X 
X 
X 
Immunogenicity 
 
 
 
 
 
 
Serology blood draw 
Section 5.1.5 
Xe 
 
X 
 
X 
Study End 
Study Completion/termination 
Section 5.6 
 
 
 
 
Xf 
* In the exceptional situation in which a clinic visit is not possible, such as in the case when a site is not able to see study subjects at the clinic due to local regulations 
related to the coronavirus pandemic, a home visit may be considered.  
Notes: a Consent form(s) should be signed prior to performing any procedures. The informed consent process may be conducted earlier, but within 10 days prior to Day 1. b 
Medical history includes existing comorbidities. c Based on specific complaints as indicated by the subject. dA pregnancy test should be done for females of childbearing 
potential in order to rule out any pregnancy. e Blood draw to be done prior to vaccination. f Subjects who terminate the study early will be requested to complete all safety-
related Study Completion procedures. Abbreviations: AE = adverse event; AESI = adverse event of special interest; n/a = not applicable; SAE = serious adverse event. 
 
 


 
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4. RANDOMIZATION AND BLINDING 
4.1 
Method of Group Assignment and Randomization 
 
Enrolled subjects will be randomized to one of the eight treatment groups in equal ratios 
(Groups A-H, see Table 1). Subjects will be stratified based on age (≥50 to 64 years and ≥65 years) 
and History of any influenza vaccination within 3 previous influenza seasons (yes/no) and site.   
The subject will be randomized in the IRT/ Randomization and Trial Supply Management (RTSM) 
system. The subject will receive a unique Subject ID that will be used for all eCRFs and associated 
study documentation for the duration of the study. After randomization, the Screening Number 
ceases to be used and remains in the Screening and Enrolment Log only. The list of randomization 
assignments is produced by the IRT service provider and approved by the Sponsor according to the 
applicable Sponsor’s Standard Operating Procedure (SOP). The randomization list is generated 
using permutated blocks assigned by site and strata. 
4.1.1 Definition of Randomization/Vaccination Errors 
 
The list below provides categories for errors that may occur during vaccination. 
Randomization errors: 
• Administered wrong kit (subject was vaccinated with a vaccine different from the one 
assigned at randomization). 
 
Vaccination errors: 
 The list below provides some examples of potential errors that may occur during vaccination: 
• Administered only part of the study vaccine. 
• Administered an incorrect dose Incorrect vaccine location. 
• Administered expired vaccine. 
• Administered temperature deviated vaccine. 
 
Stratification error: 
• Subject randomized in the wrong stratification stratum. 


 
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Randomization and vaccination errors are considered as major (CSR-reportable) protocol 
deviations. Stratification errors will not be considered as CSR-reportable PDs as there will be no 
impact on the actual dose administered. Subjects will be included in analysis according to actual 
age subgroup and previous vaccination history. 
 
4.1.2 Forced Randomization 
 
Not applicable. 
 
4.2 
Blinding and Unblinding 
 
The study is designed as an observer-blind study. Unblinded teams will be used for preparation of 
the safety reports for the Independent Safety Team (IST) and Data Monitoring Committee (DMC). 
If a subject is unblinded during the study, it is to be documented as a CSR-reportable PD, except 
for subjects unblinded by Pharmacovigilance due to suspected unexpected serious adverse 
reactions (SUSAR). The unblinding will be documented appropriately. The unblinded subject(s) 
are excluded from the PPS. Unblinded subjects will be included in the FAS and safety sets.  
A final analysis on the primary and the secondary objectives including all immunogenicity and 
safety data collected up to and including the Day 29 Visit will be conducted on cleaned and locked 
data. Access to information about study groups will be limited to biostatisticians and programmers 
in charge of statistical analysis. No individual listings and unblinding data will be generated at this 
stage until the completion of the study. 
 
 
 


 
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5. SAMPLE SIZE AND POWER CONSIDERATIONS 
 
This study is an exploratory Phase 2 study without any formal hypothesis testing. The total sample 
size of 800 subjects has been determined based on feasibility and to provide information for further 
development. The power calculations are provided to illustrate what differences can be detected. 
 
Anticipating 5% of the subjects will be non-evaluable over the course of the study (due to being 
lost to follow-up, having insufficient samples, incomplete laboratory results, or protocol violations, 
etc.), approximately 95 evaluable older adult subjects for each vaccine group will be included in 
the statistical analysis. Based on data from Seqirus study V133_01EXP (Otten et al. 2020), it is 
assumed that the within group standard deviations for the log10 HI titers for all strains are about 
0.7 or lower.  
Assuming a standard deviation (SD) of 0.7 for the log 10 HI titer at Day 29, a comparison between 
dose groups with n=95 per group has at least 80% power with a two-sided α=0.05 if the GMT ratio 
between the two groups is at least 1.93, and at least 90% power if the ratio is at least 2.14.  
 
Table 3 below describes the statistical power to detect that the GMT ratio of an aQIVc formulation 
versus QIVc is significantly higher than 1 or higher than 1.5 at a two-sided 5% level of significance 
assuming 95 evaluable subjects per formulation and a standard deviation of 0.7 for the log10 titers. 
Table 3 Power Calculations 
 
Power % to detect 
Actual GMT ratio 
Lower limit of the 95% CI of GMT 
ratio above 1 
Lower limit of the 95% CI of 
GMT ratio above 1.5 
2.00 
83.86 
23.08 
2.50 
97.37 
58.44 
3.00 
99.67 
83.86 
3.50 
99.96 
95.00 
4.00 
100 
98.65  
N=95 per group and two-sided alpha = 0.05 
 
 
 
 


 
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6. DETERMINATION OF PROTOCOL DEVIATIONS 
6.1 
Definition of Protocol Deviations 
 
