A Randomised, Double-blind, Placebo-controlled, Single-centre Phase IIb Trial as Part of the EU-funded UNISEC Project to Assess the Immunogenicity and Safety of Different Formulations and Dosing Regimens of FLU-v Vaccine in Healthy Adults
Phase: PHASE2
Conditions: Influenza
Sponsor: PepTcell Limited

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
0
THE INFORMATION CONTAINED HEREIN IS CONFIDENTIAL AND THE PROPRIETARY PROPERTY OF 
SEEK, UNIVERSITY OF GRONINGEN AND THE UNISEC CONSORTIUM. ANY UNAUTHORIZED USE OR 
DISCLOSURE OF SUCH INFORMATION WITHOUT THE PRIOR WRITTEN AUTHORIZATION OF THE 
OWNERS IS EXPRESSLY PROHIBITED 
 
 
 
 
 
A randomised, double-blind, placebo-controlled, single-centre phase IIb trial as part of 
the EU-funded UNISEC project to assess the immunogenicity and safety of different 
formulations and dosing regimens of FLU-v vaccine administered subcutaneously in 
healthy adults aged 18-60 years. 
 
Protocol version: 4.1 Dated 28 April 2017 
Sponsor code: FLU-v 003 
EudraCT number: 2015-001932-38 
 
Principal Investigator/centre   
Paul Groeneveld 
Isala Hospitals 
Isala Academy, department of Innovation and Science 
Dokter van Deenweg 1 
8052 BP Zwolle 
The Netherlands 
 
Sponsor 
PepTcell Limited (trading as SEEK) 
45 Beech Street 
London EC2Y 8AD 
UK 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
1
 
GCP Statement 
This study will be performed in compliance with Good Clinical Practice (GCP), the Declaration of Helsinki 
(with amendments) and local legal and regulatory requirements 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
2
1. Signatures and Agreement with Protocol 
Study title: A randomised, double-blind, placebo-controlled, single-centre phase IIb trial 
as part of the EU-funded UNISEC project to assess the immunogenicity and safety of 
different formulations and dosing regimens of FLU-v vaccine administered 
subcutaneously in healthy adults aged 18-60 years. 
 
We, the undersigned, agree to conduct this study according to this Study Protocol.  We 
agree that the trial will be carried out in accordance with Good Clinical Practice (GCP), 
with the Declaration of Helsinki (with amendments) and with the laws and regulations 
of the countries in which the study takes place. 
Sponsor’s 
 
Gregory Stoloff 
Representative 
CEO 
 
 
 
PepTcell Limited (trading as SEEK) 
 
 
 
45 Beech Street 
 
 
 
London EC2Y 8AD 
 
 
 
UK 
 
 
Date: _________      Signature: ______________________ 
 
Medical Monitor 
Bryan Murray, MBBS 
 
 
 
Boyd Consultants Ltd 
 
 
 
3a Station Cottages 
 
 
 
St Neots, Cambridgeshire 
 
 
 
PE19 1QF 
 
 
 
UK 
 
 
Date: ___________    Signature: _____________________ 
 
 
 
 
 
 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
3
Study title: A randomised, double-blind, placebo-controlled, single-centre phase IIb trial 
as part of the EU-funded UNISEC project to assess the immunogenicity and safety of 
different formulations and dosing regimens of FLU-v vaccine administered 
subcutaneously in healthy adults aged 18-60 years. 
 
I agree to conduct this study according to this Study Protocol.  I agree that the trial will 
be carried out in accordance with Good Clinical Practice (GCP), with the Declaration of 
Helsinki (with amendments) and with the laws and regulations of the countries in which 
the study takes place. 
Medical Monitor: 
Bryan Murray, MBBS 
Date: _________________ 
 
Signature: ________________________________________ 
 
Investigator Name: Paul Groeneveld 
Site Address: Isala Hospitals  
                          Isala Academy, department of Innovation and Science  
                          Dokter van Deenweg 1  
                          8052 BP Zwolle 
                          The Netherlands  
                          PO box 10400  
                          8000 GK Zwolle 
 
 
Date: _________________ 
 
Signature: ________________________________________ 
 
 
 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
4
 
2. Study Synopsis 
Title of the study: A randomised, double-blind, placebo-controlled, single-centre phase 
IIb trial as part of the EU-funded UNISEC project to assess the immunogenicity and 
safety of different formulations and dosing regimens of FLU-v vaccine administered 
subcutaneously in healthy adults aged 18-60 years. 
EudraCT number: 
2015-001932-38 
Protocol code: 
Sponsor: FLU-v 003 
Sponsor or sponsor’s representative in the European Union: 
PepTcell Limited (trading as SEEK), 45 Beech Street, London EC2Y 8AD, UK 
Investigators 
Paul Groeneveld 
Study Centre 
Isala Hospitals  
Isala Academy, department of Innovation and Science  
Dokter van Deenweg 1  
8052 BP Zwolle 
The Netherlands  
PO box 10400  
8000 GK Zwolle 
Planned study period: 1 Year 
Phase of Development: Phase IIb 
Objectives: To investigate the immunogenicity and safety of different formulations and 
dosing regimens of FLU-v in healthy adults. 
Study design: A randomised, double-blind, controlled single-centre study, 
Planned number of subjects: 222 
Medical condition or disease under investigation: Influenza 
Inclusion criteria 
- 
Healthy males or healthy non-pregnant females (as indicated by a negative 
blood pregnancy test done during the screening visit) between the ages of 18 
and 60 years, inclusive;  

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
5
- 
Women of childbearing potential (not surgically sterile or postmenopausal for 
greater than or equal to one year) and men must agree to practice 
appropriate contraception (a combination of barrier and hormonal methods 
for women and a condom for men) from screening and until at least 30 days 
(up to Study Day 51 for females) and 90 days (up to Study Day 111 for males), 
after the last vaccination.   
- 
A subject is in good health, as determined by a comprehensive clinical 
assessment {vital signs (heart rate, blood pressure, oral temperature)}, blood 
chemistry test (electrolytes, renal/kidney function, liver function, C-reactive 
protein, complete blood count), medical history, general physical 
examination, self-reported illness} and the clinical judgment of the 
investigator;  
- 
Able to understand and comply with planned study procedures; 
- 
Provides signed informed consent form  
Exclusion criteria 
- 
Has a known allergy to any of the components of the vaccine. 
- 
Has a history of severe reaction following immunization.  
- 
Persons with immune deficiency/disorder, whether due to genetic defect, 
immunodeficiency disease, or immunosuppressive therapy. 
- 
Women who have a positive pregnancy test during the screening visit or who 
are breastfeeding. 
- 
Has a history of any of the following (reported by subjects): 
o Acute disseminated encephalomyelitis (ADEM); 
o Neoplastic disease – current or previous; 
o Asthma or severe allergic disease; 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
6
o Bleeding disorders 
o Chronic Hepatitis B and/or C infection; 
o Chronic liver disease; 
o Diabetes mellitus; 
o Guillain-Barré syndrome; 
o HIV; 
o Rheumatoid arthritis or other autoimmune diseases; 
o Severe renal disease; 
o Transplant recipients; 
o Unstable or progressive neurological disorders. 
- 
Receipt of medicines/treatments that may affect evaluation of 
immunogenicity such as: 
o Oral or parenteral steroids, high-dose inhaled steroids (greater than 800 
micrograms/day of beclomethasone dipropionate or equivalent) or other 
immunosuppressive or cytotoxic drugs (azathioprine (Imuran), 
cyclosporine (Neoral, Sandimmune, SangCya); monoclonal antibodies such 
as basiliximab (Simulect), daclizumab (Zinbryta), infliximab (Remicade), 
rituximab (MabThera), alemtuzumab (Campath and Lemtrada), 
omalizumab (Xolair), abatacept (Orencia), adalimumab (Humira and 
Exemptia) and etanercept (Enbrel)basiliximab (Simulect), daclizumab 
(Zenapax), and muromonab (Orthoclone OKT3);  corticosteroids such as 
prednisone (Deltasone, Orasone); tacrolimus (Prograf, Advagraf, 
Protopic); Glatiramer acetate (Copaxone); Mycopehnolate (Cellcept); 
Sirolimus (Rapamune); (within 6 months of vaccination in this study) 
o Immunoglobulin or other blood products (plasma, blood cells, coagulation 
factors, haemoglobin)(within 3 months of vaccination in this study); 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
7
o An experimental agent (vaccine, drug, biologic, device, blood product, or 
medication) within 1 month of vaccination in this study, or expects to 
receive an experimental agent (during the study period). 
o Influenza antiviral medication (Amantadine (Symmetrel); Rimantadine 
(Flumadine); Zanamivir (Relenza), Oseltamivir (Tamiflu) (within 4 weeks 
of vaccination in this study). 
- 
Has received any influenza vaccine within 6 months of vaccination in this 
study. 
- 
Has influenza-like illness (a sudden onset of symptoms and at least one of the 
four systemic symptoms-fever or feverishness, malaise, headache, myalgia and 
at least one of the three respiratory symptoms-cough, sore throat, shortness of 
breath) or acute respiratory infection (a sudden onset of symptoms and at 
least one of the four respiratory symptoms-cough, sore throat, shortness of 
breath, coryza (Rhinitis) and a clinician’s judgement that the illness is due to 
an infection) within 6 months prior to vaccination in this study. These 
symptoms must have stopped the subject from carrying out their normal 
daily activities such as attending work or school for a period of at least 3 days. 
- 
Has an acute illness, including an oral temperature greater than 38 degrees 
Celsius, within 1 week of vaccination. 
- 
Has a history of alcohol or drug abuse within the last 2 years deemed 
unsuitable for inclusion by the investigator. 
- 
Any abnormal haematology values and/or serum chemistries judged by the 
Investigator as clinically significant.  
Being considered ineligible is based on the judgement of the investigator and in the 
event of uncertainty about the participant’s medical status regarding any of the 
exclusion criteria mentioned, the participant’s primary care physician will be consulted. 
Consultation of the primary care physician will only take place after having received 
written approval from the participant, and will concern medical information about 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
8
exclusion criteria only. 
Test product, dose and mode of administration: 
FLU-v vaccine in emulsion: 500 µg in WFI (water for injection, 0.25ml) and ISA-51 
adjuvant (0.25ml) on day zero. 
FLU-v vaccine in suspension: 500 µg in 0.01M HCl (0.25ml) and 0.01M NaOH (0.25 ml) 
on day zero and day 21.   
All doses administered by subcutaneous injection in the upper section of arm. 
Reference product, dose and mode of administration: 
Two reference products will be used. An emulsion (0.5ml) containing WFI (0.25ml) and 
ISA-51 (0.25ml) will be used as the reference product for FLU-v vaccine in emulsion. 
Saline (0.5ml) will be used as the reference product for FLU-v in suspension. Both 
products will be administered by subcutaneous injection in the upper section of arm. 
Duration of treatment: 21 days 
Criteria for evaluation: 
Immunogenicity 
Primary:  
To evaluate the cellular immune responses at 42 and 180 days as the change in level of 
TH1 cytokine production from baseline (day 0) following FLU-v vaccination, given as a 
suspension or as emulsion (adjuvanted), compared to placebo.   
Secondary:  
 To compare the effects of FLU-v with placebo, given as a suspension or emulsion 
(adjuvanted) on immune responses as measured using TH2 cytokines 
 To evaluate the antibody responses specific to FLU-v at 42 and 180 days from 
baseline following FLU-v vaccination. 
Exploratory: 
To assess the effect of previous influenza vaccinations on the immunogenicity of FLU-v. 
 
 
Safety 
To evaluate the incidence of solicited AEs in all groups until 21 days after the last dosing 
of study vaccine (FLU-v). (Primary) 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
9
To evaluate the incidence and nature of unsolicited AEs and SAEs in all subjects during 
the whole study period. (Primary) 
 
Clinical Efficacy 
To evaluate the efficacy of FLU-v vaccine in the reduction of the incidence of RT-PCR 
confirmed influenza A and/or B infection in all subjects during the influenza season 
2016-2017. (Exploratory) 
To evaluate the efficacy of FLU-v vaccine in the reduction of symptom scores using diary 
entries and a scoring system among RT-PCR confirmed influenza A and/or B infection 
cases during the influenza season 2016-2017. (Exploratory) 
Statistical methods: 
A detailed Statistical Analysis plan will be prepared before the data lock of the trial 
database. All measured variables and derived parameters will be listed individually by 
subject number and treatment group. The data will be summarized in appropriate tables 
of descriptive statistics by study treatment. Mixed models repeated measures analysis 
will be used to compare the effects of FLU-v with placebo, whether given as suspension 
(unadjuvanted) or as emulsion (adjuvanted) on the normalized cytokine values at days 
42 and 180. The baseline (Day 0) value will be used as a covariate and/or to adjust the 
post-treatment values. The terms in the model will include age group, time, treatment, 
formulation, and the interaction between treatment and time in order to see whether 
FLU-v has similar effects when given as a suspension or in adjuvanted form. Estimates 
for the effects of both formulations relative to the corresponding placebo will also be 
derived. The incidence of adverse events will be summarised for the Safety population in 
tables for each treatment group but no formal analysis will be conducted. The severity of 
symptom scores will be compared between treatments using analysis of variance for 
those subjects who develop flu, if numbers allow. 
In a post-hoc exploratory analysis, the effect of previous influenza vaccines on the 
immunogenicity of FLU-v will be assessed. For this purpose, subjects’ data will be 
stratified as follows: 
-subject never previously received an influenza vaccine. 
-subject received an influenza vaccine within the previous 24months. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
10
-subject received an influenza vaccine over 24 months ago. 
 
