Investigation and Comparison of the Antibody Response Initiated by Recombinant, Cell-Based and Egg-Based Influenza Vaccines
Phase: PHASE3
Conditions: Influenza Vaccination
Sponsor: Tan Tock Seng Hospital

 
 
 
 
 
PROTOCOL  
Investigation and Comparison of the 
Antibody Response initiated by 
Recombinant, Cell-Based and Egg-
Based Influenza Vaccines [VAP3] 
Protocol Number: VAP00030 
Project Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version: 2.0 
Date: 27/10/2022 
Author/s: 
Sheena Sullivan, Annette Fox, Barnaby Young, Lisa Ng, Ian Barr  
 
 
Sponsor 
Tan Tock Seng Hospital, Singapore 
 
 
ESC supported by: 
Sanofi Aventis 
 
 
CONFIDENTIAL 
 
This document is confidential. No part of it may be transmitted, reproduced, published, or used 
without prior written authorization from the authors. 
 
 
Statement of Compliance 
 
This study will be conducted in compliance with all stipulation of this protocol, the conditions of 
the ethics committee and Health Sciences Authority regulatory approval and the Note for 
Guidance on Good Clinical Practice (CPMP/ICH-135/95). 
 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 2 of 38 
Table of Contents 
 
Study Synopsis ........................................................................................................................ 6 
 
Glossary of Abbreviations & Terms .......................................................................................... 8 
 
Study Sites .............................................................................................................................. 9 
3.1 
Study Location/s ............................................................................................................... 9 
 
Introduction/Background Information ..................................................................................... 10 
4.1 
Lay Summary.................................................................................................................. 10 
4.2 
Introduction ..................................................................................................................... 10 
4.2.1 
Study overview ........................................................................................................ 11 
 
Aims and Hypotheses ............................................................................................................ 12 
5.1 
Study Aim ....................................................................................................................... 12 
5.2 
Hypotheses ..................................................................................................................... 12 
 
Objectives and Outcomes ...................................................................................................... 12 
6.1 
Primary objective ............................................................................................................ 12 
6.2 
Secondary Objectives ..................................................................................................... 12 
6.2.1 
Secondary Objective 1 ............................................................................................. 12 
6.2.2 
Secondary Objective 2 ............................................................................................. 13 
6.2.3 
Secondary Objective 3 ............................................................................................. 13 
6.2.4 
Secondary Objective 4 ............................................................................................. 13 
6.2.5 
Secondary Objective 5 ............................................................................................. 14 
6.2.6 
Secondary Objective 6 ............................................................................................. 14 
 
Monitoring of clinical symptoms and sequencing of influenza strains ..................................... 14 
7.1 
Study Design .................................................................................................................. 14 
7.2 
Justification of the study design ...................................................................................... 15 
7.3 
Study Schedule ............................................................................................................... 16 
7.4 
Vaccination ..................................................................................................................... 17 
7.4.1 
QIV-R (FluBlok®) by IM route at V1 (D0) ................................................................. 17 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 3 of 38 
7.4.2 
QIV-C (Flucelvax® Quad) by IM route at V1 (D0): ................................................... 17 
7.4.3 
QIV-E (Fluarix) by IM route at V1 (D0) ..................................................................... 17 
7.4.4 
Vaccine composition ................................................................................................ 18 
7.4.5 
Safety monitoring ..................................................................................................... 18 
 
Data collection ....................................................................................................................... 18 
8.1 
Overview of data collection ............................................................................................. 18 
8.2 
Baseline questionnaire .................................................................................................... 19 
8.3 
SMS reminders ............................................................................................................... 19 
8.4 
Blood collection ............................................................................................................... 19 
8.5 
Respiratory samples ....................................................................................................... 20 
 
Randomisation Procedures .................................................................................................... 20 
9.1 
Blinding and code breaking procedures .......................................................................... 20 
9.2 
Randomisation and allocation procedures ...................................................................... 21 
9.3 
Treatment compliance .................................................................................................... 21 
 Laboratory procedures ........................................................................................................... 22 
10.1 
Sero-response to vaccination .......................................................................................... 22 
10.2 
Influenza testing and virus characterization .................................................................... 22 
10.3 
Antibody landscapes ....................................................................................................... 22 
10.4 
Antibody analysis ............................................................................................................ 22 
10.5 
B cell analysis ................................................................................................................. 23 
 Study Population .................................................................................................................... 23 
11.1 
Inclusion Criteria ............................................................................................................. 23 
11.2 
Exclusion Criteria ............................................................................................................ 23 
11.3 
Recruitment Procedures ................................................................................................. 24 
11.3.1 
Assigning Participant IDs ......................................................................................... 24 
11.4 
Retention strategies ........................................................................................................ 24 
11.5 
Follow-up ........................................................................................................................ 24 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 4 of 38 
11.6 
Informed Consent ........................................................................................................... 25 
 Participant Safety and Withdrawal ......................................................................................... 25 
12.1 
Potential risks to participants .......................................................................................... 25 
12.1.1 
Possible Reactions to Blood Draw ........................................................................... 25 
12.1.2 
Possible Reactions to Vaccination ........................................................................... 25 
12.1.3 
Possible Reactions to respiratory swab collection .................................................... 26 
12.2 
Risk Management and Safety ......................................................................................... 26 
12.2.1 
Definitions ................................................................................................................ 26 
12.2.2 
Eliciting Adverse Event Information .......................................................................... 27 
12.2.3 
Assessment and Documentation of Adverse Events ................................................ 27 
12.2.4 
Serious Adverse Event Reporting ............................................................................ 27 
12.3 
Handling of Withdrawals ................................................................................................. 27 
12.4 
Replacements ................................................................................................................. 28 
12.5 
Potential benefits to subjects .......................................................................................... 28 
 Statistical Methods ................................................................................................................. 29 
13.1 
Randomisation ................................................................................................................ 29 
13.2 
Sample Size Estimation & Justification ........................................................................... 29 
13.3 
Power Calculations ......................................................................................................... 29 
13.4 
Statistical analyses ......................................................................................................... 31 
13.4.1 
Primary Objective .................................................................................................... 32 
13.4.2 
Secondary objective 1.............................................................................................. 32 
13.4.3 
Secondary objective 2.............................................................................................. 32 
13.4.4 
Secondary objective 3.............................................................................................. 33 
13.4.5 
Secondary objective 4.............................................................................................. 33 
13.4.6 
Secondary objective 5.............................................................................................. 33 
13.4.7 
Secondary objective 6.............................................................................................. 33 
 Storage of Blood and Tissue Samples ................................................................................... 34 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 5 of 38 
14.1.1 
Details of where samples will be stored, and the type of consent for future use of 
samples 34 
 Data Security & Handling ....................................................................................................... 34 
15.1 
Details of where records will be kept & How long will they be stored ............................... 34 
15.1.1 
Study database ........................................................................................................ 35 
15.1.2 
Data entry ................................................................................................................ 35 
15.1.3 
Data storage ............................................................................................................ 35 
15.2 
Confidentiality and Security............................................................................................. 35 
 Attachments ........................................................................................................................... 36 
 References ............................................................................................................................ 37 
 
 
 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 6 of 38 
 STUDY SYNOPSIS 
Provide brief information 
Title: 
Investigation and Comparison of the Antibody Response initiated 
by Recombinant, Cell-Based and Egg-Based Influenza Vaccines 
Short Title: 
Immunogenicity trial of 3 influenza vaccines 
Design: 
Randomized controlled trial 
Study Centre: 
National Centre for Infectious Diseases (NCID), Singapore; WHO 
Collaborating Centre for Reference and Research on Influenza 
Hospital: 
National Centre for Infectious Diseases Research Clinic 
Study Question: 
Does recombinant vaccine result in a better antibody response than 
either standard egg-grown vaccine or cell-grown vaccine? 
Primary objective: 
To assess whether vaccination with recombinant quadrivalent 
influenza vaccine (QIV-R) results in a better antibody response 
against A(H3N2) viruses than either standard egg-grown vaccine 
(QIV-E). or cell-based vaccine (QIV-C) as measured by post-
vaccination geometric mean titre from the haemagglutination 
inhibition assay 
Secondary objectives 
1. To estimate whether antibody responses to all 4 vaccine 
antigens are greatest in participants who received QIV-R and 
least in participants who received QIV-E, and if this reflects 
greater cell- to egg-grown antibody titre ratios. 
2. To estimate whether QIV-R induces antibodies against a 
greater genetic and antigenic range of A(H3N2) viruses than 
QIV-C and QIV-E.  
3. To estimate whether prior vaccination history has less effect on 
antibody responses induced against all 4 vaccine antigens by 
QIV-R compared with QIV-C and QIV-E.  
4. To estimate whether antibody titres against all 4 vaccine 
antigens are better maintained up to 12 months after 
vaccination with QIV-R compared with QIV-C and QIV-E.  
5. To estimate whether QIV-R induces higher frequencies of HA 
reactive B cells and/or B cells with different HA cross-reactivity 
profiles and phenotypes compared with QIV-C and QIV-E. 
6. To assess whether QIV-R offers better protection against 
infection compared with QIV-C or QIV-E. 
Inclusion Criteria: 
1. Aged ≥21 years and <50 years. 
2. Able to provide informed consent 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 7 of 38 
3. Willing and able to provide 4 blood samples:  
3.1. just prior to vaccination (V1),  
3.2. 14–21 days post vaccination (V2) 
3.3. 4–6 months post-vaccination (V3) 
3.4. 10-12 months post-vaccination (V4) 
4. Has not received influenza vaccine for at least 6 months before 
enrolment 
5. Willing to provide current mobile phone number for SMS 
reminders 
Exclusion Criteria:  
1. Known contraindication(s) for QIV (e.g. hypersensitivity to 
vaccine component (including eggs)). 
2. Recently (last 7 days) or currently ill or has a fever above 38°C. 
3. Cannot recall if they were vaccinated against influenza during 
more or less than two of the preceding five years. 
4. Hypogammaglobulinaemia on immunoglobulin replacement 
5. Undergoing immuno-suppressive therapies 
Number of Planned 
Subjects: 
360 
Investigational 
products: 
FluBlok® 
FlucelVax® Quad 
Fluarix® (or equivalent egg-grown QIV)  
Safety considerations: 
The risk of an adverse event associated with, influenza vaccination, 
blood collection and collection of nasal swab(s) is low. Any adverse 
event will be recorded and followed up by the project manager or 
principal investigator. 
Statistical Methods: 
Baseline comparisons: Baseline data will be summarised by 
vaccination group, with each arm.  
Analysis of the primary objective: The post-vaccination geometric 
mean titres (GMT) by vaccination regimen will be compared by using 
mixed-effects linear regression.  
Subgroups: 
A. Frequently vaccinated (vaccinated during 3-5 of the preceding 
five years) 
B. Infrequently vaccinated (vaccinated during 0 or 1 of the 
preceding five years) 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 8 of 38 
 GLOSSARY OF ABBREVIATIONS & TERMS 
 