CSR reportable PDs are defined in accordance with International Conference on Harmonization 
(ICH) E3 as important PDs related to study inclusion or exclusion criteria, conduct of the trial, 
subject management or subject assessment resulting in the potential to jeopardize the safety or 
rights of the trial subjects or the scientific value of the trial. Protocol deviations will be classified 
as CSR-reportable and non-CSR-reportable.  
Seqirus’s standard Protocol Deviation Specification Document lists all the pre-specified observable 
and programmable PDs, including their classification, categories, sub categories and impact on the 
analysis. 
CSR reportable PDs may lead to exclusion of the subject or part of the subject’s data from at least 
the PP analysis set. 
The number of subjects in any and by PD category will be summarized by study treatment and 
overall. Individual subject listings will be provided sorted by subject and by PD category. 
Prior to unblinding, all reportable PDs will be evaluated, and designated study staff will develop a 
memo that describes the PDs that led to exclusions from analysis sets. This memo will be signed 
off by at least the Lead Biostatistician, Lead Data manager,  Global Clinical Operations Lead and 
the Clinical Scientist  and will be included in the trial master file.  
 
6.2 
Determination of Protocol Deviations 
 
The source/method of identification can be either observable or programmable. Programmable PDs 
are those which can be programmed from the data recorded in the clinical database. Observable 
PDs are identified by CRAs during monitoring or other team members. 
A set of listings will be programmed following the Protocol Deviation Specification List to 
determine and categorize the protocol deviations. These listings will be provided for review on an 
ongoing basis during the study. 
This review will also include protocol deviations reported in monitoring reports. After the review, 
the Clinical Scientist and the Global Clinical Operation Lead will provide the Biostatistician with: 
• An assessment of CSR reportable PDs based on blinded clinical data review. 
 
 
 


 
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7. ANALYSIS SET 
 
7.1 
All Enrolled Set 
 
All screened subjects who provide informed consent, receive a subject ID, and provide 
demographic and/or baseline screening assessments, and are randomized  in the study.  
Demography and baseline characteristics tables as well as subject listings will be produced on the 
All Enrolled Set. 
 
7.2 
Exposed Set 
 
All subjects in the All Enrolled Set who are randomized and received study vaccination. 
 
7.3 
Full Analysis Set (FAS) Immunogenicity Set 
 
All subjects in the  Exposed Set who are randomized, received study vaccination and provided 
immunogenicity data on Day 1 and Day 29 assessment.  
In case of vaccination error, subjects in the FAS sets will be analyzed “as-treated” (i.e., according 
to the vaccine the subject received). 
If a subject is unblinded before the completion of the Day 29 visit, he/she will be included in the 
FAS, but excluded from the Per Protocol.Set. 
 
7.4 
Per Protocol Set (PPS) Immunogenicity 
 
All subjects in the FAS Immunogenicity who: 
• Correctly receive the vaccine (i.e., receive the vaccine to which the subject is randomized 
within the defined window).  
• Have immunogenicity assessments within the window of - -7 to +21 days around the Day 29 
visit, and Day 1 sample taken  
• Have no protocol deviations leading to exclusion as defined prior to unblinding / analysis. 
• Are not excluded due to other reasons defined prior to unblinding or analysis.   


 
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If a subject receives a vaccine, labelled for another subject but the same as the one the subject was 
randomized to, the subject will not be removed from the PPS. If a subject is unblinded during the 
study (except for SUSAR), he/she will be excluded from the PPS. 
 
7.5 
Safety Set 
 
Subjects will be analyzed as “treated” (i.e., according to the vaccine a subject received, rather 
than the vaccine to which the subject may have been randomized). 
Solicited Safety Set 
All subjects in the Exposed Set with any solicited adverse event data including body temperature 
measurements or use of analgesics/antipyretics recorded by the subject in the eDiary.   
The investigator assessment of the solicited adverse event will be based on the subjects with non-
missing data recorded on the investigator assessment. In case, there are subjects without any eDiary 
data recorded (so not part of the solicited safety set) but with an investigator assessment an extra 
set will be defined for the investigator evaluation – Solicited Safety Set, per investigator.  
 
Unsolicited Safety Set (unsolicited adverse events) 
All subjects in the Exposed Set for whom safety has been assessed after vaccination (including 
30-minute post-vaccination assessment). 
Overall Safety Set 
All subjects who are in the Solicited Safety Set and/or in the Unsolicited Safety Set. 
 
7.5.1 Restricted Safety Set 
 
Not applicable. 
7.6 
Other Analysis Set 
 
 
For the analysis of the immunogenicity data collected at Day 181 assessment, revised sets of the 
FAS and PPS, Day 181 immunogenicity will be used. For the FAS, Day 181, subjects will be 


 
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only included if there were Day 1 and Day 181 immunogenicity data collected. The PPS, Day 
181 will be a subset of the FAS, Day 181 with a sample visit within +/- 4 weeks around the target 
Day 181 and excluding protocol deviations related to the subjects and no disallowed concomitant 
medication impacting the results was used. 
 
 


 
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8. GENERAL ISSUES FOR STATISTICAL ANALYSES 
8.1 
Adjustment for Covariates 
 
The log-transformed antibody titers at Day 29 and Day 181 will be analyzed using an Analysis of 
Covariance (ANCOVA) model which includes the log-transformed pre-vaccination antibody titer, 
age subgroup stratification (≥50 – 64, ≥65 years) and previous vaccination history stratification (no 
in previous 3 years, yes). Summary tables will show both adjusted and unadjusted GMTs for each 
vaccine group and adjusted and unadjusted GMT ratio for the different aQIVc investigational 
vaccines versus QIVc vaccine. 
Instead of Vaccine group  as categorical variable, levels of antigen and the amount of MF-59 will 
be included as covariates to model the antibody titers at Day 29 as function of the level of antigen 
and the amount of MF-59. 
The main analysis of binary immunogenicity endpoints ((i.e., percentages of subjects with 
seroconversion and with titer ≥ 1:40) will not be adjusted for any of the covariates. Binary data will 
be summarized for each group using unadjusted estimates and will be reported together with two-
sided exact 95% CIs. Additional adjusted analysis may be done including the stratification factors 
age and previous vaccination history as covariates in a generalized linear model. 
 