Determination of sample size: 
Though a variety of immune parameters will be analyzed, the minimum sample size has 
been determined on the basis of the FLU-v-specific IFN-γ responses (assessed by ELISA) 
recorded in an earlier trial. IFN- is one of the most important markers of cell-mediated 
immunity for influenza protection. Taking the likely pattern of response and a loss to 
follow up into account, we will need to include 222 subjects in order to detect a 2.5-fold 
increase in the normalized value (change from baseline) for adjuvanted and 
unadjuvanted FLU-v relative to placebo.   
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
11
Flow chart of study design 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
12
3. Table of Contents 
1. 
Signatures and Agreement with Protocol 
2. 
Study Synopsis 
3. 
Table of Contents 
4. 
List of Abbreviations and Definitions of Terms 
5. 
Investigators and Study Administrative Structure 
5.1 Sponsor Representatives 
5.2 Sponsor Medical Monitor 
5.3 UNISEC Project Coordinator/ Coordinating Principle Investigator 
5.4 Investigator 
5.5 Qualified Physicians Who Are Responsible for (all) Trial Site-Related Medical 
(or Dental) Decisions 
5.6 Clinical Laboratories and Other Medical and/or Technical Departments 
and/or Institutions Involved in the Trial 
6. 
Summary 
7. 
Introduction 
7.1 Background 
7.2 Rationale 
8. 
Objectives 
8.1 Primary Objectives 
8.2 Secondary Objectives 
8.3 Exploratory Objective 
9. 
Study Design 
9.1 Study Groups and treatments 
9.2 Description and Justification of Control 
9.3 Description and Justification of Dosage and Administration 
9.4 Vaccination Schedule 
9.5 Duration of the Study 
9.6 Method of Assigning Subjects to Treatments 
9.7 Blinding 
9.8 Diary Cards 
9.9 Clinical Efficacy Follow-up 
9.10 Immunogenicity Follow-up 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
13
9.11 Safety Follow-up 
9.12 Data Collection and Management 
 
9.13 From Enrolment to Study Conclusion 
9.14 During the Influenza Epidemic Period 
10. 
Study Population 
10.1 Number of Subjects 
10.2 Eligibility Criteria 
11. 
Study Procedure 
11.1 Enrollment 
11.2 Outline of Study Procedures 
11.3 Detailed Description of Study Procedures 
11.4 Sample Handling and Analysis 
11.5 Withdrawal of Individual Subjects 
12.       Safety Report  
12.1 Definition of an Adverse Event 
12.2 Definition of a Serious Adverse Event 
12.3 Clinical laboratory Abnormalities, Other Abnormal Assessments 
12.4 Time Period, Frequency, and Method of Determining AEs and SAEs 
12.5 Unblinding 
12.6 Recording of AEs and SAEs 
12.7 Evaluating AEs and SAEs 
12.8 Follow-up of AEs and SAEs 
12.9 Prompt Reporting of the SARs to the Sponsor 
12.10 Regulatory Reporting Requirements for SAEs 
12.11 Post-Study AEs and SAEs 
12.12 SAEs Related to Study Participation 
12.13 Annual Safety Report 
13.       Investigational Medicinal Product 
13.1 Name and Description 
13.2 Summary of Findings from Non-Clinical Studies 
13.3 Summary of Findings from Clinical Studies 
13.4 Summary of Known and Potential Risks and Benefits 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
14
13.5 Drug Packaging and Labelling 
13.6 Product Accountability 
13.7 Study Drug Handling and Storage 
13.8 Accountability of Study Supplies 
13.9 Retention of Samples 
14.       Statistical Considerations 
14.1 Endpoints 
14.2 Estimated Sample Sizes 
14.3 General considerations 
14.4 Methods of analysis 
14.5 Handling of missing data 
14.6 Multiplicity 
15.       Ethics 
       15.1 Ethical Review Committees 
       15.2 Ethical Conduct of the Study  
       15.3 Description of Ethical Considerations Relating to the Trial 
15.4 Information and Consent 
15.5 Compensation for Injury 
16.        Provisional Time for Study Phases 
17.        Administrative Aspects, Monitoring and Publication 
17.1 Monitoring and Quality Assurance 
17.2 Amendments 
17.3 Annual Progress Report 
17.4 End of Study Report 
17.5 Public Disclosure and Publication Policy 
18.        References 
 
 
 
 
 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
15
4. List of Abbreviations and Definitions of Terms 
 
AE 
CA 
CI 
CMI 
eCRF 
CTL 
CV 
ECDC 
EU 
EudraCT 
FAS 
GCP 
GMP 
IB 
ICF 
IMP 
ITT 
MMRM 
NP 
PBMC 
Adverse Event 
Competent Authority 
Confidence Interval 
Cell Mediated Immunity 
electronic Case Report Form 
Cytotoxic T Lymphocyte 
Curriculum Vitae 
European Centre for Disease Prevention and Control 
European Union 
European Drug Regulatory Affairs Clinical Trials 
Full analysis set 
Good Clinical Practice 
Good Manufacturing Practice 
Investigator’s Brochure 
Informed consent form 
Investigational Medicinal Product 
Intention to treat 
Mixed models repeated measures 
Nucleoprotein 
Peripheral Blood Mononuclear Cells 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
16
PP 
RT-PCR 
SAE 
SC 
SOP 
SUSAR 
TCC 
Th 
Per protocol 
Reverse Transcription Polymerase Chain Reaction 
Serious Adverse Event 
Subcutaneous 
Standard Operating Procedure 
Suspected Unexpected Serious Adverse Reaction 
Trial Coordination Centre 
T helper 
 
 
 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
17
5. Investigators and Study Administrative Structure 
5.1 Sponsor Representatives 
Gregory Stoloff; 
Dr. Olga Pleguezuelos; 
Luca Landucci;  
Emma Zuccaro 
 
5.2 Sponsor Medical Monitor 
Bryan Murray, MBBS 
Boyd Consultants Ltd 
3a Station Cottages 
St Neots, Cambridgeshire 
PE19 1QF 
UK 
 
 
5.3 UNISEC Project Coordinator 
Prof. Dr. Eelko Hak (University of Groningen) 
 
5.4 Investigators 
Dr Paul Groeneveld 
Isala Hospitals  
Isala Academy, department of Innovation and Science  
Dokter van Deenweg 1  
8052 BP Zwolle 
The Netherlands  
PO box 10400  
8000 GK Zwolle 
 
5.5 Qualified Physicians Who Are Responsible for (all) Trial-site Related Medical (or 
Dental) Decisions  
The same as the Investigators. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
18
 
5.6 Clinical Laboratories and Other Medical and/or Technical Departments and/or 
Institutions Involved in the Trial 
Institution 
Role and responsibility 
Sjef van de Leur 
Isala Hospitals  
The Netherlands 
Haematology and blood chemistry tests 
Fredrik Oftung 
Norwegian Institute of Public Health  
Norway 
Antibody responses and exploratory CMI 
assays.  
Steve Norley  
Robert Koch Institute 
Germany 
Multiparametric FACS analysis and IFN-γ 
ELISA (Primary CMI endpoints)  
 
Prof. dr H.G.M Niester PhD 
University Medical Centre Groningen 
University of Groningen 
Department of Microbiology 
Division of Clinical Virology 
The Netherlands 
RT-PCR assay on influenza A and/or B 
viruses 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
19
6. Summary 
Rationale: Current seasonal influenza vaccines mainly induce immune responses 
against viral membrane glycoproteins. These proteins, however, undergo continuous 
mutations by a process called antigenic drift. To prevent immune escape, annual 
vaccination with the latest predicted viral strains is adopted. Such vaccination strategy 
not only poses inconvenience and cost-inefficiency, but also results in poor protective 
effectiveness when the vaccinated strains are mismatched with the actual circulating 
strains. The latter point is especially of concern during a pandemic outbreak, when a 
large geographical area is affected and the general population is naïve to the newly re-
assorted viral strain due to antigenic shift. 
 
Objective: To evaluate the safety and immunogenicity of the influenza vaccine (FLU-v, 
as a suspension or adjuvanted as emulsion) targeting conserved immunogenic regions of 
influenza A and B viruses in healthy adults, in particular to show that the TH1 cytokine 
response at 42 and 180 days after the first injection is greater in the adjuvanted FLU-v 
and unadjuvanted FLU-v than in the placebo. 
 
Study design: A total of 222 study participants will be recruited. The study follows a 
factorial design where the two factors are treatment (FLU-v / placebo) and formulation 
(unadjuvanted / suspension, adjuvanted / emulsion). Subjects will be randomised in 
two strata (age 18 to 40, age 41 to 60) to one of the following treatment regimens: 
 Group 1 (n=74):  FLU-v (unadjuvanted) as a suspension in pH neutral HCl/NaOH 
(0.5mL) on Day 0 and Day 21 
 Group 2 (n=74): (0.5mL) ISA-51-adjuvanted FLU-v emulsified in water for 
injection (WFI) on Day 0, saline (0.5mL) on Day 21 
 Group 3 (n=37): saline solution (0.5mL ) on Day 0 and Day 21 
 Group 4 (n=37): WFI and ISA-51 emulsion (0.5mL) on Day 0, saline (0.5mL) on 
Day 21 
 
Each administration will be given subcutaneously. Solicited and unsolicited adverse 
events (AEs) will be collected by AE questionnaire/diary card. Adverse events (AEs) 
and serious adverse events (SAEs) will be collected for the entire study period. The 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
20
treatments will be administered starting in third quarter of 2016 in order to provide 
protection for the subsequent influenza season starting in December 2016. Blood 
samples will be taken from all subjects on day 0 (before FLU-v vaccination), 42 (21 days 
after the second dosing) and 180 (159 days after the second dosing) for the evaluation 
of FLU-v-specific cellular and humoral immune responses. Clinical symptom scores to 
ascertain severity and the incidence of RT-PCR-confirmed influenza A and/or B 
infection will be recorded during the subsequent influenza season (December 2016 to 
March 2017) to decide clinical efficacy of the tested vaccines. 
 
Study population: Healthy volunteers aged 18-60 years. 
 
Intervention: FLU-v investigational influenza vaccine lyophilised product containing 
500 micrograms of total peptides reconstituted in either 0.01M HCl (0.25ml) and 0.01M 
NaOH (0.25ml) to achieve a volume of 0.5ml, or emulsified in WFI (water for injection, 
0.25ml) and ISA-51 (0.25ml) to achieve a volume of 0.5ml.  
 
Primary study parameters/endpoints: For immunogenicity: To compare the change 
from baseline (Day 0) between treatments in cellular immune responses, specifically 
TH1 cytokines, in all groups 42 and 180 days following FLU-v vaccination. For safety: (1) 
To evaluate the solicited AEs in all subjects until 21 days after the last dosing of the 
study vaccine (FLU-v); (2) To evaluate the unsolicited AEs and SAEs in all subjects for 
the entire study period after the first dosing of FLU-v.  
 
Secondary study parameters/endpoints: To evaluate the humoral immune responses 
specific to FLU-v and TH2 cytokines from baseline in all groups 42 and 180 days 
following FLU-v vaccination.  
 
Exploratory study parameters/endpoints: For immunogenicity: To evaluate the 
change from baseline in cellular immune responses based on additional CMI assays such 
as ELISPOT (Enzyme-Linked ImmunoSpot) in all groups at 42 and 180 days following 
FLU-v vaccination, in a subset of subjects chosen  After un-blinding the study a subset of 
subjects will be selected (high FLU-v responders, FLU-v non-responders and placebo 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
21
similarly represented) to assess broadness of protection against different influenza 
strains. The analytical lab will remain blinded. 
Clinical efficacy: (1) To evaluate the efficacy of FLU-v vaccine in reducing the incidence 
of RT-PCR confirmed influenza A and/or B infections in all subjects during the influenza 
season 2016-2017. (2) To evaluate the efficacy of FLU-v vaccine in the reduction of 
symptom score among RT-PCR confirmed influenza A and/or B infection cases during 
the influenza season 2016-2017. The relationship between efficacy and cellular and 
humoral response will be explored if possible. 
The effect of previous influenza vaccination on the immunogenicity of FLU-v will be 
assessed in a post-hoc exploratory analysis after stratification of the data based on 
exposure to the influenza vaccine in the previous 24 months or over. 
Exploratory endpoints may be reported separate from the main clinical study report. 
 
Nature and extent of the burden and risks associated with participation, benefit 
and group relatedness:  
The intended application of the IMP is as a prophylactic vaccine to prevent influenza 
virus infection by inducing an enhanced influenza-specific immune response. The  FLU-v 
vaccine is designed to be delivered either as a naturally particulate suspension (i.e. no 
adjuvant) or emulsified in adjuvant. Particulate and emulsified protein preparations are 
preferentially taken up by phagocytic cells (e.g. dendritic cells) responsible for inducing 
primary immune responses.  
Treatment with FLU-v of up to four doses of 263 μg/occasion or up to two doses of 553 
μg/occasion, with or without adjuvant, was well tolerated in animals with no signs of 
systemic toxicity. In pre-clinical studies, subcutaneous administration of ISA 51 resulted 
in no systemic toxicity. At the injection sites and occasionally in adjacent tissue the 
injected material remained surrounded by a mild chronic inflammatory response. As the 
IMP is provided in one presentation (500 μg of the combined peptide) in lyophilized 
form for suspension in a sealed single-use vial, overdose is highly unlikely. 
In a single centre, randomised, double blind phase I study of the safety, tolerability and 
immunogenicity of FLU-v no safety or tolerability concerns were identified at the doses 
administered (250 μg and 500 μg FLU-v) to the subjects in this clinical study and no 
safety or tolerability concerns were identified following administration of the adjuvant 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
22
to the subjects (1). FLU-v vaccine candidate was demonstrated to be immunogenic in 
humans, as measured by ex vivo γ-interferon production (1). 
Clinical experience with Montanide ISA 51 dates back to the 1990s and most trials were 
related to cancer and Acquired Immune Deficiency Syndrome (AIDS). Currently, cancer 
trials in melanoma, colorectal, prostate, cervical, brain cancer and leukemia are ongoing. 
Frequency of vaccination is often between 2 and 4 weeks, and the number of injections 
can reach up to 40. Route of immunization is most often subcutaneous and volume of 
injection can reach up to 3mL. Most common local reactions are local pain, tenderness, 
erythema and granuloma at the injection site. Less frequently, mild to moderate 
transient indurations and swelling are described. General reactions are mainly ‘flu-like 
symptoms such as chills, fever and headaches. Lethargy and nausea are also observed. 
The intensity is usually mild or moderate. No biological changes are generally observed. 
As the Placebo vaccine is provided in a sealed single-use vial, overdose is highly unlikely 
(Influenza virus (FLU-v) vaccine Investigator’s Brochure, Edition 2.0, 07 September 
2015). 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
23
7. Introduction  
7.1 Background 
Influenza is one of the major respiratory person-to-person transmittable viral infections 
in humans. The disease itself comes in two distinct forms: seasonal epidemics that occur 
every year in the winter period, and (global) pandemics that occur with unpredictable 
frequency and which can take place in any season. Over the past decade seasonal 
influenza has resulted in an estimated 80,000 to 500,000 deaths per year, depending on 
the severity of the flu outbreak. Deaths occur especially in vulnerable populations, such 
as the elderly and chronically ill subjects (2). Direct influenza-associated health care 
costs for Europe are estimated at more than €50 million per million of population per 
year (i.e. for the whole of Europe €2.5 billion/yr.), and costs are likely to increase 
further due to an ageing of the population (3).  
Pandemic influenza arises when new influenza strains enter the human population by 
transmission from farm animal species. These viruses are antigenically very different 
from seasonal influenza strains and are therefore not or very poorly recognized by pre-
existing immunity. For this reason, they can spread very easily in the entire population.  
 