Abbreviation 
Description (using lay language) 
AE 
Adverse event 
AR  
Attack rate 
ARI 
Acute respiratory infection 
At least one respiratory symptom (cough, sore throat) and one 
systemic symptom (fever, malaise, fatigue, myalgia) 
AESI 
Adverse event of special interest 
GCP 
Good clinical practice 
GMT 
Geometric mean titre 
 
Arithmetic mean of the logarithms (base 2) of the last positive 
dilution of each serum 
GMR 
GMTR geometric mean titre ratio 
 
Arithmetic mean of the logarithms (base 2) of the last positive 
dilution of each serum 
HA 
Haemagglutinin 
The surface protein of the influenza virus that is targeted by 
vaccines. 
HAI 
Haemagglutination inhibition assay 
 
Laboratory test, which measures anti-haemagglutinin 
antibodies. These antibodies inhibit attachment of the influenza 
virus to target cell membrane receptors on red blood cells. 
Frequently vaccinated 
3 or more prior influenza vaccinations during the past 5 years 
IDRL 
Infectious Diseases Research Laboratory, National Centre for 
Infectious Diseases, Singapore 
IM 
Intramuscular 
Infrequently vaccinated 
One or no prior influenza vaccinations during the past 5 years 
ITT 
Intention to treat 
NCID 
National Centre for Infectious Diseases, Singapore 
NPHL 
National Public Health Laboratory, National Centre for 
Infectious Diseases, Singapore 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 9 of 38 
PI 
Principal investigator 
PICF 
Participant informed consent form 
PP 
Per protocol 
QIV 
Quadrivalent influenza vaccine 
QIV-C 
Cell-based quadrivalent influenza vaccine  
QIV-E 
Egg-based quadrivalent influenza vaccine 
QIV-R 
Recombinant quadrivalent influenza vaccine 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
Seroconversion 
Evidence of a 4-fold increase in antibody titre post-vaccination, 
as measured using a haemagglutination inhibition or 
microneutralisation assay 
Seropositive 
Antibody titre of ≥40, as measured using a haemagglutination 
inhibition assay 
TTSH 
Tan Tock Seng Hospital, Singapore 
WHO 
World Health Organization 
WHOCCRRI 
World Health Organization Collaborating Centre for Reference 
and Research on Influenza 
 
 STUDY SITES 
3.1 
STUDY LOCATION/S  
Site 
Address 
Contact 
Person 
Phone 
Email 
WHOCCRRI  
792 Elizabeth 
St, Melbourne 
Sheena 
Sullivan  
 
Annette 
Fox 
93429317 
 
93443437 
 
Sheena.sullivan@influenzacentre.org 
Annette.Fox@influenzacentre.org 
Annette.Fox@unimelb.edu.au 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 10 of 38 
National 
Centre for 
Infectious 
Diseases  
16 Jalan Tan 
Tock Seng, 
Singapore, 
308442 
Barnaby 
Young 
81334132 
Barnaby_young@ncid.sg 
 INTRODUCTION/BACKGROUND INFORMATION 
4.1 
LAY SUMMARY 
The proposed study is a prospective randomised trial of 3 influenza vaccine formulations with 
different manufacturing processes: 1) egg-grown; 2) cell-grown; and 3) recombinant protein. The 
main objective is to compare the antibody responses following influenza vaccination among these 3 
vaccines to determine whether recombinant vaccines offer superior protection over standard egg or 
cell-based formulations. Because antibody responses to influenza vaccines can differ depending on 
how many times you have been vaccinated before, we will test these 3 vaccines in 2 groups: A) 
frequently vaccinated (3 or more vaccinations) and B) infrequently vaccinated (0 or 1 prior 
vaccinations) participants. Participants will be stratified by their prior vaccination history (frequent or 
infrequent) and randomised to 3 trial arms receiving one dose of one of the vaccines (egg-grown, 
cell-grown or recombinant influenza vaccines); i.e. 2 groups × 3 vaccines. Pre- and post-vaccination 
blood samples will be collected to assess antibody responses to vaccination and to assess changes 
to the cells that make antibodies. Responses to the 3 vaccines will be compared to assess which 
vaccine elicits the best antibody response in both infrequently and frequently vaccinated participants.  
4.2 
INTRODUCTION 
Influenza vaccine effectiveness varies from year to year and is generally poorest against A(H3N2), 
the virus subtype for which genetic and antigenic evolution among circulating strains has been 
greatest. While vaccination may stimulate robust antibody responses to vaccine antigen, the breadth 
of antibodies generated may be insufficient to protect vaccinees from infection by all circulating 
viruses. Furthermore, in persons repeatedly vaccinated vaccine-induced antibody responses may 
become blunted over time.  
In recent years, problems associated with egg-based production of vaccine strains have exacerbated 
these problems. Influenza viruses generally acquire substitutions within the hemagglutinin (HA) 
protein to adapt to growth in eggs. In the case of A(H3N2) viruses these adaptations often render 
them antigenically distinct from the wild-type virus. Subsequently, antibodies induced against egg-
adapted epitopes in the vaccine will provide limited protection against infection by circulating viruses, 
and vaccine effectiveness has been very low. The use of vaccine manufacturing processes that 
avoid growth in eggs can potentially overcome egg-focusing but may not illicit a broader antibody 
response.  
Cell-based vaccines have been developed which can overcome some of the problems associated 
with egg manufacturing. One cell-based vaccine product currently licensed in Australia is Sequiris’ 
Flucelvax Quad. As with standard dose egg-based vaccines, this product contains 15μg of each of 
the 4 recommended influenza virus antigens. Early studies suggest that cell-based vaccine may 
perform better against standard-dose egg-based vaccines (1-4).   

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 11 of 38 
Both egg and cell-based vaccines rely on the same production method for >70 years, which depends 
upon the growth and purification of live viruses that must be inactivated and split before being 
formulated into vaccines (5-7). The chemical inactivation process requires substances that cause 
significant disruption to key vaccine proteins (including HA) and have been found to alter the 
vaccines’ antigenicity via crosslinking antigens and disrupting native epitopes (5, 8-12). Disruption 
of key antigenic structure is likely to impact vaccine efficacy (9). Recombinant vaccines offer an 
alternative manufacturing pathway. The Sanofi recombinant vaccine Flublok® uses a recombinant 
technology to produce purified HA from a baculovirus overexpression system. The HA molecule 
produced in an insect cell line is in an un-cleaved form, known as HA0, i.e. a form that is unable to 
mediate endosomal and viral membrane fusion. Importantly the manufacturing process does not 
require any chemical inactivation, meaning that the HA proteins are not exposed to any potential 
cross-linking agents that may alter antigenicity of the vaccine (6).   In addition, Flublok® contains a 
higher concentration of antigen than standard-dose vaccines with 45 μg of each antigen included.  
Recent clinical evidence has shown Flublok® to have superior efficacy over traditional cell-based 
influenza vaccine (13), an immunogenicity profile comparable to Fluzone® high dose in older adults 
(14) and superior antibody driven viral neutralisation compared to other cell-based vaccines (1, 3, 
4). Another recent comparative analysis of antibody response from healthy adults (18-49 years old), 
comparing egg-based, cell-based and recombinant (Flublok®) vaccines found that Flublok® resulted 
in significantly higher titres of neutralizing antibody (1). This study used in vitro neutralisation assays 
to evaluate the antibody function and again indicates that the recombinant vaccine may have 
properties that allow for better viral neutralisation compared to traditional cell-based vaccines. As 
such clinical efficacy gains could be associated with differences between the HA in the different 
vaccines.  
4.2.1 STUDY OVERVIEW 
This study will be conducted in 360 adults, aged 21–49 years in Singapore to generate 
immunogenicity data. It will be a randomized, modified double-blind study comparing the 
immunogenicity of recombinant quadrivalent influenza vaccines (QIV-R) against egg-based (QIV-E), 
and cell-based (QIV-C) vaccines. This study is powered to primarily assess the immunogenicity (as 
assessed by hemagglutination inhibition (HAI) geometric mean titres (GMTs) at 14–21 days post-
vaccination) of QIV-R compared with QIV-E, and will also assess QIV-R compared with QIV-C. 
Given the acceptable safety data generated from the millions of doses of licensed QIV-R 
administered in the US and elsewhere and the fact that we will source this product from the US, it is 
considered that the risk/benefit ratio is appropriate for the conduct of a clinical study with QIV-R. 
 