8.2 
Handling of Dropouts, Missing Data 
 
The distribution of subjects with reasons for missing immunogenicity values will be described by 
vaccine group. Key baseline characteristics, such as age and previous vaccination history, will be 
compared between the subjects with immunogenicity values and those who have missing data. For 
immunogenicity data, it may be reasonable to consider missing immunogenicity values as missing 
completely at random (MCAR), i.e., not informative. 
Therefore, the immunogenicity analysis will comprise a complete case analysis only, without 
introducing any bias.  
Solicited adverse events are collected using electronic diaries from Day 1 to Day 7 post-
vaccination. If no data have been recorded for all 7 days, the assessment is considered missing 
and excluded from the safety analysis. In case, at least one day is filled in but other days are 
missing the presence or absence of the event will be based on the available data. If more than 5% 
of the subjects have incomplete diary data, additional tables for solicited adverse events will be 
created based on complete diary card data. The number of days and number of subjects with 
missing data will be tabulated by treatment group. 


 
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8.3 
Multicenter Studies 
 
Stratification is used on two important factors related to the immunogenicity endpoints: age and 
previous vaccination history. These factors will be included in the statistical model.  
Centers will not be included in the statistical analysis, but countries will be considered in the sub-
group analyses. 
8.4 
Multiple Comparisons and Multiplicity 
 
This a phase 2, exploratory study and no adjustment will be applied for multiple endpoints and 
multiple comparisons.  
 
8.5 
Immunogenicity/Safety/Other Subsets 
 
Not applicable. 
 
8.6 
Subgroups 
 
All analyses (safety and immunogenicity) will be done by the age subgroups ≥50-64 years or ≥65 
years. 
Unadjusted and adjusted immunogenicity analysis of the GMTs will be performed by  the following 
subgroups: 
• Influenza vaccination history previous 3 years as yes and no, where we excluding this factor in 
the analysis model; 
• Comorbidity risk (yes/no) defined as the Comorbidity Risk Score (by Hak et al.)  < 50 
(comorbidity risk = no) or ≥50 (comorbidity risk = yes); 
• Sex; 
• Country; 
• Pre-vaccination status HI or MN < 1:10 (or LLOQ) and HI or MN ≥ 1:10 (or LLOQ).. 
 
The adjusted GMT subgroup analysis will not be conducted if , the number of subjects in a 
subgroup category is less than 10% of the total population. 
Exploratory interaction analyses may be conducted to support consistency or lack of consistency 
of the results among the subgroups. 


 
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8.7 
Data Transformation 
 
Distributions of antibodies are generally skewed to the right and approximately log-normally 
distributed. Therefore, prior to any statistical analysis that assumes normally distributed 
observations, antibody titers will be log10-transformed. GMTs and their 95% CIs will be then 
computed by exponentiating (base 10) the means and 95% CIs of the log10 transformed titers.  
 
 
8.8 
Derived and Computed Variables 
 
Demographics 
In the case that Age and/or Body Mass Index must be recomputed by standard software 
Age will be calculated in years using the following formula:  
Integer [(Date of Visit 1 – Date of Birth + 1) / 365.25]  
Body Mass Index (kg/m2) will be calculated using the following formula:  
Mass (kg) / Height2 (m2)  
The total Comorbidity Risk Score will be derived by the sum of all scores (see protocol appendix 
1). The total Comorbidity Risk Score will be categorized as a score < 50 or ≥ 50.   
Immunogenicity 
Values below the lower limit of quantification will be set to half that limit. Values above the upper 
limit of quantification will be set to the value of this upper limit. 
Seroconversion based on HI antibodies is defined as binary variable for subjects with non-missing 
values pre-vaccination- and post-vaccination as: 
= 1, if seroconverted (defined as a ≥ 4-fold increase in titer post-vaccination in those with pre-
vaccination titer above the LLOQ (1:10), or a post-vaccination titer ≥ 1:40 for subjects with pre-
vaccination titer below the LLOQ (1:10))  
= 0, otherwise  
Seroconversion based on MN antibodies is defined as binary variable for subjects with non-
missing values pre-vaccination- and post-vaccination as: 


 
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= 1, if seroconverted (defined as a ≥ 4-fold increase in titer post-vaccination in those with pre-
vaccination titer above the LLOQ, or a post-vaccination titer ≥ 4 times the LLOQ for subjects 
with pre-vaccination titer below the LLOQ 
= 0, otherwise 
Fold increase is defined as the post-vaccination titer divided by the pre-vaccination titer. 
Geometric Mean Titer 
The GMT will be calculated using the following formula:  
 
( )













∑
=
n
t
n
i
i
1
10
log
10
 
where 𝑡𝑡1, 𝑡𝑡2, … , 𝑡𝑡𝑛𝑛are n observed immunogenicity titers. The 95% confidence intervals for GMT 
will be calculated as 10൛𝑀𝑀−𝑡𝑡0.975,𝑛𝑛−1𝑆𝑆𝑆𝑆)ൟ, 10൛𝑀𝑀+𝑡𝑡0.975,𝑛𝑛−1𝑆𝑆𝑆𝑆)ൟ; where M and SE are the means and 
standard error of logarithm base 10-transformed titers, respectively. 
 