7.2 Rationale 
Several strategies have been developed in recent years to broaden the response to 
vaccines, so that cross-clade recognition and neutralization of H5N1 viruses could be 
achieved. Yet, the emergence of the ‘Mexican flu’ or ‘swine flu’ caused by an H1N1 virus 
strain in 2009 demonstrated the unpredictable nature of pandemic influenza viruses. 
The Mexican Flu pandemic underlined the fact that a truly universal influenza vaccine 
will be required to tackle not only different clades generated by antigenic drift (with 
slightly changed HA), but also different virus strains generated by antigenic shift (with 
completely different HA subtypes). Consequently, universal influenza vaccines have to 
target viral proteins, or domains of viral proteins, which are highly, conserved across a 
number of (or even all known) influenza A and B virus strains.  
FLU-v vaccine developed by SEEK is a sterile equimolar mixture of four synthetic 
polypeptides that cover highly conserved regions found in the NP, M1 and M2 proteins 
of influenza A and B viruses. Please refer to the Investigator’s Brochure of FLU-v for 
detailed information. FLU-v is expected to achieve cross-protection against influenza 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
24
disease by inducing cell-mediated immunity (cytotoxic T lymphocytes (CTLs) and/or T 
helper (Th) cells) and non-neutralizing antibodies. Epitopes recognized by T and B cells 
are often highly conserved among influenza subtypes indicating that these cells have 
‘natural’ cross-reactive potential (4). In addition, recent data demonstrates that human 
CTLs elicited by seasonal influenza recognize and lyse cells infected by H5N1 or 
pdmH1N1 virus (5). Together with data indicating age-related relative protection 
against newly emerging virus strains, these results imply that CTLs (though not 
providing sterilizing immunity) have at least a disease-mitigating potential in humans.  
The aim of this randomised, double-blind, parallel group, placebo-controlled, single-
centre phase IIb study will be to evaluate the immunogenicity and safety of FLU-v 
vaccine in healthy adults (aged 18-60). The immunogenicity of FLU-v will be evaluated 
by CMI and humoral responses. The safety will be evaluated based on reported AEs and 
SAEs during the study period. In addition, an exploratory endpoint of clinical efficacy 
will be evaluated, which will be determined by reductions in occurrence of influenza and 
the clinical symptom severity in RT-PCR confirmed cases of influenza A and/or B 
infection during the 2016-2017 influenza season. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
25
8. Objectives 
Vaccination with FLU-v generates a population of T cells that can recognise conserved 
influenza antigens on the surface of infected cells. On recognition, T cells are activated 
and secrete Th1 cytokines such as INF-, TNF-α, and IL2 and release granzyme and 
perforin. In addition, FLU-v specific non-neutralising antibodies may also bind to these 
antigens on the surface of infected cells activating complement and cytotoxic responses 
lead by Natural Killer (NK) cells. The T cell and antibody actions will result in the 
destruction of infected cells.  
The objectives of this study are to determine the safety of the vaccine and assess which 
one of the two treatment groups, one dose adjuvanted vs two dose unadjuvanted, 
induces the strongest immune response which is measured by quantifying the increase 
in Th1 and non-neutralising antibody responses after vaccination compared to 
baseline and corresponding placebo vaccination. The relationship between clinical 
efficacy and the immune response will also be explored. 
 
8.1 Primary objectives 
(1) Cellular Immunogenicity 
- To compare the cellular immune responses as measured by the change from baseline 
(Day 0) in TH1 cytokines between FLU-v administered as a suspension and as 
emulsion (IFA-51 adjuvanted) at 42 and 180 days following FLU-v vaccination. 
 
(2) Safety 
- To evaluate the solicited AEs in all subjects until 21 days after the last dosing of the 
study vaccine (FLU-v). 
- To evaluate the unsolicited AEs and SAEs in all subjects during the whole study period.  
 
8.2 Secondary Objective 
- To evaluate the FLU-v specific antibody responses in all subjects from baseline at day 0 
to day 42 and day 180 following FLU-v vaccination. 
- To compare the TH2 cytokine response between treatments from baseline to day 42 
and day 180 following FLU-v vaccination. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
26
 
8.3 Exploratory objective 
(1) Cellular Immunogenicity 
- To evaluate the change from baseline in cellular immune responses based on 
additional CMI assays at 42 and 180 days following FLU-v vaccination, in a subset of 
subjects. 
- To evaluate the effect of previous influenza vaccines on the cellular immune responses 
to FLU-v. 
After un-blinding the study a subset of subjects will be selected (high FLU-v responders, 
FLU-v non-responders and placebo similarly represented) to assess broadness of 
protection against different influenza strains. The analytical lab will remain blinded. 
 
(2) Clinical Efficacy 
- To evaluate the efficacy of FLU-v vaccine, whether given as suspension or as 
adjuvanted (IFA-51) emulsion, in the reduction of the incidence of RT-PCR confirmed 
influenza A and/or B infection in all subjects during the influenza season 2016-2017. 
- To evaluate the efficacy of FLU-v vaccine whether given as suspension or as adjuvanted 
(IFA-51) emulsion, in the reduction of symptom scores among RT-PCR confirmed 
influenza A and/or B infection cases during the influenza season 2016-2017. 
- To explore the relationship between clinical efficacy and cellular and humoral response. 
 
Exploratory endpoints may be reported separately from the main clinical study report.

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
27
9. Study Design 
After providing consent, subjects will enter a screening period of approximately 7 days 
to verify eligibility for the trial. All screening evaluations will be reviewed and once 
confirmed as eligible, subjects will be considered enrolled and will progress to the 
randomisation visit.  Randomised subjects will then enter a 21-day treatment period, 
followed by 2 follow-ups on day 42 and 180, for a maximum of one year (from screening 
to study conclusion) study participation. Subjects will attend clinic visits as shown in the 
Flow Chart and detailed in section 11.3. 
 
9.1 Study Groups and Treatments 
Subjects will be randomised in a (2:2:1:1) fashion, double-blinded (see note below) and 
placebo–controlled. Randomisation will be stratified by age group (18 to 40, 41 to 60)  
to ensure that the age distribution in all treatment groups is broadly similar. (This is 
because older subjects may be more prone to infection than younger subjects though 
they are not expected to respond differently to the treatment regimens). Each subject’s 
participation will be for a maximum of one year (from screening to study conclusion) 
enrolling 222 healthy adults aged 18-60 years. The treatment groups are as follows: 
 
 Group 1 (n=74):  FLU-v (unadjuvanted) as a suspension in pH neutral HCl/NaOH 
(0.5mL) on Day 0 and Day 21 
 Group 2 (n=74): ISA-51-adjuvanted FLU-v emulsified in water for injection (WFI) 
on Day 0, saline (0.5mL) on Day 21  
 Group 3 (n=37): saline solution (0.5ml) on Day 0 and Day 21 
 Group 4 (n=37): WFI and ISA-51 emulsion on Day 0, saline (0.5mL) on Day 21 
 
9.2 Description and Justification of Control 
The placebo for arm 1 is 0.5ml saline. Its chemical composition (NaCl in water) is the 
same as when mixing HCl and NaOH in equimolar amounts. The placebo for arm 2 is 0.5 
mL emulsion of WFI (0.25 mL) and the adjuvant [Montanide ISA-51-VG; Seppic](0.25ml) 
where Montanide ISA 51 is defined as a mixture of a highly purified mineral oil (Drakeol 
6VR) and a surfactant (Mannide monooleate). When mixed with an aqueous phase at a 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
28
50/50 ratio, it renders a water in oil emulsion and is used as vaccine adjuvant to 
enhance the immune response. 
In pre-clinical studies, subcutaneous administration of ISA 51 resulted in no systemic 
toxicity. At the injection sites and occasionally in adjacent tissue the injected material 
remained surrounded by a mild chronic inflammatory response. 
No safety or tolerability concerns were identified following administration of the 
adjuvant to the subjects in a Phase 1 study (EUDRACT No: 2009-013910-28).  
 
9.3 Description and Justification of Dosage and Administration 
 
Different doses were tested in previous human studies and the dose selected for this 
study, 500ug, is the one that performed the best in the previous studies from both a 
perspective of safety/tolerability as well as for efficacy. 
 
In a previous study (EUDRACT/IND NUMBER: 2009-014716-35) FLU-v was 
administered subcutaneously as a sterile lyophilized mixture at a nominal total peptide 
dose of 500 μg/vial, resuspended in 0.5 mL sterile WFI and the suspension emulsified 
with 0.5mL of adjuvant [Montanide ISA-51-VG; Seppic] to give a 1 mL volume for 
subcutaneous injection. 
 
The dose selected (500ug) is considered both safe and well-tolerated and with the 
potential for eliciting immunogenicity based on the results of pre-clinical studies, as 
follows: 
A number of repeat dose toxicity studies, conducted in mice and rats investigated the 
systemic toxicity potential and immunogenicity of FLU-v when administered alone or in 
combination with the adjuvant ISA 51. All the toxicity studies were carried out using the 
tetravalent formulation of FLU-v used in the current study. All the studies demonstrated 
that following subcutaneous vaccination with FLU-v alone or with the adjuvant, there 
was no effect on bodyweight gain, food consumption, organ weight or ophthalmoscopy 
(where ophthalmic examination was carried out). The test substance was clinically well-
tolerated with no signs of systemic toxicity and thorough histopathological evaluation 
did not reveal any signs of systemic toxicity. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
29
In repeat dose toxicity studies in the mouse using FLU-v alone and adjuvanted FLU-v, 
cell-mediated responses were evident with enhanced responses when FLU-v was 
administered with adjuvant. Increases in total white blood cell counts were evident in 
most of the studies but were considered related to an immune response to FLU-v and, as 
such, did not represent an adverse effect. 
 
Subcutaneous injection of FLU-v alone or with adjuvant was also well-tolerated in a local 
tolerance study in rabbits, with no systemic sign of reaction to treatment. 
 
Generally, injection site responses and microscopic and macroscopic changes in animals 
receiving FLU-v were considered likely to be due to the effects of the adjuvant, ISA 51. 
In addition, treatment with FLU-v of up to four doses of 263 μg/occasion or up to two 
doses of 553 μg/occasion, with or without adjuvant, was well-tolerated in animals with 
no signs of systemic toxicity. The IMP is provided in one presentation (500 μg of the 
combined peptide) in lyophilized form for suspension in a sealed single-use vial. At this 
dose, based on the pre-clinical studies, the dose is likely to be well-tolerated and safe in 
humans. 
This protocol describes that the dose of FLU-v continues to be the same as in previous 
trials, 500ug, however the total volume has decreased from 1ml to 0.5ml in order to 
minimise injection site specific adverse effects. Additionally, rather than reconstitute 
FLU-v directly in saline as in previous trials, it will now be reconstituted in HCl first 
followed by pH neutralisation with NaOH. This method is a more time-effective way of 
reconstituting FLU-v to be administered in suspension unadjuvanted. The final result 
after mixing equal volumes of 0.01M HCl and 0.01M NaOH is NaCl in water which is the 
same composition as saline. 
 
9.4 Vaccination Schedule 
Group 
Admin 1 (day 0) 
Admin 2 (day 21) 
1 
FLU-v 
in 
pH 
neutral 
HCl/NaOH [suspension] 
FLU-v in pH neutral HCl/NaOH 
[suspension] 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
30
2 
FLU-v adjuvanted with ISA-51 
[emulsion] 
Saline 
3 
Saline 
Saline 
4 
WFI and ISA-51 [emulsion] 
Saline 
 
9.5 Duration of the Study  
The study will require 5 visits (including the screening visit) for each subject. The total 
study time is a maximum 1 year from first subject first visit to study conclusion. 
 
9.6 Method of Assigning Subjects to Treatments 
 
(1) Assignment of Subject Numbers 
The Trial Coordination Center (TCC) of University Medical Center Groningen will be in 
charge of the randomisation list. Each subject screened in Zwolle will be allocated a 3 
digit screening number representing the sequential order in which they are screened e.g. 
001, 002 etc.  On randomisation, subjects will be allocated the next available 3-digit 
randomisation number in the appropriate age group in the randomisation list.  
Randomisation numbers generated by the ALEA system will be sent out by TCC by 
courier directly to the unblinded pharmacist at Isala Clinic in Zwolle. No other person, 
except the responsible person at TCC (who generated the list) and the unblinded 
pharmacist, will have the access of the randomisation list. 
 
(2) Randomisation Method 
The randomisation system is hosted and set up by the Trial Coordination Center 
(Clinical Research Organization) in the UMCG, however the implementation of the 
randomisation itself will be performed on-site in Zwolle. The subjects will only have 
study-visits in Zwolle. 
 
In accordance with the protocol, subjects will attend a screening visit (visit 1) in Zwolle 
which will be completed [-7 ]to[ -1 ] days before the subject is randomised. Each 
screened subject will then receive a sequential identification number as described above. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
31
Subjects will retain this unique number, whether or not they are ultimately randomised 
to receive treatment. If the subject meets the inclusion/exclusion criteria, blood samples 
are taken and the subject is requested to return for a Day 0 visit (visit 2). At the day 0 
visit the blood sample results are reviewed and if the subject continues to meet the 
inclusion/exclusion criteria, the subject can be randomised. When a subject is to be 
randomised, the next randomisation number in sequence within the appropriate age 
group stratum will be allocated to the subject, and the treatment description then made 
up in accordance with the Pharmacy Instructions. 
 