 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 12 of 38 
 AIMS AND HYPOTHESES 
5.1 
STUDY AIM 
The broad aim of the proposed study is to estimate whether vaccination with recombinant 
quadrivalent influenza vaccine (QIV-R) results in a better antibody response against A(H3N2) viruses 
than either standard egg-grown vaccine (QIV-E) or cell-based vaccine (QIV-C).   
5.2 
HYPOTHESES  
1. As recombinant vaccine includes a higher dose of antigen and does not use egg-based 
manufacturing, vaccination with recombinant vaccine results in better antibody responses, 
particularly against A(H3N2) viruses, than either standard egg-grown vaccines or cell-
grown vaccines.  
2. Vaccine induced titres against influenza A(H3N2) viruses (~ wild-type), will be highest 
amongst recipients of recombinant vaccine, and lowest amongst recipients of standard egg-
based vaccine because antibody responses are miss-directed towards egg-adapted 
epitopes in the latter.  
3. The dose and form of recombinant vaccine means that it induces antibodies against a 
broader range of epitopes than the egg- and cell-grown vaccine. In turn, this will increase 
the range of strains against which vaccine-induced antibodies cross-react because some of 
these epitopes are conserved.  
4. The attenuating effects of prior vaccination (~pre-existing immunity) on vaccine 
immunogenicity are alleviated using recombinant vaccine because higher doses of antigen 
allow new B cell responses to be induced in addition to recalling memory responses.  
 OBJECTIVES AND OUTCOMES 
6.1 
PRIMARY OBJECTIVE  
To assess whether vaccination with recombinant quadrivalent influenza vaccine (QIV-R) results in a 
better antibody response against A(H3N2) viruses than either standard egg-grown vaccine (QIV-E) 
or cell-based vaccines (QIV-C) as measured by post-vaccination geometric mean titre from the 
haemagglutination inhibition assay. 
Corresponding outcome measures: 
1. Post vaccination GMT in each vaccination group, adjusted for vaccination history and 
baseline titre. 
2. Difference in log-titres between the QIV-R and QIV-E, adjusted for vaccination history and 
baseline titre. 
3. Difference in log-titres between the QIV-R and QIV-C, adjusted for vaccination history and 
baseline titre. 
6.2 
SECONDARY OBJECTIVES 
6.2.1 SECONDARY OBJECTIVE 1 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 13 of 38 
To estimate whether antibody responses to all 4 vaccine antigens are greatest in participants who 
received QIV-R and least in participants who received QIV-E, and if this reflects greater cell- to 
egg-grown antibody titre ratios.   
Corresponding outcomes: 
1. Post vaccination trend in GMT QIV-R > QIC-C > QIV-E 
2. Post-vaccination seropositivity and trend in seropositivity of QIV-R > QIC-C > QIV-E 
3. Pre- to post-vaccine mean fold-rise in GMT (~ Geometric Mean Ratio (GMR)) and trend in 
fold rise of QIV-R > QIC-C > QIV-E 
4. Post-vaccination seroconversion and trend in the proportion who have seroconverted of QIV-
R > QIC-C > QIV-E 
6.2.2 SECONDARY OBJECTIVE 2 
To estimate whether QIV-R induces antibodies against a greater genetic and antigenic range of 
A(H3N2) viruses than QIV-C and QIV-E.  
Corresponding outcomes: 
1. Breadth of antibodies induced by vaccination as measured through pre-vaccination, post-
vaccination and post-season serum samples tested against a landscape panel of 
approximately 30 influenza A(H3N2) strains that have circulated since 1968. 
6.2.3 SECONDARY OBJECTIVE 3 
To estimate whether prior vaccination history has less effect on antibody responses induced against 
all 4 vaccine antigens by QIV-R compared with QIV-C and QIV-E.  
Corresponding outcomes: 
For each vaccine type and vaccination history group (frequently versus infrequently vaccinated) 
compare: 
1. Post-vaccination GMT 
2. Post-vaccination seropositivity  
3. Post-vaccination fold-rise in geometric mean antibody titre (GMT)  
4. Post-vaccination seroconversion fraction  
5. Post-vaccination antibody breadth (proportion of landscape antigens with GMT>40) 
6.2.4 SECONDARY OBJECTIVE 4 
To estimate whether antibody titres against all 4 vaccine antigens are better maintained up to 12 
months after vaccination with QIV-R compared with QIV-C and QIV-E  
Corresponding outcomes: 
1. Post-season GMT  
2. Post-season seropositivity  
3. Proportion of landscape antigens (as per Objective 2) with GMT>40 post-season 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 14 of 38 
6.2.5  SECONDARY OBJECTIVE 5 
To estimate whether QIV-R induces higher frequencies of HA reactive B cells and/or B cells with 
different HA cross-reactivity profiles and phenotypes compared with QIV-C and QIV-E. 
Corresponding outcomes: 
1. Heterogeneity of antibody generating B cells in terms of phenotype and reactivity against a 
panel of well-defined recombinant hemagglutinin probes that represent a range of influenza 
A(H3N2) virus clades. 
6.2.6 SECONDARY OBJECTIVE 6 
To assess whether QIV-R offers better protection against infection compared with QIV-C or QIV-E. 
Corresponding outcomes: 
1. Attack rates in each vaccination group 
2. Post-vaccination GMT in infected versus uninfected participants for each vaccination group 
3. Characterisation of antibody and B cell responses 
 MONITORING OF CLINICAL SYMPTOMS AND SEQUENCING OF 
INFLUENZA STRAINS 
7.1 
STUDY DESIGN  
The proposed study is a single season randomized, modified investigator and participant blind study 
(participants & staff blinded except for clinical trial nurse) clinical trial of 360 adults aged 21-49 years 
with 3 arms comparing 3 vaccines. Randomisation will be stratified by vaccination history, frequently 
vaccinated vs. infrequently vaccinated, to compare the responses to each vaccine (Figure 1): 
Group A – frequently vaccinated: 
1. QIR-R 
2. QIV-E 
3. QIV-C 
Group B –infrequently vaccinated: 
4. QIR-R  
5. QIV-E 
6. QIV-C 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 15 of 38 
 
 
 
Infrequently 
vaccinated 
Frequently 
vaccinated 
Total 
QIV-R 
60 
60 
120 
QIV-E 
60 
60 
120 
QIV-C 
60 
60 
120 
Total 
180 
180 
360 
 
Figure 1. Randomization schema. QIV-E: egg-based quadrivalent influenza vaccine; QIV-C: cell-based quadrivalent influenza vaccine; 
QIV-R: recombinant quadrivalent influenza vaccine 
 
7.2 
JUSTIFICATION OF THE STUDY DESIGN 
The objectives of this study will be to assess whether vaccination with QIV-R results in a better 
immune response towards vaccination compared with either QIV-E or QIV-C.   
The vaccines QIV-C and QIV-R used in this study are not licensed in Singapore. However, given the 
acceptable safety data generated from the millions of doses of QIV-R administered in the USA (under 
the tradename Flublok Quadrivalent), and the fact that this product will be sourced from the US, we 
consider that the risk/benefit ratio is appropriate for the conduct of a clinical study with QIV-R.  
Some data already exist comparing QIV-R with QIV-E and QIV-C; however early formulations (pre-
2013) of QIV-R were susceptible to the formation of disulphide cross-links, resulting in decreased 
potency during storage (loss of up to 50% potency). This was rectified through the addition of low 
concentrations of a reducing agent, sodium thioglycolate (STG), in combination with citrate that 
reduced the rate of potency loss by up to 20-fold (15, 16). HA instability would have led to suboptimal 
vaccine being used in clinical trials pre-2014 and could explain the initial need for an increased 
antigen load (45ug / strain), it could also explain why earlier results failed to show any obvious 
superiority over traditional vaccines. Therefore, new data are required to assess the immunogenicity 
of this QIV-R over QIV-E and QIV-C.  
In addition, investigations to-date have largely only examined antibody titres against the vaccine 
antigen, and have mostly not considered vaccination history. We will investigate the whether the 
breadth of antibody responses varies among formulations and whether responses against each 
vaccine are similarly affected by prior vaccination. Prior studies, by ourselves and others, found that 
vaccine immunogenicity was clearly attenuated among participants who had been vaccinated during 
three or more of the preceding 5-years, not attenuated among participants who have one prior 
vaccination, and somewhat attenuated among participants with two prior vaccinations. Therefore, in 
this study, we will compare the immunogenicity of each vaccine among infrequently versus frequently 
vaccinated participants, defined as having 0-1 versus 3+ vaccinations during the preceding five-
years. Participants who have been vaccinated twice will be excluded. The number of prior years 
considered is a pragmatic choice made to minimize the number of years that participants are asked 
to recall their vaccination status, while still allowing participants to be stratified into groups that are 
anticipated to have different vaccine immunogenicity. Participants who would normally be vaccinated 
A) Highly 
vaccinated
1. QIV-R (n=60)
2. QIV-E (n=60)
3. QIV-C (n=60)
B) infrequently 
vaccinated
4. QIV-R (n=60)
5. QIV-E (n=60)
6. QIV-C (n=60)

 
 
 
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annually may have missed vaccinations during 2020-21 due to the COVID-19 pandemic, but would 
still be classified as highly vaccinated (3+) vaccines if vaccination status over 5-years is considered. 
Lower age limit for study participants will be 21 years per the age of majority in Singapore as provided 
by common law.   
 