Solicited Adverse Events  
For details see Section 13.2. 
 
Unsolicited Adverse Events 
All adverse events will be characterized according to the date of occurrence related to the 
vaccination. If the start date is before the date of injection of study vaccine or indicated as on 
injection day but before injection, these events will not be considered as unsolicited adverse events 
and mapped to the medical history. 
Note: If an adverse event start date is missing or unknown and no indication is provided on the 
timing, the adverse event will be considered as unsolicited adverse event.   
When start and/or end dates of an adverse event are only partially known, adverse events will be 
categorized as unsolicited adverse event unless the partial end date is before the vaccination date.   
All unsolicited adverse events emergent will be categorized as occurring during the treatment 
period of 28 days following vaccination (period Day 1 to Day 29) based on the start date. If start 
date is missing, events will be counted as yes during the period of 28 days following vaccination. 


 
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The maximum event severity is the greatest severity associated with a preferred term (PT) for a 
reported adverse event according to the following order: Mild < Moderate < Severe. 
Vaccination-related Adverse Events are those for which the cause has been evaluated by the 
investigator, and recorded as possibly related, probably related or unknown/ missing. 
 
Prior and Concomitant Medications 
All medications will be characterized according to the start and end date of occurrence related to 
the vaccination as follows: 
• Pre-vaccination: start date before the date of injection of study vaccine.  
• Concomitant: start date before vaccination but continued after vaccination or start date 
after vaccination. Concomitant medication during the period Day 1- 29 will be labeled if in 
addition, the start date is on or before the Day 29. If start date and/or stop date is missing 
or incomplete, medication is considered concomitant unless the information on the stop 
date is unambiguous before the vaccination date.      
Previous vaccination will be categorized by the previous year the latest influenza vaccination was 
done within the three years prior to the study vaccination. The categories year since last 
vaccination will be 1 year (if 2020 is the last year), 2 years (if 2019 is the last one)  or 3 years (if 
2018 is the last one). 
8.9 
Treatment groups 
 
The different treatment groups will be labeled as shown in the table below.  The HA antigen content 
and the MF59 content will be used as co-variates in selected statistical analyses.  
Table 4: Overview of treatment groups 
Group 
Vaccine 
Vaccine description 
HA antigen 
content/ 
strain (µg) 
MF59 
squalene 
content (mg) 
Total injected 
volume (mL) 
Sorting 
Order 
A 
aQIVc 1 
 and 
 
 
 
 
2 
B 
aQIVc 3 
 and 
 
 
 
 
6 
C 
aQIVc 6 
 and 
 
 
 
 
4 
D 
aQIVc 7 
 and 
 
 
 
 
7 
E 
aQIVc 9  
 and 
 
 
 
 
3 


 
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F 
aQIVc 10 
 and 
 
 
 
 
5 
G 
aQIVc 11 
 and 
 
 
 
 
8F 
H 
QIVc 
15 mcg  
15 
0 
0.5 
1 
 
 
8.10 Analysis Software 
 
All analyses will be performed using SAS Software version 9.4 or higher. 
 
 
 


 
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9. STUDY SUBJECTS 
9.1 
Disposition of Subjects and Withdrawals 
 
All randomized subjects will be accounted for in this study. The numbers and percentages of 
subjects in each analysis set, study withdrawals, subgroups, and major protocol deviations will be 
presented. Number of subjects per country and site will be presented by vaccine group and overall 
for All Enrolled Set. 
The time in days (i.e. date of last assessment minus date of vaccination plus 1) the subjects are 
under observation for safety will be summarized by vaccine group and overall using summary 
statistics (mean, Standard Deviation, minimum, median, maximum). 
 
All subjects who are randomized  will be accounted for in this study. The frequencies and 
percentages of subjects in each analysis set, study withdrawals, subgroups, and major protocol 
deviations will also be presented. 
 
10. DEMOGRAPHICS AND OTHER BASELINE CHARACTERISTICS 
 
In general, all tables related to baseline characteristics should include a Total column across 
vaccine groups. 
10.1 Demographics 
 
Age, height, weight, body mass index and Comorbidity Risk Score will be summarized by 
reporting the mean, standard deviation, median and range, and will be calculated by vaccine group 
and overall. 
In addition, the frequencies of age categories will be reported as 50 – 64 and ≥ 65 years old (age as 
a randomization stratum) and 50-64, 65-74, and 75-84, and ≥ 85 years old. The number and 
percentages of subjects by sex, ethnic origin, race, previous vaccination history (in past 3 years), 
country and Comorbidity Risk Score (<50 and ≥50) will be presented by vaccine group and overall. 
Demographic data will be tabulated for the All Enrolled, FAS, PPS and Safety sets. 
The distribution across all stratification factors (age subgroup * previous vaccination history) will 
be presented by vaccine group and overall. This table will be presented based on the IRT data and 
– if different – also for the actual eCRF data. 


 
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10.2 Medical History 
 
The numbers and percentages of subjects with medical history will be presented by Medical 
Dictionary for Regulatory Activities (MedDRA) system organ class (SOC) and preferred term (PT) 
by vaccine group and overall. Medical history data will be tabulated for the All Enrolled, FAS, PPS 
and Safety Sets. 
11. IMMUNOGENICITY ANALYSIS 
 
The immunogenicity analysis will be descriptive in nature and focus on the estimation of the 
treatment effects of the seven different aQIVc formulations versus QIVc. All immunogenicity 
analyses will be evaluated based on the PPS Immunogenicity. In the case that there is >5% 
difference in the total number of subjects between the PPS and the FAS Immunogenicity, additional 
analysis based on the FAS Immunogenicity will be conducted. 
11.1 Blood samples 
 
The frequencies and percentages of subjects with blood draws will be summarized overall and by 
vaccine group. Data will be tabulated for the All Enrolled Set. 
 