Randomisation will be performed using a web-based system (ALEA) by the Trial 
Coordination Centre at the University Medical Centre Groningen and which is fully 
backed up on a daily basis. Randomisation will be stratified according to age (age 18 to 
40, and age 41 to 60) with the 2:2:1:1 treatment ratio being maintained in appropriately 
sized blocks within each stratum to ensure similar age distribution between the groups. 
 
9.7 Blinding 
 
To maintain blinding, the preparation of IMP and Control will be performed by a 
qualified person other than the person administering the injection. The study will be 
double-blind although it is not possible to completely mask the presence of adjuvant in 
the injections at Day 0 since the solution and the emulsion are physically distinguishable. 
Due to the local injection site reactions likely from Montanide ISA-51 adjuvant, masking 
of syringe barrels is deemed superfluous. However, the blinded study personnel will 
remain blinded to the presence or absence of FLU-v vaccine. The label on the syringe 
will need to carry the necessary trial randomisation information. This is explained in 
detail in the pharmacy manual. 
 
The study pharmacist/designee responsible for the preparation of dosing materials is 
unblinded to the treatment each subject will receive. The unblinded study 
pharmacist/designee will prepare dosing solutions and will use the study randomisation 
code to assign each subject to the appropriate treatment group. The unblinded study 
pharmacist/designee will not reveal the treatment code to study personnel or perform 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
32
any other study related duties. The unblinded study pharmacist/designee will prepare 
syringes containing the study medication assigned by the randomisation scheme 
labelled with the subject’s randomisation number. An identical label will be attached to 
the top of the case report form. This material will be taken to the blinded study 
personnel administering the injections.  
 
The Pharmacy file, including the randomisation list, vaccine supplies and all associated 
documentation, will be stored in a locked cabinet within the Pharmacy where only 
unblinded Pharmacy personnel have granted access. 
 
9.8 Diary Cards 
 
(1) Symptom Diary Card/Diary Score 
 
A symptom diary card will be issued to all subjects which they are instructed to record 
the card daily during the influenza season (December 2016 to March 2017). Scores on a 
scale of 0 to 3 (see below for definitions) will be requested daily for the following 
symptoms: fever (≥38oC), malaise, headache, myalgia (muscle and joint pain), cough, 
sore throat, shortness of breath, runny nose, stuffy nose, sneezing and earache. The trial 
centre must be contacted immediately if subjects feel unwell for 24h, with a sudden 
onset of at least one respiratory (cough, sore throat, shortness of breath, runny nose, 
stuffy nose, sneezing and earache) and one systemic symptom (fever, malaise, headache 
and myalgia (muscle and joint pain). Nasal and tonsil swabs (2 swabs in total) should be 
taken from the reported subjects within 3 days from the trial centre being contacted or 
within 4 days of the onset of symptoms, whatever time is shorter. The completed diary 
cards should be posted/faxed/emailed back to the trial site by subjects every two weeks 
during the duration of the influenza season.  
 
The same list of symptoms in the diary card will be used in those subjects with 
confirmed influenza (by RT-PCR) to assess severity of disease. Each symptom is 
assessed on a scale of 0-3: no symptoms=0, just noticeable=1, bothersome but can still 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
33
so activities=2, bothersome and cannot do daily activities=3. The sum of all the scores 
constitutes the daily symptom score. 
 
(2) AE Diary Card 
 
To follow solicited AEs, 2 AE diary cards will be issued to all study subjects. Solicited AEs 
include fever, chills, joint pain, muscle pain, headache, backache, abdominal pain, chest 
pain, pain in arm, itching on body, swelling/tender lymph nodes, rhinorrhea, nasal 
stuffiness, difficulty breathing, sore throat, cough, fatigue, loss of appetite, vomiting, 
diarrhea and injection site reaction. The cards will need to be completed by the subjects 
and returned on day 21 and 42. 
 
9.9 Clinical Efficacy Follow-up 
 
- 
Active and periodic reports of the symptom diary card/diary score from all subjects 
during the influenza season; 
- 
Clinical sampling and laboratory confirmation by RT-PCR. 
 
9.10 Immunogenicity Follow-up 
 
Cellular and humoral immunogenicity of FLU-v vaccine as explained in section 6 and 
section 8 will be evaluated in all subjects. 60 ml of blood will be sampled on the 
scheduled visits at day 0, 42, and 180. 50 ml and 10 ml of the collected blood will be 
used for the assessment of cellular and humoral immune response, respectively. 
 
9.11 Safety Follow-up 
 
Adverse events reported after FLU-v vaccination will be collected and evaluated for all 
subjects. 
(1) 
Solicited AEs (subjects will return on day 21 (second vaccination) and 42 with AE 
diary card/questionnaire). 
(2) 
Unsolicited AEs and SAEs (collection throughout the whole study period). 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
34
 
9.12 Data Collection and Management 
 
Data management will be conducted by TCC, which is ISO 9001:2008 certified for 
"supporting researchers in the design, supervision, analysis and reporting of biomedical 
research, including project management, protocol design and review, EC/CA submission, 
data entry, data management, monitoring, biometrics, medical writing and software 
development”, hence qualified to meet the essential requirements of ICH-GCP guidelines 
for GCP according to the European directives. An interactive website will be the major 
communication instrument ensuring integration of the program communication and the 
trial study.  
(1) 
Electronic case report forms (eCRFs)  
- 
Will be optimized for scalability, in accordance with standards for electronic data 
entry and flexible data export (GCP); 
- 
Will be developed as web-based report forms integrated into the Web Content 
Management System. Windows Internet Explorer is required to support online data 
entry. 
(2) 
Data  
- 
Will be stored in the databases for consistency checks and further processing; 
- 
Will be backed up on a daily basis; 
- 
Will be subject to secure access control management to allow secure entry, access, 
analysis and export of data by users regardless of their locations; 
- 
Will be subject to plausibility and consistency check during the entry process to 
enforce high data integrity, authentication and data protection according to the relevant 
user access levels and server and communication security mechanisms will be used;  
- 
All nominal subject data will be anonymised to ensure personal data protection. If 
several reports taken at different times have to be correlated, pseudo-anonymization 
instead of complete anonymization will be used.   
We will ensure that the data protection authority of the trial participating nation accepts 
the security and privacy procedures.  
 
9.13 From Enrolment to Study Conclusion 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
35
 
(1) 
Scheduled visits at day 0 and 21 for vaccination.  
(2) 
Scheduled visits at day 0, 42 and 180 for cellular and humoral immunogenicity 
follow-up. 
(3) 
Scheduled visit at day 21 and 42 for solicited AE follow-up. 
(4) 
Unsolicited AEs and SAEs will be followed throughout the study period for all 
subjects. 
 
9.14 During the Influenza Period (December 2016 to March 2017) 
A symptom diary card will be issued to all subjects. For details please refer to point (1) 
Symptom Diary Card/Diary Score under section 9.8 Diary Cards. 
In the event of missing data for any timepoint in the influenza symptom diary card, the 
study doctor will ask the subject about any symptoms during the missing day(s). 
However, no protocol deviation will be recorded for missing influenza symptom scores.

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
36
10. Study Population 
10.1 Number of Subjects  
A total of 222 healthy adults aged 18-60 years will be randomised.  
 
10.2 Eligibility Criteria 
 
Subject eligibility should be reviewed and documented by an appropriate qualified 
member of the Investigator’s study team before subjects are included in the study. 
(1) Inclusion Criteria 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria: 
- 
Healthy males or healthy non-pregnant females (as indicated by a negative blood 
pregnancy test during the screening visit) between the ages of 18 and 60 years, inclusive;  
- 
Women of childbearing potential (not surgically sterile or postmenopausal for 
greater than or equal to one year) and men must agree to practice appropriate 
contraception (a combination of barrier and hormonal methods for women and a 
condom for men) from screening and throughout the study treatment and for at least 30 
days (up to Study Day 51 for females) and 90 days (up to Study Day 111 for males).  
After the last dose of the IMP); 
- 
Is in good health, as determined by a comprehensive clinical assessment {vital 
signs (heart rate, blood pressure, oral temperature), blood chemistry test (electrolytes, 
renal/kidney function, liver function, C-reactive protein, complete blood count), medical 
history, general physical examination, self-reported illness} and the clinical judgment of 
the investigator;  
- 
Able to understand and comply with planned study procedures; 
- 
Provides signed informed consent form. 
(2) Exclusion Criteria 
A potential subject who meets any of the following criteria will be excluded from 
participation in this study: 
- 
Has a known allergy to any of the components of the vaccine. 
- 
Has a history of severe reaction following immunization.  

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
37
- 
Persons with immune deficiency/disorder, whether due to genetic defect, 
immunodeficiency disease, or immunosuppressive therapy. 
- 
Females (child bearing potential) having a positive blood pregnancy test during 
the screening visit or women who are breastfeeding. 
- 
Has a history of any of the following (reported by subjects): 
o 
Acute disseminated encephalomyelitis (ADEM); 
o 
Neoplastic disease – current or previous; 
o 
Asthma or severe allergic disease; 
o 
Bleeding disorders 
o 
Chronic Hepatitis B and/or C infection; 
o 
Chronic liver disease; 
o 
Diabetes mellitus; 
o 
Guillain-Barré syndrome; 
o 
HIV; 
o 
Rheumatoid arthritis or other autoimmune diseases; 
o 
Severe renal disease; 
o 
Transplant recipients; 
o 
Unstable or progressive neurological disorders. 
- 
Receipt of medicines/treatments that may affect evaluation of immunogenicity 
such as: 
o Oral or parenteral steroids, high-dose inhaled steroids (greater than 
800 micrograms/day of beclomethasone dipropionate or equivalent) 
or other immunosuppressive or cytotoxic drugs (azathioprine 
(Imuran), cyclosporine (Neoral, Sandimmune, SangCya); monoclonal 
antibodies such basiliximab (Simulect), daclizumab (Zinbryta), 
infliximab (Remicade), rituximab (MabThera), alemtuzumab (Campath 
and Lemtrada), omalizumab (Xolair), abatacept (Orencia), adalimumab 
(Humira and Exemptia) en etanercept (Enbrel);  corticosteroids such 
as prednisone (Deltasone, Orasone); tacrolimus (Prograf, Advagraf, 
Protopic); Glatiramer acetate (Copaxone); Mycopehnolate (Cellcept); 
Sirolimus (Rapamune)  (within 3 months prior to vaccination); 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
38
o Immunoglobulin or other blood products (plasma, blood cells, 
coagulation 
factors, 
haemoglobin)(within 
3 
months 
prior 
to 
vaccination in this study); 
o 
An experimental agent (vaccine, drug, biologic, device, blood product, or 
medication) within 1 month prior to vaccination in this study, or expects to receive an 
experimental agent (during the study period). 
o Influenza antiviral medication (Amantadine (Symmetrel); Rimantadine 
(Flumadine); Zanamivir (Relenza), Oseltamivir (Tamiflu) (within the 4 
weeks prior to vaccination in this study). 
- 
Has received any influenza vaccine within 6 months prior to vaccination in this 
study. 
- 
Has influenza-like illness (a sudden onset of symptoms, and at least one of the 
four systemic symptoms-fever or feverishness, malaise, headache, myalgia and at least 
one of the three respiratory symptoms-cough, sore throat, shortness of breath) or  acute 
respiratory infection (a sudden onset of symptoms and at least one of the four 
respiratory symptoms-cough, sore throat, shortness of breath, coryza and a clinician’s 
judgement that the illness is due to an infection) within 6 months prior to vaccination in 
this study. These symptoms must have stopped the subject from carrying out their 
normal daily activities such as attending work or school for a period of at least 3 days. 
- 
Has an acute illness, including an oral temperature greater than 38 degrees C, 
within 1 week prior vaccination. 
- 
Has a history of alcohol or drug abuse within the last 2 years deemed unsuitable 
for inclusion by the investigator 
- 
Any abnormal haematology values and/or serum chemistries judged by the 
Investigator as clinically significant.  
- 
Ineligible subject based on the judgement of the investigator. 
- 
In the event of uncertainty about the participant’s medical status regarding any of 
the exclusion criteria mentioned, the participant’s primary care physician will be 
consulted. Consultation of the primary care physician will only take place after having 
received written approval from the participant, and will concern medical information 
about exclusion criteria only. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
39
11. Study Procedure 
11.1 Enrollment 
Individuals volunteering to participate into the study will be invited for a study 
screening visit. During this visit,  
 Subject Information Sheet ‘SIS’ and Informed Consent Form ‘ICF’ will be 
explained and given to the subjects.  
 Subject eligibility will be assessed and a signed/dated informed consent will be 
obtained.  
 Baseline information including demographics and self-reported medical and 
medication use history, influenza vaccination, alcohol, drug and cigarette 
consumption history/habits will be collected 
 A blood sample (15 ml) will be taken to examine a standard blood picture, 
pregnancy test (females) and antibodies against circulating influenza viruses.  
Only subjects showing no abnormalities/non pregnancy (females) with the entry 
blood tests will be accepted for the study and later randomised to the study 
treatments.  
 