7.3 
STUDY SCHEDULE 
Assessment/Procedure Screening 
Visit 1 (V1) 
(prior to 
vaccination) 
Visit 2 (v2) 
(14 days 
post 
vaccination) 
Visit 3 (V3) 
(6 months 
post 
vaccination) 
Visit 4 (V4) 
(12 months 
post 
vaccination) 
Study timeline 
Day 0 
Day 1 
Day 14 
Day 150 
Day 330 
Time window 
-28 to 1d 
NA 
+7d 
±30d 
±30d 
Informed Consent 
X 
 
 
 
 
Collection of 
demographic 
information 
X 
 
 
 
 
Weight and height 
measurement 
X 
 
 
 
 
History of seasonal 
influenza vaccination 
x 
 
 
 
 
Randomization 
x 
 
 
 
 
Blood Collection 
xa 
xa 
x 
x 
x 
Vaccination  
 
X 
 
 
 
Immediate surveillance 
(15 min) 
 
X 
 
 
 
ARI surveillanceb 
 
D14-D360 
Swab collectionb 
 
D14-D360 
Study termination 
record  
 
 
 
 
x 
Collection of SAEs and 
AESIsd 
Throughout study 
AESI = adverse event of special interest, SAE = serious adverse event; ARI: acute respiratory infection 
a. 
Collection of the first blood sample to occur before vaccination, and may be taken at Screening or Visit 1 if the 
blood draw is not possible for one reason or another. The study team will make every effort to complete all 
blood-taking in one seating to avoid pricking the participants more than once. 
b. 
Subjects will be contacted weekly by SMS to record information about acute respiratory infections and prompted 
to collected a nasal swab if symptomatic.  
c. 
AESIs will have the same detailed information collected as SAEs. These include new onset of Guillain-Barré 
syndrome, encephalitis/myelitis (including transverse myelitis), Bell’s palsy, optic neuritis, and brachial neuritis. 

 
 
 
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7.4 
VACCINATION  
Participants will be randomized as they are recruited to receive a single injection of one of the 3 
vaccines. QIV-R is currently only manufactured to contain influenza virus strains recommended for 
use in northern hemisphere Influenza Vaccines. Therefore, all vaccines used in this study (QIV-R, 
QIV-E, QIV-C) will be northern hemisphere vaccines. 
7.4.1 QIV-R (FLUBLOK®) BY IM ROUTE AT V1 (D0)  
FluBlok is manufactured by Sanofi-Aventis. In the United States, it is registered for use in people 
aged ≥18 years. Administration is by intramuscular injection. The vaccine antigens are manufactured 
synthetically and recombined with a baculovirus that infects insects. The recombinant virus is grown 
in bulk in a qualified insect cell line. Each 0.5mL monodose pre-filled syringe contains 45µg 
haemagglutinin of each of antigen.   
7.4.2 QIV-C (FLUCELVAX® QUAD) BY IM ROUTE AT V1 (D0): 
Flucelvax Quad is a registered vaccine manufactured by Seqirus for use in people aged ≥9 years. 
Administration is by intramuscular injection. The vaccine antigens are prepared in a qualified cell 
line. Each 0.5 mL monodose pre-filled syringe contains 15µg haemagglutinin of each of the 4 
recommended influenza virus strains.  Product advice also indicates it may contain traces of 
propiolactone, cetyltrimethylammonium bromide and polysorbate 80.  
7.4.3 QIV-E (FLUARIX) BY IM ROUTE AT V1 (D0) 
Fluarix is a registered vaccine manufactured by GlaxoSmithKline (GSK) in Germany for use in 
people aged ≥6 mths and older. Administration is by intramuscular injection. The vaccine antigens 
are prepared in a pathogen-specific free embryonated hens eggs. Each 0.5 mL monodose pre-filled 
syringe contains: 
• 
15 µg haemagglutinin of each of the 4 recommended influenza virus strains  
Other ingredients: 
• 
polysorbate 80 • octoxinol 10 • dl-alpha-tocopheryl acid succinate • sodium chloride • dibasic 
sodium phosphate dodecahydrate • monobasic potassium phosphate • potassium chloride • 
magnesium chloride hexahydrate • water for injections • ovalbumin (≤0.05 micrograms) • 
formaldehyde (≤5 micrograms) Traces of: hydrocortisone (trace) • gentamicin 
sulphate(trace) • sodium deoxycholate (trace) 
 
 

 
 
 
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7.4.4 VACCINE COMPOSITION 
Influenza antigens included in these vaccines will be:  
QIV-E 
QIV-C and QIV-R 
A/Victoria/2570/2019 (H1N1)pdm09-like virus  
A/Wisconsin/588/2019 (H1N1)pdm09-like virus  
A/Darwin/9/2021 (H3N2)-like virus 
A/Darwin/6/2021 (H3N2)-like virus 
B/Austria/1359417/2021 (B/Victoria lineage)-like 
virus  
B/Austria/1359417/2021 (B/Victoria lineage)-like 
virus  
B/Phuket/3073/2013 (B/Yamagata lineage)-like 
virus  
B/Phuket/3073/2013 (B/Yamagata lineage)-like 
virus  
7.4.5 SAFETY MONITORING 
Participants will be observed for 15 minutes after vaccination for adverse events (AEs), consistent 
with usual vaccination practices. Participants are asked to contact the project manager to report an 
AE / SAE throughout the study period. Additionally, participants will be asked if they have 
experienced an AE at each study visit.  See Section 12 for further information about the collection of 
AE data.  
 DATA COLLECTION 
8.1 
OVERVIEW OF DATA COLLECTION 
The data collected from participants includes: 
1. Baseline questionnaire 
2. Follow-up questionnaire 
3. Additional information – participant contact information 
4. Blood samples 
5. Respiratory swab samples 
6. Symptoms diaries 
 
Content 
Indicators 
Baseline 
questionnaire 
(Attachment 3) 
Demographics 
Year of birth, sex, co-morbid conditions, current 
medications, prior confirmed influenza or COVID-19 
infection 
Vaccination 
history 
Years vaccinated for seasonal influenza, dates and 
types of COVID-19 vaccines 

 
 
 
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Figure 3. Summary of data to be collected 
8.2 BASELINE QUESTIONNAIRE 
A baseline questionnaire will be used to collect information about the participant including 
demographics, height, weight, vaccination history, and relevant medical history (Attachment 3). The 
past 5-years’ vaccination history of the participant will be determined through self-report.  
Participants will provide their email and mobile phone number, to allow the study team to send weekly 
SMS reminders from day 14 post-vaccination.  
8.3 SMS REMINDERS 
Throughout the study, participants will be sent a weekly short message service (SMS) asking 
whether they have experienced an ARI (at least one acute respiratory symptom or sign and one 
systemic symptom) in the previous 7 days (Attachment 5). Responses will be recorded in the study 
database and used to calculate ARI risk in the cohort at the end of the study.  
Participants will also receive SMS reminders about any follow-up appointments. The use of SMS 
reminders has been shown to improve retention in research studies [16]. The SMS reminder will 
include their appointment time and the location. Participants failing to attend their appointment will 
be contacted by phone to reschedule the appointment.    
8.4 
BLOOD COLLECTION 
Participants will be requested to provide blood samples at 4 time-points: 
1. Pre-vaccination at Screening or V1 (D-28 to D1) 
2. Post-vaccination at V2 (D14 [+7 days]) 
3. Post-season at V3 (D150 [+/-30 days]) 
SMS reminders 
(Attachment 5) 
Symptoms diaries 
Weekly SMS to document any ARI events  
Biodata 
Blood sample 
Serology: HI assay to detect antibodies to most recent 
circulating strains of influenza, including the 2022 
influenza vaccine strains. Microneutralization assay 
may be performed for some A(H3N2) and B viruses if 
HI assay is found to have relatively poor sensitivity to 
detect titre rise. 
B-cell analysis: abundance and heterogeneity of 
memory B-cells induced by vaccination. 
Nasal swab 
RT-PCR to identify influenza A, B and other respiratory 
viruses. 

 
 
 
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4. Post-vaccination at V4 (D330+/-30 days), and prior to receiving the following years annual 
vaccination, 
Blood collection will take place at the study sites. At each visit blood samples will be taken from a 
vein in the arm. Up to 30mls of blood will be drawn. This will always include 10ml in serum tubes, 
and for 1/3 of participants (20/arm) will also include 20ml in Sodium Heparin tubes. The serum tubes 
will be processed at the IDRL at NCID and will consist of centrifuging serum tubes to separate sera 
from clotted blood. Sodium Heparin tubes will be processed at A*Star Infectious Disease labs with 
whole blood leukocyte subsets analysis on NA Heparin blood, followed by separation of plasma and 
peripheral blood mononuclear cells (PBMCs). Sera and plasma will be stored at -20°C and four 
aliquots of PBMCs per sample will be stored in liquid nitrogen. Stored samples will be shared 
between NCID and WHOCCRRI. 
8.5 
RESPIRATORY SAMPLES 
Participants will be provided with swab kits (swab plus vial of universal transport medium) at 
enrolment with instructions on how to use them (Attachment 6). During the 1-year follow-up period, 
if participants develop an ARI, they should perform a COVID-19 antigen rapid test (ART) and inform 
the study team within 24 hours of their ARI and COVID-19 ART result. Participants will be instructed 
to collect respiratory swab samples only if the COVID-19 ART is negative (Attachment 9). The study 
team will follow up with them to arrange for the respiratory swabs to be sent to the laboratory for 
influenza testing. Influenza-positive swabs will then be forwarded to the National Public Health 
Laboratory at NCID for virus characterization. Participants should store the respiratory samples in 
the home refrigerator until the study team collects them.  
 RANDOMISATION PROCEDURES 
9.1 
BLINDING AND CODE BREAKING PROCEDURES 
To ensure that objective data are obtained, the study is designed as a double-blind study: 
• 
The PI and the study staff who collect the AE data will not know which vaccine was 
administered; 
• 
The subjects will be blinded with an eye mask during administration; 
• 
The laboratory personnel who analyse the blood samples will not know which vaccine was 
administered; 
• 
The research fellow performing the randomisation will be unblinded and will be responsible 
for performing emergency unblinding. 
• 
The study pharmacist and nurse-vaccinator administering the vaccine will be unblinded.  
Prior to randomization a dose number will be generated, which will be used to identify each vaccine 
for the purpose of randomization, vaccination and the recording of the vaccine administered. Dose 
numbers will be randomly assigned to QIV-E, QIV-C and QIV-R.  
The code may be broken in the event of an AE only when the identification of the vaccine received 
could influence the treatment of the subject. Code-breaking should be limited to the subject(s) 
experiencing the AE. The blinding can be broken by the PI or a delegate. Once the emergency has 