11.2 Primary Objectives Analysis 
 
The primary endpoints in terms of HI antibody response against homologous vaccine strains are 
defined as: 
• Geometric Mean Titer (GMT): Geometric mean of HI antibodies at Day 1 and Day 29. 
• Geometric Mean Fold Increase (GMFI): The Geometric mean of the fold increase in 
serum HI titer post-vaccination (Day 29) compared to pre-vaccination (Day 1). 
• Percentages of subjects with HI titers ≥ 1:40 at Day 1 and Day 29. 
• Percentage of subjects with seroconversion (defined as a ≥4-fold increase in titer post-
vaccination in those with pre-vaccination titer above the LLOQ (1:10), or a post-vaccination 
titer ≥1:40 for subjects with baseline titer below the LLOQ (1:10) for HI antibodies at Day 
29). 
Note that the HI assay could be replaced by the MN assay in case of a lack of agglutination. 
 
In addition, reverse cumulative distribution plots will be generated to display the distribution of 
the antibody responses at Day1 and Day 29 for each of the vaccine groups. The x-axis represents 
the immunogenicity values, and the scale of the axis is logarithmic. The y-axis represents the 


 
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percentage of subjects having at least that immunogenicity value. Due to the discrete values of 
antibody response, the plot will show a step-wise function. The figures begin at 100%, and then 
descends to the lowest point on the curve, which is the percentage of subjects having an 
immunogenicity value equal to the highest observed value. 
 
All statistical analyses will be descriptive in nature. No hypothesis testing will be applied. 
 
Geometric Mean Titer 
Summary statistics (geometric mean, 95% confidence interval of GMT, minimum, median, 
maximum) of the titers will be presented by strain, assessment (Day 1 or Day 29) and vaccine 
group.   
The analysis model used for the comparison of the HI (or MN) GMT between each of the seven 
aQIVc formulations and QIVc will be done using a general linear model on log-transformed (base 
10) Day 29 titers as the outcome variable and as covariates: treatment groups as categories 
(aQIVc1, aQIVc3, aQIVc6, aQIVc7, aQIVc9, aQIVc10, aQIVc11 and QIVc), log-transformed pre-
vaccination titer, age subgroup and previous vaccination history. From this model, adjusted 
differences in the least square means (on the log scale) will be produced with 95% confidence 
limits for aQIVc1 versus QIVc, for aQIVc3 versus QIVc, for aQIVc6 versus QIVc, for aQIVc7 
versus QIVc, for aQIVc9 versus QIVc, for aQIVc10 versus QIVc, and for aQIVc11 versus QIVc. 
The estimated difference and the confidence limits will be back-transformed to obtain an adjusted 
GMT ratio with 95% confidence limits. Each of the four strains will be analyzed separately. 
In addition, a second analysis model will be applied – similar as the model described above but 
using the amount of antigen and MF-59 dose as factors instead of the treatment groups as category, 
but with the same covariates age group, previous vaccination history and log-transformed pre-
vaccination titer. Model fit will be assessed, and alternative models may be considered.  
Potential interaction between age subgroup and treatment effect will be examined by including an 
interaction term in the model and present all adjusted GMT ratios with 95% confidence intervals 
for each age subgroup.  
Unadjusted GMT ratio will be based on a simple ANOVA model on the log-transformed titers and 
the treatment group.  
 
Geometric Mean Fold Increase 
Summary statistics (geometric mean, 95% confidence interval of GMT, minimum, median, 
maximum) of the relative increase in titers will be presented by assessment (Day 29), strain and 
vaccine group.   


 
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The analysis model for the fold increase in titers will be done using the same models as mentioned 
above on log-transformed (base 10) (Day 29 titers/Day 1 titers) as the outcome variable but 
excluding the log-transformed pre-vaccination titer. 
 
 
Analysis of binary endpoints 
The number and proportion of subjects achieving the binary endpoints (seroconversion or HI titer 
>=1:40) will be summarized by assessment (Day 29), strain and vaccine group. 
For each of the influenza vaccine strains and for each of the vaccine groups, summaries will 
include the associated two-sided 95% confidence intervals according to Clopper-Pearson. 
The binary endpoints (i.e. seroconversion or titer ≥1:40) will be compared between each aQIVc 
formulation and QIVc using the Miettinen and Nurminen method without adjustment for the age 
subgroup. The results will be presented as the difference in the percentage of subjects with 95% 
confidence intervals.  
Additional supportive analyses may be done using generalized linear models with factors for 
vaccine group, age subgroup, pre-vaccination titer as well as previous vaccination history. 
Adjusted differences between vaccine groups with 2-sided 95% CI will be calculated based on the 
model and potential interaction effects will be examined. 
 
11.3 Secondary Objectives Analysis 
 
The secondary endpoints in terms of HI antibody response against homologous cell-derived 
vaccine strains (A/H1N1, A/H3N2, B/Yamagata and B/Victoria) at Day 181, are: 
• GMT: Geometric mean of HI antibodies at Day 181; 
• GMFI: The Geometric mean of the fold increase in serum HI GMTs post-vaccination (Day 181) 
compared to pre-vaccination (Day 1); 
• Percentages of subjects with HI titers ≥ 1:40 on Day 181. 
In addition, the humoral immune response in terms of MN antibody response against homologous 
cell-derived vaccine strains (A/H1N1, A/H3N2, B/Yamagata and B/Victoria) at Days 1, 29 and 
181: 
• GMT: Geometric mean of MN antibodies at Days 29 and 181; 
• GMFI: The Geometric mean of the fold increase in serum MN GMTs post-vaccination (Day 29 
and Day 181) compared to pre-vaccination (Day 1); 


 
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• Percentages of subjects with seroconversion (defined as ≥ 4-fold increase for subjects with pre-
vaccination MN titers ≥ Lower Limit of Quantitation (LLOQ) or as ≥ 4*LLOQ for subjects 
with pre-vaccination MN titer <LLOQ) at Day 29. 
The analysis of the secondary immunogenicity endpoints will be conducted in the same way as 
described for the primary immunogenicity endpoints.  
 