11.2 Outline of Study Procedures (see next page) 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
40
Time 
 
Screening 
Day 0 
Day 21 
Day 42 
Day 180 
Unscheduled 
Contact 
Number of 
subjects 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 
Phone  
call 
I. Study enrollment  
Check inclusion criteria 
300 
√ 
√ 
 
 
 
 
 
Check exclusion criteria 
222 
√ 
√ 
 
 
 
 
 
General physical examination  
222 
√ 
 
 
 
 
 
 
Medical and medication history 
222 
√ 
 
 
 
 
 
 
History of influenza vaccination 
(within 6 months) 
222 
√ 
 
 
 
 
 
 
Alcohol and drug usage history/habits 
222 
√ 
 
 
 
 
 
 
Smoking history/habits 
222 
√ 
 
 
 
 
 
 
Demographic data 
222 
√ 
 
 
 
 
 
 
Informed consent 
222 
√ 
 
 
 
 
 
 
Blood sampling (15 ml for baseline 
blood profile, for all participants) 
222 
√ 
 
 
 
 
 
 
Blood pregnancy test (only for 
female) part of the 15ml baseline 
sample 
Depends1 
√ 
 
 
 
 
 
 
II. Study procedure 
Randomisation 
222 
 
√ 
 
 
 
 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
41
Pre-vaccination body temperature 
(judged by clinical investigator) 
222 
 
√ 
√ 
 
 
 
 
Blood sampling (60 ml) 
222 
 
√ 
 
√ 
√ 
 
 
Vaccination 
222 
 
√ 
√ 
 
 
 
 
30-minute post-vaccination 
observation 
222 
 
√ 
√ 
 
 
 
 
Issue symptom diary card/diary score 
222 
 
 
√ 
 
 
 
 
Issue AE diary card/questionnaire  
222 
 
√ 
√ 
 
 
 
 
III. Study follow-up 
Examination of AE diary 
card/questionnaire 
222 
 
 
√ 
√ 
 
 
 
Reporting of severe/high-grade 
solicited AEs by the subjects 
Physical examination of severe/high-
grade AEs 
depends1 
 
 
 
 
 
√ 
 
Evaluation of severe AEs (soon after 
reporting by the subject until 
resolution of the event) 
depends1 
 
 
 
 
 
 
√ 
Reporting of unsolicited AEs and SAEs  
222 
Reported throughout the entire study period 
Reporting of diary card/symptom 
score 
222 
Self-recording daily during the influenza period.  
Self-reporting when reached the symptom criteria for swab sampling. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
42
ILI (nasal and tonsil swabs) sampling 
depends1 
 
 
 
 
 
√ 
 
ILI follow-up (till resolution of the 
event) 
depends1 
 
 
 
 
 
 
√ 
Recording of (urgent) 
hospitalization/care visit of all cause 
depends1 
 
 
 
 
 
 
√ 
 
Note:  
1. The number of subjects depends on the actual needs/reporting cases. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
43
11.3 Detailed Description of Study Procedures 
 
During influenza season: 
All subjects will be instructed and taught to complete the symptom diary card on a daily 
basis and to call the trial site(s) immediately when reaching the pre-defined criteria 
(the presence of one respiratory and one systemic symptom). Nasal and tonsillar swab 
samples (2 in total) should be collected from the reported subjects within 3 days from 
the centre being contacted or within 4 days of the onset of symptoms, whichever time is 
shorter. The completed diary cards will then be posted/emailed/faxed back to the trial 
site(s) every two weeks during the entire influenza season. If the cards are not 
returned, the trial site personnel will call those participants to remind them. In the 
event of missing data for any timepoint in the influenza symptom diary card, the study 
doctor will ask the subject about any symptoms during the missing day(s). However, no 
protocol deviation will be recorded for missing influenza symptom scores. 
 
Screening, visit 1 
- 
Check inclusion and exclusion criteria. 
- 
Obtain written informed consent. 
- 
Collect subject baseline information including medical and medication history, 
influenza vaccination history, alcohol, drug usage and smoking history/habits 
and demographic data (age, gender and ethnicity). 
- 
General physical examination. 
- 
15 ml of blood will be collected for baseline blood profile (haematology, serum 
chemistry and antibodies against circulating influenza viruses) and pregnancy 
test (females) 
 
Day 0, visit 2 (Within 1 week of visit 1) 
- 
Review results from haematology values and blood chemistries. 
- 
Examine pre-vaccination body temperature, judged by investigators. 
- 
Check protocol violations from subjects. Obtain and record on-going medications. 
- 
Randomise subjects as per protocol. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
44
- 
60 ml Blood sampling prior to vaccination. (50 ml for CMI measurements and 10 
ml for the antibody response analysis) 
- 
Vaccinate subjects. (One dose administered subcutaneously) 
- 
The subjects will be observed for at least 30 minutes.  
- 
Issue AE diary card/questionnaire. Instruct subjects how to use the diary.  
- 
Instruct subjects to contact investigators immediately if they develop signs of 
SAEs. 
- 
Record any AEs and SAEs. 
- 
Report any SAEs to the sponsor 
 
Day 21, visit 3 
- 
Check protocol violations from subjects. Obtain and record on-going medications. 
- 
Collect and verify completed AE diary card. The information will be transcribed 
to eCRF in English. The trial site should contact the participants before the visit 
and remind them to bring the AE diary card. No replacement of the card will be 
made. 
- 
Check pre-vaccination body temperature. 
- 
Vaccinate subjects. (One dose administered subcutaneously) 
- 
The subject will be observed for at least 30 minutes.  
- 
Issue AE diary card/questionnaire.  
- 
Issue symptom diary card/diary score to complete during the influenza season 
2016-2017. Instruct subjects when to call the study site to report a possible 
influenza-like illness (ILI) and to arrange nasal and tonsil swabs sampling. 
- 
Record any AEs and SAEs. 
- 
Report any SAEs to the sponsor 
 
Day 42, visit 4 
- 
Check protocol violations from subjects. Obtain and record on-going medications. 
- 
Collect and verify completed AE diary card. The information will be transcribed 
to eCRF in English. The trial site should contact the participants before the visit 
and remind them to bring the AE diary card. No replacement of the card will be 
made. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
45
- 
60 ml of blood will be collected from all subjects. (50 ml for CMI measurements 
and 10 ml for antibody response analysis) 
- 
Record any AEs and SAEs. 
- 
Report any SAEs to the sponsor 
 
Day 180, visit 5 
- 
Check protocol violations from subjects.  
- 
60 ml of blood will be collected from all subjects. (50 ml for CMI measurements         
and 10 ml for antibody response analysis) 
- 
Record any AEs and SAEs. 
- 
Report any SAEs to the sponsor 
 
From Visit 5 to the end of study (1st April 2017) 
- 
Collect AE/SAEs from subjects until the end of the study 
- 
Collect patient symptom diaries from the subjects every two weeks 
- 
Arrange clinic appointments for any subjects that feel unwell for 24 hours with a 
sudden onset of a least one respiratory (cough, sore throat, shortness of breath, 
runny nose, stuffy nose, sneezing or earache) and one systemic symptom (fever, 
malaise, headache and myalgia (muscle and joint pain)). Nasal and tonsil swabs 
will be taken. 
 
 
 
11.4 Sample Handling and Analysis 
Biological samples including blood, nasal and tonsil swabs that are drawn for the trial 
study will be stored and accessed according to national guidelines. The storage duration 
is 10 years for the data and samples. Samples will be destroyed and data will be 
permanently removed from the computer network. When transferring biological 
samples from one country to another, trial participants will confirm and make sure that 
they comply with the Oviedo convention and the Recommendation Rec (2006) of the 
Committee of Ministers to member states on research on biological materials of human 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
46
origin (Adopted by the Committee of Ministers on 15th March 2006 at the 958th meeting 
of the Ministers' Deputies). 
 
(1) 
Treatment and Storage of Biological Samples 
 
PBMC isolated form subject’s blood will be stored at -80oCelsius until shipped to the 
central laboratory where they will be stored in liquid nitrogen. 
Serum samples will be stored at -20oCelcius before and after shipment. 
Tonsil and nasal swab samples will be stored at -80 oCelsius. 
 
(2) 
 Shipment of Biological Samples     
         
PBMCs, serum samples and nasal and tonsil swabs must be placed at in a container 
containing 2.5 -5 kg dry ice per 24 hours expected transport time (plus 24 hours in case 
of delays) complying with International Air Transport Association (IATA) requirements 
if shipment by air or complying with ADR or local regulation if transport by road. The 
completed Biological Specimen Listing Form should always accompany the shipment. 
The box must be clearly labelled with a Dry Ice Shipping Label. 
 
(3) 
Laboratory Assays 
 
At visit 1 (screening visit), 15 ml blood will be collected from all subjects for laboratory 
tests (haematology, serum chemistry, pregnancy test (females) and antibodies against 
circulating influenza viruses). Values for the haematology and serum chemistry 
parameters are to be within the normal ranges and pregnancy test negative, screening 
laboratory values outside the normal limits will be accepted into the study only after the 
Investigator or his/her designee has determined that the abnormal values are “not 
clinically significant”. Haematology and serum chemistry parameters for analysis are 
outlined below: 
Haematology 
Serum Chemistry 
Haemoglobin/Haematocrit/Total Total bilirubin/ Uric acid/ Creatinine/ Total 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
47
and differential leukocyte count/ 
Red blood cell count/ Platelet 
count/ MCH/ MCHC/ MCV 
protein/ 
Albumin/ 
AST/ 
ALT/ 
Alkaline 
phosphatase/ 
GGT/ 
Glucose/ 
Urea/ 
Sodium/ 
Potassium/ LDH/ Calcium/ Phosphate/ Cholesterol/ 
Triglycerides/ C reactive Protein 
Serum samples from the collected blood will be stored at -20 oCelsius. At the end of the 
trial, the serum samples will be applied to serology tests to detect antibodies against the 
influenza virus strains that circulated whilst efficacy was monitored. This will help us to 
identify subjects who had pre-existing immunity to the circulating strains prior to the 
trial start. Endpoint analyses will be sub-analyzed with or without these subjects to 
account for subjects with pre-existent antibody responses to the strains of influenza 
circulating during the 2016-2017 season. 
 
At visit 2, 4 and 5 (day 0, 42 and 180), 60 ml blood will be collected from all subjects for 
cellular immunogenicity measurement and FLU-v specific antibody responses. 50 ml of 
the collected blood will be used to isolate PBMCs and the isolated cells should be stored 
for later analysis. 10 ml of the collected blood will be used to measure antibody 
responses specific to FLU-v. Primary CMI measurements will be performed at the Robert 
Koch Institute (Germany). The Norwegian Institute of Public Health (Norway) will 
conduct additional exploratory CMI measurements. At RKI, IFN- ELISA and 
multiparametric FACS analysis will be performed according to the standardized 
procedures developed by the UNISEC consortium with adequate controls.  
All swab samples will be stored at -80° Celsius and later posted to University Medical 
Centre Groningen Laboratory of Clinical Virology on dry ice. The samples will be tested 
for both influenza A and B viruses by generic RT-PCR assay targeting matrix gene. 
Multiplex and strain-specific primers will be used for typing and subtyping respectively.  
Method 
Marker 
Laboratory 
Multi-parametric 
FACS 
analysis 
(Primary CMI) 
Staining 
panel 
Marker 
Purpose 
A 
Viability 
dye 
Exclude 
dead cells 
CD3 
Define 
lineage 
CD4 
Robert 
Koch 
Institute 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
48
CD8 
IFN-γ 
Cytokine 
response 
IL-2 
IL-4 
TNF-α 
 
CD107a 
Cytotoxicity 
Marker 
IFN-γ 
ELISA 
(Co-
primary CMI) 
IFN-γ  
Additional 
CMI 
assays (exploratory 
CMI) 
Cytokine markers such as IFN-γ and 
granzyme B by ELISpot 
Norwegian Institute 
of Public Health 
Antibody assays  
Flu-v-specific antibody responses  
Norwegian Institute 
of Public Health 
RT-PCR 
Influenza A or B 
University Medical 
Centre 
Groningen 
Laboratory 
of 
Clinical Virology 
 
 
 
 
 
(4) 
Laboratory Readouts 
Sampling time 
Assay 
Sample 
Day 
Visit 
number 
Nasal 
and 
tonsil 
swabs sampling 
within 3 days of subject 
symptom report  
RT-PCR 
Blood sampling for 
222 subject 
0 
2 
Multi-parametric FACS analysis (222 
samples), IFN-γ ELISA (222 samples), 
additional CMI assays (a subset of 
samples1) and antibody responses (222 
samples) 
42 
4 
180 
5 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
49
1 All blood samples taken for CMI measurement will be analyzed by multi-parametric 
FACS analysis and IFN-γ ELISA, which are the primary measurements for the CMI 
endpoint. Additional CMI assays (exploratory measurements for the CMI endpoint) will 
only be carried once the results from FACS analysis are complete and validated. The 
exploratory CMI assays will be performed on a subset of samples randomly selected 
from those available for both day 0 (reference) and day 42 and/or day 180.  
 
11.5 Withdrawal of Individual Subjects 
 
A window of ±3 days is allowed for the study visits.  
Subjects will be advised that they are free to withdraw from the study at any time for 
any reason. In addition, the Investigator may withdraw a subject from the study in order 
to protect the subject’s health. Investigators will withdraw subjects according to the 
following criteria: 
 
Withdrawal from the study is, in the Investigator’s judgment, in the subject’s 
best interest 
 
The development of any medical condition excluded in section 10.2  
 
Intolerable/unacceptable adverse experiences 
 
Major violation of study protocol procedures 
 
Subject unwilling to proceed and/or consent is withdrawn 
 
In the event that a subject is withdrawn from the study due to a serious adverse event 
(SAE), the Investigator will evaluate the urgency of the event. If the situation warrants, 
the Investigator will take appropriate diagnostic and therapeutic measures. If the 
situation is not an immediate emergency, the Investigator at the clinical study facility 
will attempt to contact the Medical Monitor for consultation. No medical help, diagnosis, 
or advice will be withheld from the subject due to an inability to contact the Medical 
Monitor. The subject will be encouraged to remain available for follow-up monitoring by 
the Medical Monitor. 
 
The Sponsor will be notified as soon as possible of any subject withdrawals. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
50
 
Subjects who are withdrawn or discontinued after the start of study dosing will not be 
replaced. The investigator will ask the subject to complete the Study Termination Visit. 
The reasons for withdrawal will be recorded on the CRF and included in the final report, 
along with any AEs and any necessary medical treatment. 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
51
12. Safety Report 
The Investigator and site staff are responsible for detection, recording and reporting of 
events that meet the criteria and definition of an AE or SAE (described below). 
 