 
 
 
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been addressed by the site, the PI will notify the HREC and the Sponsor if a subject’s code was 
broken.   
A request for the code to be broken may also be made by the HREC in the case of an SAE. In this 
case, the code will be broken only for the subject(s) in question. The information resulting from code-
breaking (i.e., the subject’s vaccine or group assignment) will not be communicated to either the PI 
or the immediate team working on the study. 
The HREC will be notified of the code-breaking. All documentation pertaining to the event will be 
retained in the site’s study records and in the Sponsor files. Any intentional or unintentional code-
breaking must be reported, documented, and explained, and the name of the person who requested 
it must be provided to the Sponsor. 
9.2 
RANDOMISATION AND ALLOCATION PROCEDURES 
Randomisation to vaccine group is stratified based on history of vaccination. Consenting participants 
will be identified as being in either the infrequently vaccinated group (0 or 1 prior vaccinations) or the 
frequently vaccinated group (3 or more prior vaccinations) and subsequently randomized within each 
stratum (see Figure 1) 
A computer-generated randomization list will be used for labelling and packaging of doses.  
Within each vaccination history stratum (frequently and infrequently vaccinated), subjects who meet 
the inclusion/exclusion criteria and provide informed consent will be randomly assigned to one of the 
3 vaccine groups (QIV-R, QIV-E or QIV-C) in a 1:1:1 ratio. A second round of randomization will be 
used to assign participants in each of the 6 groups for collection of additional blood tubes in a ratio 
of 1:2 (1/3) until a target of 20 per group. 
Site staff will enter the identification information into REDCap and confirm a minimal amount of data. 
The unblinded research fellow will perform the randomisation on REDCap. Both the unblinded 
research fellow and vaccination nurse will  have access to the randomisation results on REDCap. 
Before administering the vaccine, the nurse vaccinator will check the vaccine group allocated to the 
participant by referring to the REDCap randomisation results and have the site administrator confirm 
it. The full detailed procedures for group allocation will be described in the manual of procedures.  
If a subject is not eligible to participate in the study, then the information will only be recorded on the 
screening/enrolment log. 
Subject numbers that are assigned by REDCap will be unique for each screened potential 
participant. Subject numbers will not be reassigned for any reason. The randomization codes will be 
stored securely in REDCap. 
9.3 
TREATMENT COMPLIANCE 
Compliance is only measured at the time of vaccination. Any non-compliance will be documented so 
that it can be accounted for in the data analyses. To ensure compliance: 
• 
All vaccinations will be administered by a qualified nurse-vaccinator; 

 
 
 
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• 
The project manager will maintain accountability records of product delivery to the study site, 
product inventory at the site, dose given to each subject, and the disposal of unused or 
wasted doses. 
 LABORATORY PROCEDURES  
10.1 
SERO-RESPONSE TO VACCINATION 
Pre-vaccination, post-vaccination and post-season serum samples will be tested for antibodies to 
the 4 vaccine strains in QIV-R, QIV-C and QIV-E vaccines. We will primarily use haemagglutination 
inhibition (HI) assays to measure antibody titres against the 4 vaccine antigens. This assay 
measures the ability of antibodies in the blood to prevent haemagglutination—the attachment of 
influenza virus particles to red blood cells. Samples are serially diluted (titrated) and the highest 
dilution of serum that prevents haemagglutination is the HI titre. Reciprocal titres of 40 or higher are 
generally accepted as indicating protection against infection (“seropositivity”), with very high titres 
suggestive of recent infection (17). The HI assay is the standard assay used to measure antibody 
response to both infection and vaccination, and to assess the sensitivity of circulating influenza 
viruses to the vaccine.  
For some A(H3N2) and B viruses, we will also use a microneutralisation (MN) to assess antibody 
responses to vaccination. This is a functional assay that measures the ability of antibodies to 
neutralize virus infectivity. Serum is mixed with virus and residual infectivity of the virus is assessed 
by adding the mixture to cells. The highest dilution of serum that neutralizes virus infectivity is the 
MN titre. Concordance between the HI and MN is high (18). 
10.2 
INFLUENZA TESTING AND VIRUS CHARACTERIZATION 
Respiratory swabs collected from participants reporting ARI symptoms will be tested using reverse 
transcription real-time polymerase chain reaction (RT-PCR), according to standard operating 
procedures in each hospital laboratory. Influenza-positive samples will be forwarded to the National 
Public Health Laboratory for virus characterization.  The virus subtype (for influenza A) or lineage 
(for influenza B) will be identified. Viruses will be isolated and tested by HI/MN or similar assay to 
assess antigenic match to vaccine, and sequenced to assess genetic match to the vaccine and to 
identify any genetic clusters. 
10.3 
ANTIBODY LANDSCAPES 
We will build antibody landscapes (19) by testing sera in HI assay (as described in 8.1) against a 
range of influenza A antigens.  Landscapes will consist of a panel of approximately 30 influenza 
A(H3N2) viruses that have circulated since 1968. Panels will include currently-circulating strains and 
candidate vaccine viruses to assess “future” protection, and will probably include 3-4 viruses from 
each antigenic cluster.  
10.4 
ANTIBODY ANALYSIS  
Antibody analysis: 
HI, MN, competitive antibody binding assays will be used to investigate differences in conformational 
vs non-conformational epitope recognition and stem / cleavage site reactivity. A panel of well-defined 

 
 
 
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monoclonal antibodies that span key antigenic sites will be used to investigate differences in antibody 
response to the vaccines. Any key differences between vaccines to be confirmed with genetically 
modified antigen. Any differences in stem / cleavage site reactivity to be further investigated using 
fusion inhibition assays. 
ADCC assays will be used to investigate differences in non-neutralising antibody functionality.   
10.5 
B CELL ANALYSIS 
PBMC will be thawed and stained with up to four fluorescent labelled recombinant HA probes 
representing the vaccine strain and prior A(H3N2) vaccine strains, together with monoclonal 
antibodies against B cell activation and differentiation markers and isotypes (IgG, IgG3, IgM, IgA, 
IgD). Stained samples will be analysed on a five laser Cytek Aurora. The magnitude of total HA-
reactive B cell response and HA cross-reactivity profiles will be compared.  
 STUDY POPULATION 
11.1 
 INCLUSION CRITERIA  
Eligible participants will be recruited from staff members of NCID, TTSH and the general public by 
hospital/University newsletters, social media posts/advertising, emails and the study website, as 
permitted. Participants will meet the following criteria: 
• 
Aged ≥21 years and ≤ 49 years. 
• 
Able to provide informed consent 
• 
Willing and able to provide 4 blood samples:  
a. just prior to vaccination (V1),  
b. 14–21 days post vaccination (V2) 
c. 4–6 months post-vaccination (V3) 
d. 10-12 months post-vaccination (V4). 
• 
Has not received influenza vaccine for at least 6 months1  
• 
Willing to provide current mobile phone number for SMS reminders 
 
1 To maximise the recruitment of healthcare workers before the year-end flu vaccination exercise (Oct-Dec period), 
participants who have received their influenza vaccine less than 6 months ago may be enrolled but their vaccination visit 
and blood sample collection will be scheduled at least 6 months after their last influenza vaccine dose. 
 
11.2 EXCLUSION CRITERIA  
• 
Known contraindication(s) for QIV (e.g. hypersensitivity to vaccine component (including 
eggs)). 
• 
Recently (last 7 days) or currently ill or has a fever above 38°C. 
• 
Cannot recall if they were vaccinated against influenza during more or less than two of the 
preceding five years. Vaccinated during two of the preceding five years. 
• 
Hypogammaglobulinaemia on immunoglobulin replacement 
• 
Undergoing immuno-suppressive therapies including corticosteroids2 

 
 
 
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2 Received systemic immunosuppressants or immune-modifying drugs for >14 days in total within 6 months prior to 
Screening (for corticosteroids >/= 20 milligram per day of prednisone equivalent). Topical tacrolimus is allowed if not used 
within 14 days prior to Day 1. Individuals who are immunocompromised (e.g. active leukemia or lymphoma, generalised 
malignancy, aplastic anaemia, solid organ transplant, bone marrow transplant, current radiation therapy congenital 
immunodeficiency, HIV/AIDS with CD4 lymphocyte count < 200). 
 