11.4 Exploratory Objectives Analysis 
 
Additional analyses may be done on the immunogenicity endpoints as measured on Day 29 by HI 
or MN assay using cell-derived target viruses or cell-derived heterologous target viruses. Details 
on this exploratory analysis will be included in a separate statistical analysis plan. The results 
may be presented in an addendum to the CSR. 
 
12. EFFICACY ANALYSIS 
 
 
Not applicable. 
 
 
13. SAFETY ANALYSIS 
 
The analysis of safety assessments in this study will include summaries of the following categories 
of safety data collected for each subject: 
• Vaccine exposure; 
• Solicited local and systemic adverse events as recorded by the subject; 
• Solicited systemic events and injection site pain assessed using the investigator evaluation; 
• Unsolicited adverse events; 
• Serious AEs, MAAEs, AESIs and AEs leading to withdrawal from study.  
 
13.1 Analysis of Extent of Exposure 
 


 
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The frequencies of subjects with vaccinations will be summarized overall and by vaccine group 
and by age group. Data will be tabulated for the All Exposed Set. 
 
13.1.1 Safety Completeness 
 
Analysis Solicited Adverse Events 
The safety completeness analysis on solicited adverse events aims to identify subjects who 
completed the electronic diaries. The analysis will show the number of subjects with valid results 
by solicited adverse event and day.   
Three summaries will be produced: 
1. The frequencies of subjects who provided any data on the electronic diary cards by vaccine 
group. 
2. For each solicited adverse event – including analgesic use, the frequencies of subjects with   
data will be presented by vaccine group and day   
3. For each solicited adverse event – including analgesic use, frequency of the number of days 
with data on the eDiary by vaccine group.  
For the corresponding percentages, the denominator will be the respective number of subjects 
vaccinated (All Exposed Set).   
All analyses will be based on the ‘as treated’ analysis set. 
 
13.2 Solicited Local and Systemic Adverse Events 
 
Each solicited AE is to be assessed by the subject for Day 1 to Day 7 according to a defined severity 
grading scale; see specifics of the solicited event and grading system below in Table 3. 
Table 5 Severity Grading for Solicited Local and Systemic Adverse Events 
 
 
Any Event 
Type 
Solicited Event 
Grade 1/Mild 
Grade 2/Moderate 
Grade 3/Severe 
Local 
Injection site pain 
No interference with 
daily activity 
Interferes with daily 
activity 
Prevents daily 
activity 
 
Erythema 
25-50 mm 
51-100 mm 
> 100 mm 
 
Induration 
25-50 mm 
51-100 mm 
> 100 mm 
Systemic 
Loss of appetite 
Eating less than 
usual with no effect 
on normal activity 
Eating less than 
usual /interfered 
with normal activity 
Not eating at all 


 
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Any Event 
Type 
Solicited Event 
Grade 1/Mild 
Grade 2/Moderate 
Grade 3/Severe 
 
Nausea 
No interference with 
daily activity 
Interferes with daily 
activity 
Prevents daily 
activity 
 
Fatigue 
No interference with 
daily activity 
Interferes with daily 
activity 
Prevents daily 
activity 
 
Myalgia 
No interference with 
daily activity 
Interferes with daily 
activity 
Prevents daily 
activity 
 
Arthralgia 
No interference with 
daily activity 
Interferes with daily 
activity 
Prevents daily 
activity 
 
Headache 
No interference with 
daily activity 
Interferes with daily 
activity 
Prevents daily 
activity 
 
Chills 
No interference with 
daily activity 
Interferes with daily 
activity 
Prevents daily 
activity 
 
Fever 
38.0 - 38.4 °C 
100.4 – 101.1 °F 
38.5 – 38.9 °C 
101.2 – 102.0 °F 
≥39.0 °C 
≥102.1 °F 
Note: presence of an event on a day is defined as grade 1, 2 or 3; absence is defined as non-missing 
and not present.  Erythema and Induration: grading will be derived from the actual measurements 
in mm; Fever will be derived from the actual temperature. 
Other Indicators of Reactogenicity 
The use of analgesics/antipyretics will be captured as “absent” or “present” separately by reason 
“for treatment” or “for prevention”. 
 
The analyses will be based on the solicited safety set and encompass various summaries of the data 
by vaccine group, overall and by age subgroup.  
1. Overall summary of subjects with solicited adverse events  
2. Local Solicited adverse events, maximum event severity by event and time interval (i.e. 
Day 1-7, Day 1-3, Day 4-7) 
3. Systemic Solicited adverse events, maximum event severity by event and time interval (i.e. 
Day 1-7, Day 1-3, Day 4-7) 
4. Number of days of solicited adverse events, including ongoing AE after Day 7  
5. Daily reports of subjects with solicited adverse events. 
6. Day of first onset of solicited adverse events  


 
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7. Ongoing adverse events at Day 7 
8. Distribution of maximum temperature  
9. Other use of analgesics/antipyretics. 
10. Exploratory analyses to investigate the effect of amount of antigen and MF-59 levels. 
For each of the time points or time intervals presented in the summaries, only subjects with at least 
one valid observation (i.e., any non-missing values) for the solicited adverse events in the interval 
of interest will be considered. Subjects without valid data will be removed from the denominator 
to prevent a downward bias (towards zero). 
All tables are run by vaccine group, overall and by age subgroup.  
 