12.1 Definition of an Adverse Event 
Any untoward medical occurrence occurring after the subject has given informed 
consent, whether or not considered related to the investigational product. 
An AE can therefore be any unfavourable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease (new or exacerbated) occurring after the subject 
has given informed consent. 
Examples of an AE include: 
 Exacerbation of a chronic or intermittent pre-existing condition, including either an 
increase in frequency and/or intensity of the condition 
 A new condition detected or diagnosed after informed consent, even though it may 
have been present prior to this 
 Signs, symptoms, or the clinical sequelae of a suspected interaction 
 Signs, symptoms, or the clinical sequelae of a suspected overdose of either 
investigational product or a concurrent medication. 
Examples of an AE do NOT include: 
 A medical or surgical procedure (e.g. endoscopy); the condition that leads to the 
procedure is an AE 
 Situations where an untoward medical occurrence did not occur (social and/or 
convenience admission to hospital) 
 Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present 
or detected before informed consent was obtained, that do not worsen. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
52
 
12.2 Definition of a Serious Adverse Event 
An SAE is any untoward medical occurrence that: 
 Results in death 
 Is life threatening: this refers to an event in which the subject was at risk of death at 
the time of the event. It does not refer to an event, which hypothetically might have 
caused death, if it were more severe 
 Requires hospitalization or prolongation of an existing hospitalization. In general, 
hospitalization signifies that the subject has been detained (usually involving at least 
an overnight stay) at the hospital or emergency ward for observation and/or 
treatment that would not have been appropriate in the physician's office or out-
subject setting. Complications that occur during hospitalization are AEs. If a 
complication prolongs hospitalization or fulfils any other serious criteria, the event is 
serious. When in doubt as to whether hospitalization occurred or was necessary, the 
AE should be considered serious. Hospitalization for elective treatment of a pre-
existing condition that did not worsen from baseline is not considered an AE 
 Results in disability/incapacity. The term disability means a substantial disruption of 
a person’s ability to conduct normal life functions. This definition is not intended to 
include experiences of relatively minor medical significance such as uncomplicated 
headache, nausea, vomiting, diarrhoea, influenza, and accidental trauma (e.g. 
sprained ankle), which may interfere or prevent everyday life functions but do not 
constitute a substantial disruption; 
 Is a congenital anomaly/birth defect 
 Medical or scientific judgment should be exercised in deciding whether reporting is 
appropriate in other situations, such as important medical events that may not be 
immediately life threatening or result in death or hospitalization but may jeopardize 
the subject or may require medical or surgical intervention to prevent one of the 
other outcomes listed in the above definition. These should also be considered 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
53
serious. Examples of such events are invasive or malignant cancers, intensive 
treatment in an emergency room or at home for allergic bronchospasm, blood 
dyscrasias or convulsions that do not result in hospitalization, or development of 
drug dependency or drug abuse 
 All pregnancies will be reported and followed up. The Investigator will notify the 
Medical Monitor of any women partners who become pregnant during the study 
within 24 hours, who will then notify the Sponsor within 1 business day. Any 
resulting offspring will be monitored for up to 6 months post-partum, unless 
otherwise medically indicated. 
 
12.3 Clinical Laboratory Abnormalities, Other Abnormal Assessments 
Abnormal laboratory findings (e.g. clinical chemistry and haematology) or other 
abnormal assessments (e.g. ECGs, vital signs) that are judged by the Investigator as 
clinically significant will be recorded as AEs or SAEs if they meet the definition of an 
AE or SAE (as defined in Sections 12.1 and 12.2, respectively). In addition, any clinical 
symptom or diagnosis or abnormal laboratory test thought to represent an AIDS 
defining illness will also be classed as a medically significant event and reported as an 
SAE.  
The Investigator will exercise his or her medical and scientific judgment in deciding 
whether an abnormal laboratory finding or other abnormal assessment is clinically 
significant. 
 
12.4 Time Period, Frequency, and Method of Determining AEs and SAEs 
As a consistent method of soliciting AEs, the subject should be asked a non-leading 
question such as "How do you feel?” 
Any pre-existing conditions or signs and/or symptoms present in a subject prior to the 
start of the study (i.e. before informed consent is obtained) should be recorded as 
Medical/Surgical History. Any medical occurrence, which is reported after informed 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
54
consent is obtained but prior to starting active or randomised treatment, will be 
documented as an AE. 
Any signs and symptoms present at the time of informed consent will be documented as 
baseline signs and symptoms. 
All AEs occurring after administration of the dose of study medication and on or before 
the final visit must be reported as AEs. All AEs must be recorded, irrespective of whether 
they are considered drug related. 
At each visit/assessment in the period defined above, AEs will be evaluated by the 
Investigator and recorded. 
Any AEs already documented at a previous assessment and designated as ongoing, 
should be reviewed at subsequent visits as necessary. If these have resolved, this should 
be documented. 
 
12.5 Unblinding 
It is unlikely that knowledge of a subject’s treatment assignment will influence the 
nature of any immediate treatment necessary should a serious adverse event, such as 
anaphylaxis, occur. Where unblinding is deemed appropriate, the Principal Investigator 
should first contact SEEK’s trial medical expert (please refer to 5.2 “Sponsor’s Medical 
Expert for the Trial”) before the blind is broken and the pharmacist or designee should 
not be contacted until unblinding has been agreed. 
In the event of unblinding, a note to file should be prepared and signed by the Principal 
Investigator to include the date and reason for unblinding and personnel involved. The 
original file note should be given to the Unblinded Pharmacist for inclusion in the 
Pharmacy file and copies included in the subject’s notes and site study file. 
To comply with ICH E2A guidance and EU Clinical Trial Directive, any SAE associated 
with 
the 
use 
of 
the 
investigational 
product, 
which 
is 
unexpected 
and 
attributable/suspected is to be unblind prior to regulatory reporting. SEEK’s medical 
expert is responsible for unblinding the treatment assignment in accordance with 
specified time frames for expedited reporting of SAEs. 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
55
12.6 Recording of AEs and SAEs 
 
When an AE/SAE occurs, it is the responsibility of the Investigator to review all 
documentation (e.g. hospital progress notes, laboratory, and diagnostics reports) 
relative to the event. The Investigator will then record all relevant information 
regarding the AE/SAE on the eCRF. It is not acceptable for the Investigator to send 
photocopies of the subject’s medical records to the Sponsor in lieu of completion of the 
appropriate AE/SAE eCRF pages. However, there may be instances when the Sponsor 
requests copies of medical records for certain cases. In this instance, all subject 
identifiers will be blinded on the copies of the medical records prior to submission to the 
Sponsor. 
The Investigator will attempt to establish a diagnosis of the event based on signs, 
symptoms, and/or other clinical information. In such cases, the diagnosis should be 
documented as the AE/SAE and not the individual signs/symptoms. All SAEs that occur 
anytime after informed consent is given until study conclusion or early termination visit 
are considered reportable to the sponsor or sponsor’s designee. Serious adverse events 
occurring after a subject is discontinued from the study will not be reported unless the 
Investigator feels that the study drug or a protocol procedure may have caused the event. 
 
12.7 Evaluating AEs and SAEs 
(1) Assessment of Intensity 
The Investigator will make an assessment of intensity for each AE and SAE reported 
during the study. The assessment will be based on the Investigator’s clinical judgment. 
The intensity of each AE and SAE recorded in the eCRF should be assigned to one of the 
following categories: 
 Mild: an event that is easily tolerated by the subject, causing minimal discomfort 
and not interfering with everyday activities 
 Moderate: an event that causes sufficient discomfort as to interfere with normal 
everyday activities 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
56
 Severe: an event that prevents normal everyday activities. 
An AE that is assessed as severe should not be confused with a SAE. Severity is a 
category utilized for rating the intensity of an event; and both AEs and SAEs can be 
assessed as severe. An event is defined as ‘serious’ when it meets one of the pre-defined 
outcomes, as described in Section 12.2 “Definition of a SAE”.  
(2) Assessment of causality 
The Investigator is obliged to assess the relationship between investigational product 
and the occurrence of each AE or SAE. The Investigator will use clinical judgment to 
determine the relationship. Alternative causes, such as natural history of the underlying 
diseases, concomitant therapy, other risk factors, and the temporal relationship of the 
event to the investigational product will be considered and investigated. The 
Investigator will also consult the IB in the determination of his/her assessment. 
The Investigator will provide the assessment of causality as per the instructions on the 
AE or SAE form. Causality will be assessed by the Investigator according to the following 
definitions:  
 Unrelated: where an event is not considered related to the study drug 
 Unlikely: although the relationship to study drug cannot be completely ruled out, 
the nature of the event, the underlying disease, concomitant medication or 
temporal relationship make other explanations more likely 
 Possibly related: the temporal relationship and the absence of a more likely 
explanation suggest the event could be related to the study drug 
 Probably related: the known effects of the study drug or its therapeutic class, or 
based on challenge testing, suggest the study drug is the most likely cause 
 Definitely related: this category applies to those AEs that are clearly a 
consequence of administration of the drug. It is likely that such events will be 
widely documented and generally accepted as having association with the study 
medication. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
57
The steps taken as a result of an AE/SAE will be recorded, including any treatment 
administered because of the event, any adjustments of the dose of study drug or 
withdrawal of the subject. 
 
12.8 Follow-up of AEs and SAEs 
After the initial AE/SAE report, the Investigator is required to proactively follow each 
subject and provide further information to the Sponsor on the subject’s condition. 
All AEs and SAEs documented at a previous visit/contact and are designated as ongoing, 
will be reviewed at subsequent visits/contacts. 
All AEs and SAEs will be followed until resolution, until the condition stabilizes, until the 
event is otherwise explained, or until the subject is lost to follow-up. Once resolved, the 
appropriate AE/SAE eCRF page(s) and SAE form will be updated. The Investigator will 
ensure that the follow-up includes any supplemental investigations as may be indicated 
to elucidate the nature and/or causality of the AE or SAE. This may include additional 
laboratory tests or investigations, histopathological examinations, or consultation with 
other health care professionals. 
The Sponsor may request that the Investigator perform or arrange for the conduct of 
supplemental measurements and/or evaluations to elucidate as fully as possible the 
nature and/or causality of the AE or SAE. The Investigator is obligated to assist. If a 
subject dies during participation in the study or during a recognized follow-up period, 
the Sponsor will be provided with a copy of any post-mortem findings, including 
histopathology. 
New or updated information will be recorded on the originally completed SAE form, 
with all changes signed and dated by the Investigator. The updated SAE form should be 
resent to the Sponsor. 
All SAEs must be reported to the SEEK Safety Team within 24 hours (or sooner if 
possible) of the Investigator (or designee) becoming aware of the situation. 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
58
12.9 Prompt Reporting of the SAEs to the Sponsor 
All SAEs will be reported promptly to the SEEK Safety Team Medical Monitor:  
Bryan Murray, MBBS 
Boyd Consultants Ltd 
3a Station Cottages 
St Neots, Cambridgeshire 
PE19 1QF 
UK 
 
The SEEK Safety Team will coordinate SAE reporting for all sites participating in this 
study. A form will be used for the collection of SAE data. The SAE form must be 
completed in English.  
The SAE form should be completed as soon as possible from the time of the initial report, 
including the Investigator’s assessment of causality. If the Investigator does not have all 
information to hand regarding an SAE, he/she should not wait to receive additional 
information before notifying the Medical Monitor of the event and completing the form. 
The form can be updated when additional information is received.  
Investigators must notify the Medical Monitor and pharmacovigilence of all SAEs 
(whether considered treatment-related or not and whether expected or not) by email to: 
bryan.murray@boydconsultants.comUpon receipt of an SAE form, the Medical Monitor 
will review the form for completeness and accuracy and for confirmation of the serious 
criteria, expectedness and causality. The Medical Monitor will notify the Sponsor in 
writing of all SAEs within 1 business day of being notified by the Investigator.  
A business day is defined as 1 calendar day, excluding weekends and national holidays. 
The local clinical monitor will be notified by fax/email at the same time. 
The Safety Team will generate queries as needed for all SAEs. If the Sponsor requires 
additional follow up information they will contact the Safety Team by e-mail or fax. The 
Safety Team will contact the Investigator for missing information and for the 
clarification of discrepancies on the initial report. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
59
The Sponsor, in conjunction with the Safety Team, will decide on the need for expedited 
reporting to regulatory authorities, Investigators and the Independent Ethics 
Committees (IECs). The Sponsor is responsible for forwarding reportable SAEs to the 
appropriate regulatory authorities. Reports expedited to regulatory authorities will be 
forwarded to the Project Manager for distribution to all Investigators. The Safety Team 
will ensure that the Investigators notify their IEC as appropriate. 
For medical queries, Investigators should contact the Medical Monitor of the SEEK 
Safety Team: 
 Bryan Murray, MBBS 
 
 
 
Boyd Consultants Ltd 
 
 
 
3a Station Cottages 
 
 
 
St Neots, Cambridgeshire 
 
 
 
PE19 1QF 
 
 
 
UK 
 
12.10 Regulatory Reporting Requirements for SAEs 
The Investigator will promptly report all SAEs to the Sponsor in accordance with the 
procedures detailed in Section 12.9, "Prompt Reporting of SAEs to the Sponsor." Prompt 
notification of SAEs by the Investigator to the appropriate project contact for SAE 
receipt is essential, so that legal obligations and ethical responsibilities towards the 
safety of other subjects are met. 
The Sponsor (or responsible person, according to local requirements) will comply with 
the EU directive 2001/20/EU and the applicable local regulatory requirements related 
to the reporting of SAEs to regulatory authorities.  
 
12.11 Post-Study AEs and SAEs 
Any unresolved AE and SAEs will be followed for up to 30 days after the subject has 
completed the study. Following this time, any suspected unexpected serious adverse 
reactions (SUSARs) will be recorded. If the Investigator learns of any SAE, including a 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
60
death, at any time after a subject has been discharged from the study, and he/she 
considers the event reasonably related to the investigational product, the Investigator 
will promptly notify the Sponsor.  
 
12.12 SAEs Related to Study Participation 
An SAE considered related to study participation (e.g. procedures, invasive tests, a 
change in existing therapy), even if it occurs during the pre- or post-treatment period, 
will be reported promptly to the Sponsor (see Section 12.9, "Prompt Reporting of SAEs 
to the Sponsor").  
 
12.13 Annual Safety Report 
The duration of the trial, from the visit 1 (screening) to study conclusion, is 
approximately a maximum of 1 year for each subject. The Clinical Study Report (or End 
of Study Report) contains a review of safety data gathered. Please refer to 17.4 “End of 
Study Report” for details. A separate safety report will be provided to the ethical 
committee and regulatory authority in the Netherlands. 
 