11.3 
RECRUITMENT PROCEDURES 
The study will seek to recruit hospital staff working at NCID, TTSH and the general public. It will be 
promoted via poster advertisements, as well as sharing of digital copies of the poster via emails and 
on NCID/TTSH and/or A*STAR websites (Attachment 1 and 2). Participants who are interested in 
participating in the study may directly contact the study team via the contact details provided on the 
advertising poster.  
A study team member will go through the eligibility criteria and informed consent procedures. 
Baseline data collection and blood draws will take place in a private location located in or adjacent 
to the clinic. To facilitate recruitment, the baseline blood draw may occur up to four weeks prior to 
vaccination. 
The recruitment of infrequently vaccinated people will be reinforced by using the “snowball sampling” 
strategy in which existing study participants can refer other potential participants from among their 
acquaintances. 
Participants will be reimbursed 50$ at each visit for their time and travel costs. 
11.3.1 
ASSIGNING PARTICIPANT IDS 
At baseline, participants will be requested to provide their name, surname and their date of birth to 
enable linkage to clinical records. Once the necessary data has been abstracted, identifying 
information, including name and date of birth, will be removed from the working data file and records 
will only be identifiable by their participant ID. A master list linking participant IDs and clinical records 
will be stored by the PI.       
11.4 
RETENTION STRATEGIES 
To retain participants during the study period, we will use the following strategies:  
1. 
Weekly reminders through SMS or email to report ARI symptoms;  
2. 
MS Outlook calendar invites and SMS reminders for scheduled follow-up appointments;  
3. 
Provide prompt feedback about the results of influenza testing. 
11.5 
FOLLOW-UP 
At enrolment, participants will be requested to provide a mobile phone number and email address. 
Follow-up appointment reminders will be sent via email and/or SMS.   
In addition, participants will be given a participant ID card at enrolment (Attachment 8). This is so 
that participants will have at hand their participant ID, the date of influenza vaccination (specific to 
them) and contact numbers for questions/concerns.  
 

 
 
 
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11.6 
INFORMED CONSENT  
A copy of the participant information and consent form (ICF; Attachment 4) will be provided to all 
participants and written informed consent will be sought from all participants prior to enrolment in the 
study. Participation in the study will be voluntary. Participants will be informed that they are free to 
withdraw from the study at any time. The concept of voluntary participation will be clearly explained, 
and ample time will be given to ensure that participants understand the content and have all of their 
questions answered. Willing participants will sign the informed consent form to indicate assurance 
of their understanding and voluntary participation in the research. Informed consent will be 
administered by the PI or delegated Co-I who will also sign their name. A copy of the signed and 
dated consent form will be given to the participant. This process will be documented in the 
participant’s study file. Personal information will not be released without written permission of the 
participant, except as necessary for monitoring by the Human Research Ethics Committee. The 
project manager and site PI’s contact details will be made available to participants should they have 
any queries or concerns during the study. Signed informed consent forms will be stored in the study 
office in a locked filing cabinet. 
 PARTICIPANT SAFETY AND WITHDRAWAL  
12.1 
POTENTIAL RISKS TO PARTICIPANTS 
Study investigators and institutions are committed to protecting personal health information through 
the maintenance of privacy and security of each subject’s personal information in this study. To 
protect confidentiality, we will use a study assigned number instead of personal information on study 
forms and we will store data in locked files and/or secured computers. If information from this study 
is presented publicly or published in a medical journal, results will be presented using aggregate 
statistics; individuals will not be identified by name or by any other personally identifiable information. 
Only specific and authorized researchers (e.g. project manager) in this study may see personal 
health information but will not disclose personally identifying information about individual participants 
to others. A participant’s general practitioner will be notified the participant is enrolled in the study.  
12.1.1 
POSSIBLE REACTIONS TO BLOOD DRAW 
There are no major risks associated with giving blood. Having a blood sample taken may cause 
some discomfort when the needle is inserted, and some people experience bruising, minor infection 
or bleeding. Blood will be collected by trained healthcare staff using a butterfly and small gauge 
needle. Any complications related to blood sample collection will be reported to the project manager, 
and investigated by study staff. 
12.1.2 
POSSIBLE REACTIONS TO VACCINATION 
Vaccine injection into the deltoid muscle may cause transient discomfort. Immediate and potentially 
life-threatening allergic reactions to the vaccine could be manifested by adverse events (AEs) such 
as laryngeal edema, asthma, or hypotension. These types of reactions are exceedingly rare and 
would most likely occur in persons with a severe reaction to influenza vaccine in the past. Therefore, 
people with known contraindications, including prior allergic reaction to vaccine, will be excluded 
from the study. All participants will be required to wait for 15 minutes after vaccination to document 
any AEs, including AEs of special interest (AESIs), arising during this period.  

 
 
 
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12.1.3 
POSSIBLE REACTIONS TO RESPIRATORY SWAB COLLECTION 
Collection of respiratory swabs can be associated with some pain and discomfort, especially if the 
nostril is already sore or irritated due to illness. Clear instructions will be provided to participants on 
how to self-collect the swab to minimise discomfort. Any complications related to respiratory swab 
collection should be reported to the project manager, and will be investigated by study staff.   
12.2 
RISK MANAGEMENT AND SAFETY 
12.2.1 
DEFINITIONS 
Adverse Event (AE): Any untoward medical occurrence in a participant enrolled into this study 
regardless of its causal relationship to study treatment. An adverse event can therefore be any 
unfavourable and unintended sign, symptom, or disease temporally associated with the use of a 
medicinal (investigational) product, whether or not related to the medicinal (investigational) product. 
Adverse events are classified as serious or non-serious. 
Serious Adverse Event (SAE): An SAE is defined as any event that: 
• 
results in death; or 
• 
is immediately life threatening; (the term “life-threatening” refers to an event/reaction in 
which the participant was at risk of death at the time of the event/reaction; it does not refer 
to an event/reaction which hypothetically might have caused death if it were more severe); 
or 
• 
requires inpatient hospitalisation or results in prolongation of existing hospitalisation; or 
• 
results in persistent or significant disability/incapacity; or 
• 
is a congenital anomaly/birth defect; or 
• 
is a medically important event or reaction.  or 
• 
requires inpatient hospitalisation; or 
• 
results in persistent or significant disability/incapacity. 
Important medical events that might not be immediately life-threatening or result in death or 
hospitalisation may be considered an SAE when, based upon appropriate medical judgement, they 
may jeopardize the participant and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition. 
New Safety Finding: means any (other than reportable individual case safety report (ICSR)) safety 
issue that may require expedited reporting because providing information that may lead to a change 
in the known risk-benefit balance for the product and as mentioned, but not limited to, in the following 
regulatory texts: Europe: Good Pharmacovigilance Practices, modules VI and VII; and US: FDA: 21 
CFR Parts 312 Investigational New Drug Application- Section 312.32, (c) (1) IND safety reports. 
Adverse Event of Special Interest (“AESI”) means an adverse event (serious or non-serious) of 
scientific and medical concern specific to the product or program, for which ongoing monitoring and 
rapid communication by the Investigator may be appropriate.  Such events may require further 
investigation in order to characterise and understand them.  AESIs may be added or removed during 
a Study by protocol amendment.   
 

 
 
 
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12.2.2 
ELICITING ADVERSE EVENT INFORMATION  
Adverse events will be recorded from the time the participant signs the Informed Consent Form until 
14 days after each visit. 
12.2.3 
ASSESSMENT AND DOCUMENTATION OF ADVERSE EVENTS  
The project manager is responsible for recording all adverse events, regardless of their relationship 
with the exposure, with the following exceptions: 
• 
Conditions that are present at screening and do not deteriorate will not be considered 
adverse events. 
• 
Abnormal laboratory values will not be considered adverse events unless deemed 
clinically significant by the investigator and documented as such. 
Adverse events will be recorded using the Adverse Events Report Form, which will record: 
• 
A description of the AE; 
• 
The onset date, duration, date of resolution; 
• 
Severity (mild, moderate or severe); 
• 
Seriousness (i.e. is it an SAE?) 
• 
Any action taken (e.g. treatment, follow-up tests); 
• 
The outcome (e.g. recovery, death, continuing, worsening); 
• 
The likelihood of the relationship of the AE to the exposure being investigated (e.g. 
Unrelated, Possible, Probable, Definite). 
• 
Whether and when reported to the HREC 
All AEs will be followed to adequate resolution. 
12.2.4 
SERIOUS ADVERSE EVENT REPORTING  
Any SAE occurring in a study participant will be reported to study site. The SAE reporting form will 
be completed, signed and submitting by an investigator. All SAE reporting forms will be reported to 
the HREC by email to HrecCorrespondence@mh.org.au in line with the Melbourne Health Research 
policy.  
All SAEs and AESIs, pregnancy and overdose, regardless of the investigator’s causality must be 
reported to Sanofi within one (1) business day of becoming aware of the event. 
Sanofi Global Pharmacovigilance 
Email: PV.outsourcing@sanofi.com or Fax: to +33 (0) 4 37 37 71 32 
 
And copy in the Australian Sanofi Product Safety Department: 
Email: ae@sanofi.com 
All SAEs should be submitted to the appropriate Regulatory Authority (TGA) in accordance with the 
applicable timelines and reporting requirements.  
12.3 
HANDLING OF WITHDRAWALS  
Participants may withdraw from the study at any time for any of the following reasons: participant 
has chosen to withdraw from the study, protocol violation, or participant has experienced an adverse 
event. 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 28 of 38 
Information collected to that point will be kept unless the participant explicitly requests otherwise.  
This information will be used for comparison with participants who complete all study procedures. 
All participants will be included in the study analyses, so it is important to have information on as 
many participants as possible.  If a participant wishes to withdraw from the study, every reasonable 
effort will be made to complete a final evaluation of participants who exit the study early and the 
reason(s) for withdrawals will be recorded in the participant’s study records.  
12.4 
REPLACEMENTS 
The sample recruited allows for some attrition. However, we will attempt to replace participants who 
fail to attend their first follow-up (V2). 
12.5 
POTENTIAL BENEFITS TO SUBJECTS 
All participants will receive a quadrivalent influenza vaccination. Participants should benefit from 
coverage against influenza and may be less likely to catch influenza or develop complications during 
the study period.  
Participants will be vaccinated against the influenza A/H1N1, A/H3N2 and the B strains from both 
the Victoria and Yamagata lineages chosen and recommended by WHO for the composition of QIVs 
for the 2022–2023 NH season. 
Regarding immunogenicity, QIV-R is expected to induce a higher immune response against the 4 
influenza strains compared to QIV-C or QIV-E. Therefore, QIV-R is likely to bring an increased 
benefit versus QIV-C or QIV-E in terms of immunogenicity against the 4 influenza strains with a risk-
benefit profile that is expected to be favourable. 
 