Overall summary of subjects with solicited adverse events. 
Any solicited adverse event presence is defined as at least one day recorded a presence of a local 
or a systemic adverse event. No solicited adverse event is defined as for all days ‘No’ for all pre-
defined solicited adverse events.  
Note that, where applicable, missing values for all days for all events will be excluded from % 
calculations.  
The use of analgesics/antipyretics will be considered in this summary as a separate category under 
“other”, however will be considered as part of reactogenicity. 
 
Local Solicited adverse events, maximum event severity by event and time interval 
The maximum event severity will be defined if there is at least one non- missing observation 
within this time interval. Each subject’s data will be aggregated across the time points of the 
interval and summarized according to the maximal severity observed for each local adverse event, 
followed by a summary across subjects for each vaccine. Subjects with missing values at each of 
the requested time points, will be excluded.  
The time intervals will be Day 1 to Day 7, Day 1 to Day 3 and Day 4 to Day 7. A summary table 
will be created with the frequency using only any local solicited AE and severe local solicited 
events. 
 
Systemic adverse events, maximum event severity by event and interval   
The analysis on the maximum severity of the systemic adverse events will be done along the 
same methods as for the local adverse events.  
 
Number of days with solicited adverse events 
The number of days with the adverse event is defined irrespective of severity. If a solicited adverse 
event continues beyond day 7 the period after day 7 is added.  


 
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The frequency distribution of the number of days (including mean and median values) will be 
provided in a summary table by vaccine and by adverse event. As reference the number of missing 
days will be presented in a frequency table. 
 
Daily reports of solicited adverse event 
For each of the time points only subjects with at least one non-missing observation for the solicited 
adverse event in the interval of interest will be considered. Subjects with missing values will be 
removed from the denominator to prevent a downward bias (towards zero). Data collected will be 
summarized (frequencies and percentages of subjects) by vaccine group, solicited adverse event, 
and time point (i.e. by day). 
 
 
Time of first onset of solicited adverse events 
The time of first onset is defined, for each subject, for each solicited adverse event, as the day at 
which the respective solicited adverse event first occurred. The summary will provide the 
frequencies and percentages of subjects with first onset of each solicited adverse events by vaccine 
group and by each time point, as well as mean and median day of onset. 
 
Adverse events ongoing at Day 7 
For each of the solicited adverse events, the number and proportion of subjects in the solicited 
safety set reported the event ongoing at Day 7 will be summarized. 
 
Distribution of maximum temperature 
Body temperature will be summarized by 0.5 °C increments from 36.0 °C up to ≥40 °C by 
frequency tables. 
 
Use of analgesics/antipyretics 
The use of antipyretics and analgesics will be summarized as “other” by type of use (prophylactic 
versus treatment) as the number and percentage of subjects reporting use.  
Exploratory analyses to investigate the effect of amount of antigen and MF-59 levels. 
A logistic regression analysis will be applied with the binary response (presence of solicited 
adverse reaction or no reaction), to explore the relation with antigen and MF59 levels as factors 
as well as with age and previous vaccination history. This model will be applied for both 
reported solicited local or systemic adverse events (AEs) for 7 days following vaccination and 
reported severe solicited local or systemic AEs for 7 days following vaccination (Day 1 through 
Day 7). 


 
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13.3 Investigator assessment of Solicited Adverse Events 
 
The solicited systemic events and injection site pain will also be assessed by the investigator. 
For each subject, the investigator will assess if the subject’s solicited symptoms are consistent 
with the data reported in the e-diary. If no, the investigator will record the evaluation of the 
solicited systemic events and injection site pain for consistency with eDiary and if not presence 
or absence, and if present the maximum severity and start and stop date. If yes, the subject’s 
eDiary data will be mapped to the investigator’s assessment.  
 
A frequency table will be presented on the consistency assessment of the investigator (yes/no) by 
vaccine group and total.  
Furthermore, the overall occurrence during Day 1 to 7 after vaccination for each of the solicited 
systemic events and injection pain based on the investigator assessment will be tabulated by 
vaccine group for total and by age group. The same summaries will be presented by the 
maximum severity including  the occurrence of severe events according to the investigator. All 
subjects for which an investigator assessment has been done will be included in these analyses. 
 
13.4 Unsolicited Adverse Events 
 
This analysis applies to all AE’s occurring during the study, judged either as probably related, 
possibly related, or not related to vaccination by the investigator, recorded in the AE eCRF.   
The original verbatim terms used by investigators to identify adverse events in the eCRFs will be 
mapped to preferred terms using the MedDRA dictionary.  
The adverse events will then be grouped by MedDRA preferred terms into frequency tables 
according to system organ class. All reported AEs, as well as AEs judged by the investigator as at 
least possibly related to study vaccine, will be summarized according to system organ class and the 
preferred term within system organ class. These summaries will be presented by vaccination group. 
When an AE occurs more than once for a subject, the maximum severity and strongest relationship 
to the vaccine group will be counted. 
The assignment to safety follow-up time intervals will be done by day of onset and not by days 
ongoing/persisting. 


 
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The summaries will be presented by period of onset (Day 1 to Day 29, Day 30 to end of study or 
Day 1 to end of study). An overview summary will be created by time -period with number and % 
of subjects with the following categories: 
- 
AE  
- 
AE by maximum severity 
- 
Related (i.e. at least possible) AE 
- 
SAEs  
- 
Related SAEs 
- 
AESIs  
- 
MAAEs 
- 
AEs leading to withdrawal from study 
- 
Deaths. 
 