 
 
 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
61
13. Investigational Medicinal Product 
13.1 Name and Description 
 
(1) FLU-v 
FLU-v is an equimolar mixture of 4 synthetic polypeptides. These sequences represent 
predicted immunoreactive conserved regions identified in silico through multiple 
sequence (ClustalW) and immunogenicity analysis of all Influenza A and B protein 
sequences available at the National Centre for Biotechnology Information (NCBI) 
taxonomy database5. Each polypeptide represents a region of the consensus sequence 
where every consecutive amino acid is present in ≥70% of the sequence population 
tested and where >5 potential human T cell epitopes are found.  
Chemical synthesis is a simpler and more easily scalable manufacturing alternative to 
biological systems (e.g. eggs or cell culture). Also, the final product is safer as the risks of 
contamination with infectious agents (e.g. Campylobacter, Salmonella, etc.), egg allergy 
in the population and increased incidence of Guillain–Barré syndrome are eliminated 
(6,7). Polypeptides are synthetically manufactured by American Peptide Company (USA) 
and 500ug of an equimolar mix will be vialed by Symbiosis (Scotland, UK) under sterile 
conditions in accordance with current Good Manufacturing Practice. 
 
(2) 0.01M HCl and 0.01M NaOH suspension 
FLU-v will be reconstituted in 0.01M HCl and later neutralised with the same volume of 
0.01M NaOH. Both solutions are manufactured by Tiofarma (The Netherlands) under 
sterile conditions in accordance with current Good Manufacturing Practice.  
 
(3) ISA-51 
Montanide ISA-51 is a mixture of a highly purified mineral oil (Drakeol 6VR) and a 
surfactant (Mannide monooleate). When mixed with an aqueous phase in a 50/50 ratio, 
it renders a water in oil emulsion. This water in oil emulsion is used as vaccine adjuvant. 
It is manufactured by Seppic (Air Liquide, France) under sterile conditions in accordance 
with current Good Manufacturing Practice. 
 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
62
13.2 Summary of Findings from Non-Clinical Studies 
 
(1) FLU-v 
A series of pharmacology, safety and toxicology studies have been carried out in two 
animal species, the mouse and the rat. Pharmacology studies have demonstrated that 
FLU-v is able to generate both a cell mediated and humoral response in vaccinated 
animals and that the cell mediated response is enhanced when FLU-v is injected 
together with an adjuvant. A safety pharmacology study demonstrated that FLU-v had 
no biologically relevant effects on cardiovascular or respiratory parameters when 
administered subcutaneously alone or in combination with the adjuvant, in rats. 
Toxicology studies have demonstrated evidence of injection site responses and 
microscopic and macroscopic changes in animals receiving FLU-v and it is thought that 
these were likely to be due to the effects of the adjuvant used in the studies, ISA-51. In 
general, FLU-v was clinically well tolerated with no signs of systemic toxicity. FLU-v was 
also well tolerated in a local tolerance study in rabbits, with no local or systemic sign of 
reaction to treatment.  
 
A safety pharmacology study in rats, under Good Laboratory Practice (GLP) principles, 
was carried out to measure potential side-effects of FLU-v on cardiovascular and 
respiratory parameters. FLU-v was administered subcutaneously alone or in 
combination with the adjuvant, ISA 51, to rats and was found to have no biologically 
relevant effects on cardiovascular or respiratory parameters. 
Study  
Species  
N  
Dose  
Adj  
Methods  
Results  
TR008  
C57BL/6 
mice  
8/ group  
2 doses of 2.5, 5 & 
10nmol (27.6, 
55.3 or 110.6ug) 
FLU-v (tetrav) or 
5nmol NRP-v on 
Days 1 & 15  
None  
Measuremen
t of IFN-γ  
No dose 
response. 
Immune response 
higher with larger 
dosing volume.  
TR009  
C57BL/6 
mice  
8/ group  
2 doses of 2.5, 5 & 
10nmol (27.6, 
55.3 or 110.6 ug) 
FLU-v (tetrav)  
None ISA 
51  
Measuremen
t of IFN-γ. 
Weight loss 
following 
intranasal flu 
challenge  
Greatest IFN-γ 
response in 
lowest dose 
group without 
adjuvant.  
Lowest weight 
loss in lowest 
dose group with 
ISA 51.  

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
63
TR010  
C57BL/6- 
mice  
10/ group  1 dose of 2.5nmol 
(27.6 ug) FLU-v 
(tetrav), NRP-v, 
commercial or live 
flu virus  
ISA 51 
None for 
live virus  
Weight loss 
following 
intranasal flu 
challenge  
FLU-v gave 
equivalent 
protection to 
commercial 
vaccine. Sub-
lethal dose of live 
virus gave best 
protection.  
TR015  
C57BL/6-
Tg HLA-
A*201 
mice  
8/ group  
1 dose of 10nmol 
(110.6 ug) FLU-v 
(tetrav) or NRP-v  
None  
ISA 51  
ISA 720  
Measuremen
t of IFN-γ 
and  
IL-4 
response.  
Increased IFN-γ 
&  
human MHC-
restricted Th1 
immune response 
in FLU-v. Higher 
in both 
adjuvanted 
groups.  
No IL-4 response 
in any group.  
 
Key messages from FLU-v non-clinical studies: 
 FLU-v generates both cell mediated and Ab responses in mice and rats and cell-
mediated responses are enhanced when FLU-v is given with adjuvant. 
 Injection site responses and microscopic and macroscopic changes in animals 
receiving FLU-v likely to be due to the effects of the adjuvant, ISA 51. 
 FLU-v was well tolerated with no signs of systemic toxicity. 
 
(2) HCl/NaOH solution 
Non-clinical studies on HCl/NaOH solution are irrelevant to the study endpoints 
(immunogenicity and safety). 
 
(3) ISA-51  
A number of potential adjuvants for use with the FLU-v vaccine were tested for their 
ability to enhance the immune response. In a number of experiments, the adjuvants ISA 
51, Montanide® ISA 720 (ISA 720) and Quill saponin (QS 21) all boosted the cellular 
immune response to FLU-v, as measured by IFN- γ, although they had no effect on the 
antibody response. 
 
ISA 51 in a final vaccine dose, prepared as instructed, is an emulsion of oil in water in 
which the micelles have an average diameter of approximately 4 μm. As particles 
between 2 and 5 μm are preferentially taken up by phagocytic cells and most antigen 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
64
presenting cells (e.g. dendritic cells) have phagocytic capacity, this peptide adjuvant 
combination increases the delivery of antigen to cells capable of triggering an immune 
response. In addition, these micelles contain, in themselves, both hydrophilic and 
hydrophobic environments and thus can accommodate, and efficiently carry, peptides of 
very different hydrophilic potentials. Therefore ISA 51 was chosen to test the ability of 
FLU-v as an adjuvant to enhance the immunogenicity of the FLU-v vaccine in clinical 
studies in humans. 
 
13.3 Summary of Findings from Clinical Studies  
 
(1) FLU-v 
FLU-v is the only synthetic influenza vaccine that has been clinically shown to elicit a T 
cell response that (a) correlates with reduced viral shedding by 3 logs (r=-0.821; p=0.01) 
and morbidity scores by half (r=-0.786; p=0.02) and (b) recognizes widely divergent 
influenza strains5. For full information please refer to the IB.  
 
(2) HCl/NaOH solution 
Clinical studies on HCl/NaOH solution are irrelevant to the study endpoints 
(immunogenicity and safety). 
 
(3) ISA-51  
Please refer to the ISA-51 IB. 
 
13.4 Summary of Known and Potential Risks and Benefits 
 
(1) FLU-v 
In study EUDRACT No. 2009-014716-35, all subjects reported AEs. 32 subjects reported 
mild AEs (16 FLU-v: 16 Placebo), 10 reported moderate AEs (6 FLU-v: 4 Placebo) and 1 
subject had a severe adverse event (AE)(presyncope- Placebo). There were no serious 
adverse events (SAEs). Injection site AEs related to the investigational medicinal 
product (IMP) were frequently reported. Pain, swelling and erythema were most 
commonly reported by the FLU-v group within 24 hours post-vaccination. AEs related to 
the injection site corresponded with those reported for Montanide ISA-51-VG (Seppic), 
used as an adjuvant in the vaccine and Placebo preparations. 1 FLU-v subject had an 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
65
injection site lump removed 6 months following the study. 4 FLU-v (25%) and 3 Placebo 
(18.8%) subjects reported rhinorrhea which was considered IMP related. In general, 
changes in complete physical examination, clinical chemistry, haematology and 
urinalysis, vital signs, ECG, oral temperature or concomitant medications were not 
clinically significant and were resolved by the end of the study. 2 Placebo subjects had 
abnormal spirometry results at the Day 28 follow-up which were followed up post-study. 
(2) HCl/NaOH solution 
There are no potential risks and benefits expected from HCl/NaOH solution. 
(3) ISA-51 
Refer to 13.4 (1) FLU-v 
 
13.5 Drug Packaging and Labelling 
The study drug product will be supplied by SEEK. The study drug product will be 
manufactured and packaged in accordance with current Good Manufacturing Practice 
(GMP).  Study drug supplies will be stored securely under the appropriate conditions at 
study sites according to the applicable local laws.  Study drug product will be supplied to 
the study site after receipt of required documents in accordance with all applicable 
regulatory requirements and SEEK procedures. Labels will be in accordance with all 
applicable regulatory procedures.  
 
13.6 Product Accountability 
 
The trial site pharmacies will be responsible for recording the receipt of all vaccine 
supplies and for ensuring the supervision of the storage and allocation of these supplies. 
When a shipment is received, an assigned qualified person verifies the quantities 
received and the accompanying documentation and returns the acknowledgment of 
receipt to the Sponsor.   
 
Drug administration will be recorded in the source documents, in the eCRFs and in the 
Drug Administration Record form. The latter includes the subject identification, quantity 
(volume) and date of administration. The containers from which the vaccine was 
administered to the subjects will be retained for dose confirmation. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
66
At the end of the study, delivery records of study vaccine will be reconciled with used / 
unused stocks and appropriate forms will be completed, to verify that all used, unused 
or partially used supplies have been returned and that no study supplies remain in the 
Investigator’s possession.  
All unused vaccine supplies, partially used and empty containers will be returned to the 
Sponsor.  
 
13.7 Study Drug Handling and Storage 
Only subjects enrolled in the study may receive the study drug, in accordance with all 
applicable regulatory requirements. 
 Only authorized site staff may supply or administer the study drug. The study drug must 
be stored in a secure area with access limited to the Investigator and authorised staff 
only. 
Authorised study site staff (e.g. a pharmacist) will verify and dispense each subject’s 
study treatment supply and equipment. Subjects will receive their study dose under 
clinical supervision and observation.  
 
13.8 Accountability of Study Supplies 
 
All materials supplied are for use only in this clinical study and should not be used for 
any other purpose. 
 
The Investigator is responsible for the investigational product accountability, 
reconciliation and record maintenance. In accordance with all applicable regulatory 
requirements, the Investigator or designated site staff must maintain investigational 
product accountability records throughout the course of the study. The Investigator or 
designated site staff will document the amount of investigational product received from 
SEEK and the amount administered to subjects. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
67
 
A Drug Dispensing Log must be kept current and will contain the following information: 
 The identification of the subject to whom the drug was dispensed 
 The date(s) of the drug dispensed to the subject. 
 
The inventory must be available for inspection by the study monitor at any point during 
the study. Drug supplies, excluding empty containers, will either be collected at the end 
of the study by the study monitor or returned by the Investigator or designee to SEEK. 
When requested in writing by the Sponsor, unused drug supplies may be destroyed by 
the Investigator provided such disposition does not expose humans to risks from the 
drug. Records will be maintained by the Investigator of any such alternate disposition of 
the test drug. These records must show the identification and quantity of each unit 
disposed of, the method of destruction (taking into account the requirements of local 
law), and the person who disposed of the test substance. Such records must be 
submitted to the Sponsor. 
 
13.9 Retention of Samples 
 
It will be the responsibility of SEEK to ensure adequate samples of all study drug are 
retained in accordance with the regulatory guidelines. 
 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
68
14. Statistical Considerations 
14.1 Endpoints 
(1) Primary 
- Cellular immunogenicity: 
Cellular immune responses based on change from baseline (Day 0) in TH1 cytokines 
(IFN-γ, TNF-α, IL-2) on days 42 and 180. 
- Co-Primary Safety 
To evaluate the occurrence and intensity of all AEs (i.e. solicited and unsolicited, local 
and systemic) and their relationship to vaccination based on the AE diary 
card/questionnaire returned on day 21 and 42 
To evaluate the occurrence of SAEs and their relationship to vaccination based on 
reports collected during the entire study period.  
(2) Secondary 
 Change from baseline (Day 0) in cellular immune TH2 cytokine responses (IL-4) 
on day 42 and 180  
 Humoral immune responses (specific to FLU-v) on days 0, 42 and 180  
(3) Exploratory 
- Cellular immunogenicity: 
Cellular immune responses will be evaluated by additional CMI assays on day 0, 42 and 
180 in all groups, in a subset of subjects. 
Effect of previous influenza vaccines on the immunogenicity of FLU-v will be assessed in 
a post-hoc analysis after stratifying the subjects’ data based on whether they have ever 
been exposed to other influenza vaccines and if so, if it was in the previous 24 months or 
longer. 
- Clinical efficacy 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
69
The efficacy of FLU-v vaccine in the reduction of the incidence of RT-PCR-confirmed 
influenza A and/or B infections and the reduction of symptom score among the 
confirmed influenza infection cases. 
The relationship between clinical efficacy and immune responses will be explored. 
Exploratory endpoints may be reported separately from the main clinical study report. 
 