 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 29 of 38 
 STATISTICAL METHODS 
13.1 
RANDOMISATION 
Subjects will be stratified based on history of vaccination and randomised into the three vaccine 
groups using a block-randomisation scheme. 
13.2 
SAMPLE SIZE ESTIMATION & JUSTIFICATION   
The sample size is calculated considering the primary objective to test whether QIV-R offers 
superior immunogenicity independent of vaccination history. Our estimates for post-vaccination HI 
titres are based on a study among elderly adults in Hong Kong, comparing QIV-R and QIV-E (20) 
and is similar to the estimated sample size based on similar HI titres in a study of 18-49 year olds 
(21). 
A sample was simulated under the following model, informed by the published estimates:  
𝑡𝑖𝑡𝑟𝑒𝑡=2 = 𝑡𝑖𝑡𝑟𝑒𝑡=1 + 𝑣𝑎𝑥𝑅+ 𝑣𝑎𝑥𝐶+ 𝑝𝑟𝑖𝑜𝑟𝑣𝑎𝑥+ 𝑣𝑎𝑥𝑅× 𝑝𝑟𝑖𝑜𝑟𝑣𝑎𝑥+ 𝑣𝑎𝑥𝐶× 𝑝𝑟𝑖𝑜𝑟𝑣𝑎𝑥 
(titret=2 = post vaccination log titre; titret=1 =  post-vaccination log titre for QIV-E infrequent vaccination; 
vaxR = QIV-R vaccinated; vaxC = QIV-C vaccinated) 
For the test of a superior A(H3N2) response among vaccinees receiving QIV-R versus QIV-E or 
QIV-C (one-sided test for the parameter vaxrecomb) and assuming α=0.05, β=0.2, the sample 
needed to test these effects is around 55 per arm per vaccination history. Accounting for 10% 
attrition, the target sample for recruitment will be 60 per arm per vaccination history.  
 
Infrequently vaccinated 
Frequently vaccinated Total 
QIV-R 
60 
60 
120 
QIV-E 
60 
60 
120 
QIV-C 
60 
60 
120 
Total 
180 
180 
360 
 
13.3 
POWER CALCULATIONS  
Power calculations were simulation-based. The following model was used for the simulations: 
logtitret=2 = b0 + bbaseline logtitret=1 + bpriorvax priorvax + bC vaxC + bR vaxR + bC-add vaxC priorvax + bR-add vaxR 
priorvax + e 
The following table contains the interpretation of all model terms. 
Term 
Interpretation 
logtitret=2 
Post-vaccination log-titre. 
b0 
Expected post-vaccination log-titre for baseline log-titre 0, infrequent prior 
vaccination, vaccinated with QIV-E. 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 30 of 38 
bbaseline 
Expected increase in the post-vaccination log-titre for a 1-unit increase in the pre-
vaccination log-titre. Adjusted for prior vaccination frequency and vaccine 
received. 
logtitret=1 
Pre-vaccination log-titre. 
bpriorvax 
Expected difference in the post-vaccination log-titre between the frequently and 
infrequently vaccinated. Adjusted for baseline titre. 
priorvax 
0=Infrequent prior vaccinations. 1=Frequent prior vaccinations and vaccine 
received. 
bC 
Expected difference in post-vaccination log-titre between QIV-E and QIV-C. 
Adjusted for baseline titre and prior vaccination frequency. 
vaxC 
0=Not vaccinated with QIV-C. 1=Vaccinated with QIV-C. 
bR 
Expected difference in post-vaccination log-titre between QIV-E and QIV-R 
vaccine. Adjusted for baseline titre and prior vaccination frequency. 
vaxR 
0=Not vaccinated with QIV-R. 1=Vaccinated with QIV-R. 
bC-add 
Expected additional effect of QIV-C in the frequently vaccinated. 
bR-add 
Expected additional effect of QIV-R in the frequently vaccinated. 
e 
Random error 
 
The following table contains the values used for the parameters in the model (subsequent tables will 
show deviations if there are any). 
Term 
Value 
b0 
1.1 
bbaseline 
0.8 
bpriorvax 
-0.2 
bC 
0.25 
bR 
0.5 
bC-add 
0 
bR-add 
0.2 
 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 31 of 38 
In each simulation there was an equal number of subjects in each arm and vaccination history group. 
Baseline log-titres were simulated from N(3.7, 1) which produced a distribution representative of the 
baseline titre data of published studies. The error term was sampled from N(0, 1) which produces 
post-vaccination titre distribution representative of published studies. 
The model simulates a subject’s post-vaccination titre given their pre-vaccination titre, prior 
vaccination status (frequent or infrequent) and the vaccine they were given (QIV-R, QIV-C, QIV-E). 
The effect of the vaccines was allowed to vary between frequently and infrequently vaccinated. 
Simulations were performed with different per-group sample sizes and different effects of QIV-R (bR). 
For the purposes of fitting the model to the simulated data, all titres were censored to the appropriate 
interval (<10, 10-20, 20-40 up to 5260-10240 on the original titre scale) to simulate real 
measurements. The model was then fit using the midpoints of the intervals except <10 which was 
censored to 5 (as it would be for real data). 
There were 5000 simulations for each sample size and value of bR. Each simulated dataset was 
used to fit a model (the same one that was used to simulate it). The primary outcome measure was 
taken to be the effect of the QIV-R as compared to the QIV-E (i.e., the expected difference in post-
vaccination log-titres between the QIV-R and QIV-E,) which corresponds to the bR parameter in the 
model. A one-sided and a two-sided test was performed on the bR estimate for each simulation. The 
“power” is the proportion of simulated studies that showed that bR is significantly greater than 0. The 
results are in the table below. 
Per-arm 
per-
vaccine history 
sample size 
True 
bR 
(expected 
ratio between QIV-R 
and QIV-E) 
Proportion 
with 
significant (one-sided) 
positive bR 
Proportion 
with 
significant 
(two-sided) positive bR 
50 
0.5 (1.65) 
0.772 
0.665 
55 
0.5 (1.65) 
0.810 
0.712 
60 
0.5 (1.65) 
0.832 
0.741 
70 
0.5 (1.65) 
0.889 
0.807 
80 
0.5 (1.65) 
0.920 
0.858 
200 
0.25 (1.28) 
0.775 
0.676 
220 
0.25 (1.28) 
0.810 
0.717 
240 
0.25 (1.28) 
0.834 
0.742 
Power of 80% can be achieved (for a one-sided test) with approximately 55 subjects per arm/per 
vaccine history given the true effect of 0.5 (true expected ratio 1.65). The total sample required would 
be approximately 330 subjects. This equates to 74% power for a two-sided test. 
 
13.4 
STATISTICAL ANALYSES            

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 32 of 38 
The stratification variable of vaccination history will be included as a covariate in models for all 
primary and secondary objectives.  
13.4.1 PRIMARY OBJECTIVE 
To assess whether vaccination with recombinant quadrivalent influenza vaccine (QIV-R) results in a 
better antibody response against A(H3N2) viruses than either standard egg-grown vaccine (QIV-E) 
or cell-based vaccine (QIV-C) as measured by post-vaccination geometric mean titre from the 
haemagglutination inhibition assay 
Post-vaccination GMTs will be calculated for each group using the model described in 13.3, in which 
the outcome is an individual’s post-vaccination titre, estimated given their pre-vaccination titre, prior 
vaccination status (frequent or infrequent) and the vaccine they were given (QIV-R, QIV-C, QIV-E). 
A t-test (on a model parameter) will be used to assess statistical significance of the effects of QIV-R 
versus QIV-E and QIV-C. 
13.4.2 SECONDARY OBJECTIVE 1 
To estimate whether antibody responses to all 4 vaccine antigens are greatest in participants who 
received QIV-R and least in participants who received QIV-E, and if this reflects greater cell- to 
egg-grown antibody titre ratios.   
The post-vaccination vs pre-vaccination log-titre difference (geometric mean ratio) will be 
compared using linear regression with the explanatory variable being the type of vaccine (reference 
QIV-E, separate parameter for QIV-R and QIC-C). A t-test will be used to assess statistical 
significance of the effects of QIV-R and QIC-C (as compared to reference) as well as the difference 
in those effects. 
The post-vaccination seropositivity (coded as 1 if the post-vaccination titre is at least 40, 0 
otherwise) will be compared using logistic regression with the explanatory variable being the type of 
vaccine (reference QIV-E, separate parameter for QIV-R and QIC-C). A t-test will be used to assess 
statistical significance of the effects of QIV-R and QIC-C (as compared to reference) as well as the 
difference in those effects. 
Seroconversion (coded as 1 if the post/pre vaccination titre ratio is at least 4, 0 otherwise) will be 
compared using logistic regression with the explanatory variable being the type of vaccine (reference 
QIV-E, separate parameter for QIV-R and QIC-C). A t-test will be used to assess statistical 
significance of the effects of QIV-R and QIC-C (as compared to reference) as well as the difference 
in those effects. 
The trend will be determined using the estimate of the effect of the cell vaccine and the estimate of 
the effect of the recombinant vaccine for each of these outcomes. The expected trend is the cell 
vaccine producing better outcomes than the egg vaccine (estimate of the effect of the cell vaccine 
should positive) and the recombinant vaccine producing better outcomes than the cell vaccine (the 
difference of estimates of the effect of recombinant and cell vaccines should be positive). 
13.4.3 
SECONDARY OBJECTIVE 2 
To estimate whether QIV-R induces antibodies against a greater genetic and antigenic range of 
A(H3N2) viruses than QIV-C and QIV-E.  