The following summaries will be produced by SOC and Preferred Term and by time-period (Day 1 
to Day 29, Day 30 to end of study or Day 1 to end of study): 
• AEs  
• AEs by maximum severity 
• AEs that are possibly or probably related to the vaccine 
• SAEs 
• SAEs that are possibly or probably related to the vaccine 
• AESIs 
• MAAEs 
• AEs leading to withdrawal 
• Deaths. 
 
Data listings of all adverse events will be provided by subject. In addition, adverse events in the 
categories above will be provided as listed data. 
All tables will be produced overall and by age subgroup.  


 
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13.5 Combined Solicited and Unsolicited Adverse Events 
 
A summary of subjects with all combined solicited (regardless of their duration) and unsolicited 
adverse events will be provided. Solicited adverse events will be coded by MedDRA. For 
clinicaltrials.gov posting purposes, a summary of combined solicited and unsolicited non-serious 
adverse events will be produced by System Organ Class and according to occurrence of each event 
based on the Overall Safety set.   
.  
 
13.6 Clinical Safety Laboratory Investigations 
 
Not applicable. 
 
13.7 Concomitant Medication 
 
Medications and vaccines taken prior or during the study are categorized as pre-trial and/or 
concomitant. In addition, a subset of concomitant medications is defined for the period Day 1 to 
29 (see section 8.7 for definition). 
Medications (generic drug name) will be coded using the WHODRUG dictionary.  
The frequencies and percentages of subjects reporting pre-trial, concomitant medications (Day 1 
– 29 and Day 1 to end of study) will be tabulated by vaccine group.  
 
14. INTERIM ANALYSIS 
 
There are no planned interim analyses for this study.  
The statistical analyses will be performed stepwise in two parts, as follows: 
1. A final analysis including all immunogenicity and safety data collected from Visit 1 to Visit 3 
(Day 29) and associated primary and secondary objectives will be conducted on cleaned and locked 
data. No individual listings and unblinding data will be generated at this stage. Access to 
information about study groups will be limited by a biostatistician in charge of statistical analysis. 


 
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2. The analysis of immunogenicity and safety data collected between Visit 3 and Visit 5 will be 
performed after all data is available. The results of this analysis will cover the entire study duration. 
All results will be presented in the clinical study report and will include individual data listings and 
unblinded information.  
 
15. DATA MONITORING COMMITTEES 
 
An internal safety team (IST) – independent of the study team - will be installed to monitor the 
safety data collected during the enrollment of the subjects in the study. Details are included in 
the IST charter. In addition, upon request an independent DMC will be asked to review the 
safety data and make recommendations for the conduct of the trial. Further details are described 
in the DMC charter. 
 
 
Alerting and pausing rules 
A summary table will be created with number and % of subjects who met one of the criteria by 
treatment based on the solicited or unsolicited Safety set. 
• Any SAE that cannot be reasonably attributed to a cause other than vaccination, according 
to the Investigator’s assessment, within 7 days post-vaccination (i.e. probable). 
• Severe systemic hypersensitivity such as anaphylaxis, within 24 hours after study 
vaccination. 
• Any Grade 3 (severe) local solicited AE lasting ≥2 consecutive days, within 7 days 
postvaccination. 
• Any Grade 3 (severe) systemic solicited AE lasting ≥2 consecutive days, within 7 days 
postvaccination. 
• Any severe unsolicited AE that cannot be reasonably attributed to a cause other than 
vaccination (i.e. probable), within 7 days postvaccination. 
 
 
16. CHANGES TO PLANNED ANALYSIS 
 
Compared to the final protocol, an additional safety set may be defined for the investigator 
assessment of the solicited adverse events. This will only be applicable if there are subjects without 
any eDiary data, but with an investigator assessment of the solicited adverse events. 


 
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The SAP, version 1.0, 16 April 2021 has been amended with a few changes mainly related to the 
programming of data sets. 
• The window around the Day 29 sample visit has been extended to -7 and + 21 days with 
respect to the Day 29 visit to be included in the PP analysis. 
• The Day 181 FAS and PPS have been defined in more detail 
• Last vaccination prior to study will be categorized as 1 year before, 2 years or 3 years before 
• The investigator assessment will be derived both on the overall consistency as well as the 
particular symptom. 
 
17. LIST OF FINAL REPORT TABLES, LISTINGS AND FIGURES 
 
The list of tables, listings and figures will be developed based on the final version 1.0 of this 
statistical analysis plan. This will be documented as part of the TLF shells. 
Numbering will follow ICH E3 guideline on clinical study report. 
 
18. 
REFERENCES 
 
Clinical Study Protocol V201_01 FINAL VERSION 2.0: JAN 2021SEPT 2020  
 
ICH. Statistical Principles for Clinical Trials, Guideline E9, 1998. 
 
Sasaki S and Sullivan M. Limited efficacy of inactivated influenza vaccine in elderly individual 
is associated with decreased production of vaccine-specific antibodies. J Clin Invest. 
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U.S. Department of Health and Human Services, Food and Drug Administration, CBER (2007): 
Guidance for Industry Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers 
Enrolled in Preventive Vaccine Clinical Trials.  
 
Miettinen O., Nurminen M. Comparative analysis of two rates. Statistics in Medicine 1985; 
4(2):213-226.  
 


 
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Wong SS and Webby RJ. Traditional and new influenza vaccines. Clin Microbiol Rev. 
2013;26:476-492. 
Otten G, Matassa V, Ciarlet M, Leav B. A phase 1, randomized, observer blind, antigen and 
adjuvant dosage finding clinical trial to evaluate the safety and immunogenicity of an adjuvanted, 
trivalent subunit influenza vaccine in adults ≥65 years of age. Vaccine. 2020;38:578-587. 
 
Reber AJ, Chirkova T, Kim JH, Cao W et al. Immunosenescence and challenges of vaccination 
against influenza in the aging population. Aging and Disease. 2012;3:68-90.