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 33 of 38 
The breadth of antibodies induced by vaccination will be measured using pre-vaccination and post-
vaccination serum samples tested against a landscape panel of approximately 30 influenza 
A(H3N2) strains that have circulated since 1968. A metric for response breadth (e.g., the 
proportion of viruses with post/pre vaccination titre ratio of at least 4) will be calculated for each 
individual and compared using linear regression as for secondary objective 1. 
13.4.4 
SECONDARY OBJECTIVE 3 
To estimate whether prior vaccination history has less effect on antibody responses induced against 
all 4 vaccine antigens by QIV-R compared with QIV-C and QIV-E.  
All analyses for objective 1 and 2 will be repeated including a covariate to indicate the vaccination 
history group (0=infrequently vaccination; 1=frequently vaccinated). An interaction term between 
vaccine received and prior vaccination will be included to allow vaccine effect to vary by history of 
vaccination. Infrequently vaccinated is defined as 0 or 1 prior vaccinations; frequently vaccinated is 
defined as 3 or more prior vaccinations with any influenza vaccine.  
13.4.5 
SECONDARY OBJECTIVE 4 
To estimate whether antibody titres against all 4 vaccine antigens are better maintained up to 
12months after vaccination with QIV-R compared with QIV-C and QIV-E  
Post-season log-titres and seropositivity will be compared the same way post-vaccination log-titres 
and seropositivity are compared in objective 1.  
Post-season response breadth will be using the same methods described for objective 2. 
13.4.6 
SECONDARY OBJECTIVE 5 
To estimate whether QIV-R induces higher frequencies of HA reactive B cells and/or B cells with 
different HA cross-reactivity profiles and phenotypes compared with QIV-C and QIV-E. 
The percentage of B memory phenotype (CD27+ and/or IgD-) cells that are HA probe reactive on 
d14 will be estimated for each vaccine arm, accounting for pre-vaccination percentage and 
vaccination history as described in 13.3. The HA probe cross-reactivity, isotype, and phenotype of 
HA reactive B cells for each vaccine group and time-point will be visually compared using t-
distributed stochastic neighbor embedding (t-SNE), a statistical method for visualizing high-
dimensional data by giving each datapoint a location in a two or three-dimensional map. 
13.4.7 
SECONDARY OBJECTIVE 6 
To assess whether QIV-R offers better protection against infection compared with QIV-C or QIV-E. 
Evidence of influenza infection will be based on RT-PCR-confirmed infection, only, as serological 
evidence may be biased in vaccinees who elicit a good antibody response to vaccination (22).  
Attack rates will be calculated for each vaccination group as the number of cases during the person-
time at risk.  If there are sufficient cases, the proportion testing positive for each vaccine will be 
compared using logistic regression with influenza infection as 1 if test-positive and 0 if test-negative 
or not tested and no ARI. The explanatory variable with be the type of vaccine (1=QIV-R, 2=QIC-C, 
3=QIV-E). Samples size permitting, we will also explore whether attack rates were associated with 
the post-vaccination GMT.  

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 34 of 38 
 STORAGE OF BLOOD AND TISSUE SAMPLES 
14.1.1 
DETAILS OF WHERE SAMPLES WILL BE STORED, AND THE TYPE OF CONSENT 
FOR FUTURE USE OF SAMPLES 
14.1.1.1 
Serum samples storage and shipping 
Blood samples collected in serum tubes will be centrifuged within 24 hours of collection to separate 
the clotted blood from the serum and the serum removed to a clean tube. The clotted blood may be 
discarded. Sera will be aliquoted into separate tubes, the number of which will depend on the volume 
recovered but is expected to be around 3 aliquots of approximately 500-1000μl each. One serum 
aliquot (~1 ml will be shipped to WHOCCRRI for serology). One will be stored at -20°C, while the 
remaining aliquots will be stored in a -70°C freezer or in liquid nitrogen. All freezers are locked and 
located in secured areas.  
Written consent will also be obtained from participants for use of their samples for future studies. All 
participant information will be de-identified and a Human Research Ethics Committee would have to 
approve such future studies before stored samples can be analysed.  
14.1.1.2 
Peripheral Blood Mononucleocytes (PBMCs) storage and shipping 
Blood samples collected in Sodium Heparin tubes will be processed for recovery of PBMCs on the 
same day or within 24 hours of collection. The recovered cells will be aliquoted in to ~4 tubes. Each 
aliquot will be labelled and cryogenically stored in liquid nitrogen at the recruitment site until the end 
of each study year. The cryogenic storage facility is in secured area at A*Star Infectious Disease 
Labs. Half of the aliquots for each sample will be shipped to WHOCCRRI for B cell analysis. 
Respiratory swabs will be tested for influenza at the National Public Health Laboratory at NCID. 
Media from swabs that test positive for influenza virus will be aliquoted into two tubes and stored at 
-70C. One aliquot will be inoculated into established cell lines to obtain influenza isolates 
Influenza isolates will be shipped to the WHOCCRRI for antigenic and genetic testing. Isolates will 
be assessed using antigenic assays such as the HI assay (see 10.2). Both original specimens and 
isolates may be genetically sequenced (note that viral RNA will be sequenced, not human DNA). 
Both the original specimen and any isolates recovered will be stored in -70°C freezers, according to 
NATA-approved SOPs at the WHOCCRRI. All freezers are locked and located in secured areas. 
Samples will be stored indefinitely using study numbers.  
14.1.1.3 
Consent to storage of specimens for future related research 
Samples will be stored indefinitely and may be retested as new technologies for understanding 
immunological responses to respiratory virus infection and/or vaccination become available. During 
the informed consent process participants will be asked if their samples can be stored for future 
studies.   
 DATA SECURITY & HANDLING 
15.1 
DETAILS OF WHERE RECORDS WILL BE KEPT & HOW LONG WILL THEY BE 
STORED 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 35 of 38 
15.1.1 
STUDY DATABASE 
A study database will be established and managed using the REDCap electronic data capture tool 
developed by Vanderbilt University and hosted at NCID. REDCap provides detailed information 
about 
the 
security 
measures 
they 
have 
taken 
to 
secure 
data 
storage 
https://projectredcap.org/software/mobile-app/. Briefly, data are transmitted using secure, encrypted 
transmission (SSL/HTTPS). Additional validation checks are also used when communicating with 
the server. The REDCap Mobile App employs encryption-at-rest on the mobile device’s hard drive 
so that all important data and information stored on the device is properly protected from 
unauthorized or malicious users. 
If during the course of the study it is determined that other software may better meet the needs of 
the study, the team may migrate the study database to different software.  
15.1.2 
DATA ENTRY 
Data collected in questionnaire and generated from laboratory assays will be collected in paper form 
or electronically. Participants will be able to enter responses to the questionnaire directly using a 
mobile device. This will avoid the need for transcribing data from paper questionnaires and enable 
the use of validity checks to ensure that impossible or improbable entries are avoided. Paper 
questionnaires may also be used if there are technical issues at the study site. The project manager 
will enter the data into the study database.  
15.1.3 
DATA STORAGE 
Hard copies of any information collected (e.g. informed consent) will be stored in locked filing 
cabinets in the PI’s office. Electronic copies of data collected will be stored using the password 
protected study database.  
All research data will be stored for a minimum of 15 years after completion of the project.  After 
fifteen years electronic records will be erased unless further approval for retention is obtained. 
Individual participants will not be identifiable from the presented or published material. The PIs will 
be responsible for all data management. 
15.2 
CONFIDENTIALITY AND SECURITY  
Participant confidentiality is strictly held in trust by the participating investigators, research staff, and 
the sponsoring institution and their agents. This confidentiality is extended to cover testing of 
biological samples in addition to the clinical information relating to participants. The study protocol, 
documentation, data and all other information generated will be held in strict confidence. No 
information concerning the study, or the data will be released to any unauthorized third party, without 
prior written approval of the sponsoring institution. Authorized representatives of the sponsoring 
institution may inspect all documents and records required to be maintained by the PI, including but 
not limited to, medical records (office, clinic or hospital). The clinical study site will permit access to 
such records. All laboratory specimens, evaluation forms, reports and other records that leave the 
site will be identified by the participant ID number only to maintain participant confidentiality. Clinical 
information will not be released without written permission of the participant, except as necessary 
for monitoring by HREC or regulatory agencies.  

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 36 of 38 
 ATTACHMENTS 
 
Document Name 
Version 
Number 
Date  
1. Poster 
V1.0 
08 Jun 2022 
2. NCID poster 
V1.0 
05 Aug 2022 
3. CRF 
V4.0 
16 Sep 2022 
4. ICF 
V4.0 
27 Oct 2022 
5. Symptoms questionnaire 
V1.0 
15 Aug 2022 
6. Nasal swab instructions 
V1.0 
 10 Aug 2022 
7. Acknowledgement form  
V1.0 
15 Aug 2022 
8. Participant ID card 
V1.0 
06 Sep 2022 
9. ARI reporting guide 
V1.0 
28 Sep 2022 
 
 
 

 
 
 
Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
Page 37 of 38 
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Study Name: Immunogenicity trial of 3 influenza vaccine [VAP3] 
Protocol Number: 2021.247 
DSRB Ref Number: 2022/00275 
Version & date: version 2.0, dated 27 Oct 2022 
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