14.2 Estimated Sample Size 
Vaccination with FLU-v generates a population of T cells that can recognise conserved 
influenza antigens on the surface of infected cells. On recognition, T cells are activated 
and secrete Th1 cytokines such as INF-, TNF-α, and IL2 and release granzyme and 
perforin. In addition, FLU-v specific non-neutralising antibodies may also bind to these 
antigens on the surface of infected cells activating complement and cytotoxic responses 
lead by Natural Killer (NK) cells. The T cell and antibody actions will result in the 
destruction of infected cells.  
The objectives of this study are to determine the safety of the vaccine and assess which 
one of the two treatment groups, one dose adjuvanted vs two dose non-adjuvanted, 
induces the strongest immune response which is measured by quantifying the increase 
in Th1 and non-neutralising antibody responses after vaccination compared to baseline 
and corresponding placebo vaccination. The relationship between clinical efficacy and 
the immune response will also be explored. 
Although a variety of immune parameters will be analysed, the minimum sample size 
has been determined on the basis of influenza-specific IFN-γ responses, one of the most 
important cell mediated immunity for influenza protection. The calculations were 
conducted based on results from the earlier Phase I trial, FLU-v-001 and full details are 
in a separate sample size document (13th March 2015).  
The primary analysis objective of the study is to assess whether the active treatment 
(vaccine), in particular the adjuvanted version (administered as emulsion), produces a 
greater TH1 cytokine response than when given as a suspension (i.e. no adjuvant). It is 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
70
therefore necessary to power the study to detect an interaction between the effects of 
treatment (i.e. vaccine versus placebo) and delivery method (adjuvanted or emulsified 
versus saline or solution). If there is such an interaction i.e. if the effect of the adjuvanted 
vaccine compared to placebo is greater than the effect of the vaccine when given as a 
solution compared to placebo then the study should be able to detect this. Conversely, if 
there is no such effect, then it is possible to test the overall effect of the vaccine relative 
to the control, irrespective of whether it is delivered as an emulsion or as a solution. 
Another requirement for the study is to estimate the difference between the adjuvant 
and non-adjuvant administration of the vaccine. 
 
The proposed plan is to do a weighted allocation of patients to treatments, with twice as 
many patients allocated to the active treated group than to the placebo. Since there are 
two factors namely treatment and formulation, the study will follow a factorial design, 
so there are effectively 4 groups of patients. Three scenarios were used to derive the 
sample size and to investigate the power, of which the first (see below) was used as the 
model to drive power calculations. All calculations assumed that the final data will be 
log-transformed since the data is likely to be very skewed with some high values. 
Furthermore, analysing logged data allows estimates to be produced of fold-increases in 
cytokine responses, which are perhaps more readily interpretable. The assumptions are 
set out in Table 1. 
 
Table 1 Scenario used for power calculations (Numbers in cells contain loge response) 
 
 
Scenario 
Relevant tests 
Treatment 
Adjuvant therapy 
No 
Yes 
 2-fold increase 
Active non-adj vs. 
placebo non-adj 
 5-fold increase 
Active adj vs. 
placebo adj. 
 2.5-fold Increase 
adj. vs. non-adj 
(active) 
 
 
Interaction, 
Active vs placebo 
(adj) 
Adj vs non-adj 
(active) 
Placebo 
2.5 
2.5 
Active 
3.2 
4.1 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
71
 
 
The sample size calculations were conducted using PROC GLMPOWER in SAS® v9.4. 
This allows you to vary the input assumptions as well as investigating specific tests for 
the various treatment combinations. The analysis would be conducted using analysis of 
variance. The inclusion of baseline information may be performed using analysis of 
covariance. 
A 2-sided Type I error (alpha) of 5% was assumed throughout. The target power was 
80%. A standard deviation of 1 was assumed, and calculations have been done for equal 
allocation and also 2:1 allocation (active relative to placebo). 
The most important test is that of the interaction between the effects of treatment and 
adding the adjuvant therapy. If this is non-significant then it is possible to conduct an 
overall test of the treatment effect. In particular, for Model 1, where there is an 
interaction, it is important to be able to detect this. Table 2 shows the total number of 
patients required for the study, in order to achieve 80% power for the various tests.  
Table 2: Total sample size for study 
 
s.d.=1.0 
Test 
1:1 allocation 
2:1 allocation 
Interaction 
160 
180 
Active adj vs. 
placebo adj. 
132 
150 
Adj. vs. non-adj 
(active) 
80 
66 
 
The sample sizes in Table 2 do not allow for loss to follow up, assay problems or any 
other major protocol deviations. If a withdrawal rate of 15% is assumed, the figures 
need to be divided by 0.85. So, for example, 188 patients would need to be recruited to 
achieve 160 evaluable patients. 
The final choice of sample size needs to be made considering the relative importance of 
the various tests and the likelihood of the proposed treatment differences occurring. As 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
72
a first step the interaction test is the most important: if there is no interaction the 
numbers required on each treatment to test for an overall treatment effect are relatively 
small. To test for interaction, equal allocation is more efficient than unequal allocation. 
Based on all the information provided, a sample size of 222 subjects was chosen, 
assuming loss to follow up of approximately 20%, to provide 80% power to test the 
interaction between formulation and treatment based on the assumptions using a 2:1 
allocation and a s.d = 1. 
 
14.3 General considerations 
1). General 
A Statistical Analysis Plan (SAP) will be developed according to the CONSORT guidelines 
for the conduct and report of trials. This will provide full details of the analyses to be 
conducted, as well as methods of handling missing data. It will also provide table shells 
for the summary tables in the report. All subjects enrolled in the trial will be accounted 
for using a flowchart or table: this will provide numbers of subjects recruited, 
randomized and followed up as well as details of withdrawals. All subject data will be 
listed in data listings according to treatment group. Tables will be produced containing 
descriptive statistics, by treatment group, of the efficacy and safety variables, 
demographic variables, extent of exposure and other relevant outcomes. For continuous 
variables these will comprise mean (arithmetic or geometric as appropriate), median, 
standard deviation, minimum and maximum as well as the number of observations. For 
categorical variables counts and percentages will be shown. Graphs will be used where 
appropriate to complement the use of summary tables. 
Any deviation from the original statistical plan will be described and justified in the 
study report. 
 
2). Analysis populations 
Three analysis populations will be defined as follows: 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
73
 Intention to treat (ITT): comprising all subjects randomized to receive treatment, 
irrespective of whether they receive any injections. The Full Analysis Set (FAS) 
will be derived from this population to include any subject with at least one set of 
post-vaccination data (i.e. at either Day 42 or Day 180)  
 Per Protocol (PP): comprising those subjects who receive both injections, as per 
the randomisation schedule who provide a blood sample for ELISA determination 
of IFN-γ on Day 0, Day 42 and Day 180, and who do not have any major protocol 
violations regarding to eligibility criteria or study procedures 
 Safety: comprising all subjects who received at least one injection 
The primary efficacy analysis (TH1 cytokine response) will be conducted on the FAS, 
with the PP population providing supporting evidence of efficacy. All secondary analyses 
will be conducted on the ITT population; an additional analysis of the clinical efficacy of 
the vaccine based on flu symptom scores may also be conducted on the PP population if 
numbers allow. 
Safety analyses will be conducted using the safety population. 
All analysis populations will be determined at the end of the study when the database is 
complete before breaking the randomisation code in order to avoid any bias. 
 
14.4 Methods of analysis 
1) Primary outcome 
The change from baseline in TH1 cytokines will be compared between the treatment 
groups using mixed models repeated measures analysis (MMRM), with the baseline (Day 
0) level as a covariate. This takes into account the correlation between repeated 
observations made on the same individual. The model will include subject effects as 
random and fixed effect terms for age group (corresponding to the randomisation 
strata), time (day 42, 180), treatment (FLU-v or placebo), formulation (solution or 
emulsion) and the interaction between treatment and formulation. The terms 
comprising treatment formulation and their interaction together allow for the 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
74
evaluation of the overall difference between the four treatments. The significance of the 
interaction effect tests whether the effect of FLU-v relative to placebo differs according 
to whether it is administered as a solution or as an emulsion (i.e. adjuvanted). Additional 
terms may also include the interaction between time and each of the 
treatment/formulation terms to see whether the response over time differs.  Since the 
data are likely to be skewed with some very high values, the data will be loge 
transformed before analysis. 
The analysis will provide estimates of the difference between groups at each time (Day 
42 and 180) on the loge scale which can be back-transformed to give ratios of geometric 
means. These therefore provided estimates of the fold-increases in response. The 
absolute means will give estimates of the ratio relative to baseline. All estimates of 
treatment differences will be accompanied by 95% confidence intervals. Summary 
tables and graphs will also be produced. 
2) Secondary outcomes 
The change from baseline in humoral response and TH2 cytokine (IL-4) will be handled 
in a similar manner to that described in 1).  
3) Safety 
For each of the following the incidence rate (number and percentage of subjects) will be 
presented for each vaccine group with associated 95% confidence intervals where 
appropriate:   
(i) At least one AE (solicited and unsolicited);  
(ii) At least one systemic AE (solicited and unsolicited);  
(iii) With each individual solicited AE (local and systemic) and  
(iv)  Any AE (by preferred term, MedDRA- see below)) 
All adverse events will be coded according to the coding dictionary (MedDRA version 
17.1 or higher) for System Organ Class (SOC) and Preferred Term (PT). 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
75
Formal hypothesis testing will not be conducted on the adverse event data since the 
study is not powered to detect differences in adverse event incidence. The absence of 
statistically significant differences would not mean that there is no difference in the 
safety profile. Estimates and confidence intervals therefore provide a more useful means 
of evaluating any difference in the safety profile. Graphs will be used to complement 
tables where appropriate. 
- SAEs  
For each vaccine group the percentage of subjects with at least one SAE will be reported 
with 95% CI. SAEs and withdrawals due to AEs will be described in detail.  
 4) Clinical efficacy 
For each vaccine group, the incidence rate of subjects with RT-PCR confirmed influenza 
A and/or B infection will be presented with 95% CI.  
The severity of the influenza symptoms (based on the diary card with 1-3 score) among 
the laboratory-confirmed influenza cases will be summarised in tables of descriptive 
statistics but no formal statistical analysis will be performed as it is likely that the 
numbers of such subjects will be small (approximately 10-20% of subjects).  
An exploratory analysis looking at the relationship between clinical efficacy (i.e infection 
rates and reductions in symptom scores) and the cellular and humoral responses will be 
performed. This will include graphical summaries of the data. 
14.5 Handling of missing values 
The MMRM method of analysis planned for the cellular and humoral responses is fairly 
robust to missing data under certain assumptions related to whether data are missing at 
random. The pattern of missing data in the four treatment arms will be assessed by 
plotting data according to withdrawal time to see whether the pattern of time differs 
accordingly. Sensitivity analysis will be conducted, for example using multiple 
imputation, and from the PP analysis to see whether the conclusions are similar. Note 
that missing data may arise through problems with assays: these are expected to be 
random occurrences and so the MMRM analysis should provide good estimates of 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
76
treatment effects even if some values are unusable. Note that every effort should be 
made to ensure that subjects are followed up and attend clinic visits in order to 
minimize the amount of missing data. 
Sensitivity analyses may be conducted excluding any values which appear to be outliers 
to investigate their influence on results. If there is scientific evidence or explanation for 
one or more outliers, this will be presented and the results of the analyses will be 
discussed in the light of such explanations. However, the primary analysis will be that 
based on all data in the FAS. Note that by log transforming the data prior to analysis, the 
number of outliers may be reduced.  
All summary tables will show the number of cases with missing data. 
14. Multiplicity 
Since there are several primary outcome variables, namely the four TH1 cytokines, there 
is a risk of inflation of the Type 1 error (i.e. obtaining false positive results). No formal 
adjustment of significance levels will be performed. However, the discussion of the 
results and the conclusion being drawn will be made in the light of the actual 
significance levels from the tests. In addition, the results of the analyses of each cytokine 
will be shown in a Forest plot to aid interpretation and to see whether results are 
consistent for all cytokines measured. A 2-fold increase in TH1 cytokine production 
would represent an important treatment effect, so this will also be taken into account 
when discussing the study results. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
77
15. Ethics 
15.1 Ethical Review Committees 
The trial study protocol will be submitted to and approved by the Central Commission 
Research Involving Human Subjects in the Netherlands (Centrale Commissie 
Mensgebonden Onderzoek, CCMO).  
 
The protocol will also be submitted to the Minister of Health, Welfare and Sport for the 
marginal review. If requested by medical ethical review committees and the Dutch 
health authority access may be given to medical records related to the program as stated 
by national law.  
 
The following international conventions and declarations will be respected: 
- 
Helsinki Declaration in its latest version; 
- 
Convention of the Council of Europe on Human Rights and Biomedicine signed in 
Oviedo on 4 April 1997, and the Additional Protocol on the Prohibition of Cloning 
Human Beings signed in Paris on 12 January 1998; 
- 
Beneficiaries should take into account to the opinions of the European Group of 
Advisers on the Ethical Implications of Biotechnology (1991 -1997) and the 
opinions of the European Group on Ethics in Science and New technologies (as 
from 1998); 
- 
The Council of Europe additional Protocol to the European Convention on Human 
Rights and Biomedicine on Biomedical Research (CETS No.:195). 
 
All relevant national and international provisions on data protection, subject’s right and 
informed consent will be met, with the most up-to-date regulations. An ethics evaluation 
team is established for periodic discussion about relevant ethical issues, monitoring of 
ethical standards and a common statement on all ethical issues. 
 
15.2 Ethical Conduct of the Study 
The study will be conducted in accordance with the ethical principles that have their 
origins in the Declaration of Helsinki. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
78
All existing ethical and safety provisions of the Netherlands and of the EU will be strictly 
adhered. Relevant EU legislations include: 
- 
The Charter of Fundamental Rights of the EU; 
- 
Repeal of Directive 2001/20/EC of the European Parliament and of the Council of 
4 April 2001 on the approximation of the laws, regulations and administrative 
provisions of the Member States relating to the implementation of good clinical 
practice in the conduct of clinical trials on medicinal products for human use; 
- 
Commission Directive 2005/28/EC of 8 April 2005 laying down principles and 
detailed guidelines for good clinical practice as regards investigational medicinal 
products for human use, as well as the requirements for authorization of the 
manufacturing or importation of such products; 
- 
Directive 95/46/EC of the European Parliament and of the Council of 24 October 
1995 on the protection of individuals with regard to the processing of personal 
data and on the free movement of such data and the revision of this directive 
once come into force; 
- 
Council Directive 83/570/EEC of 26 October 1983 amending Directives 
65/65/EEC, 75/318/EEC and 75/319/EEC on the approximation laid down by 
law, regulation, or administrative action relating to proprietary medicinal 
products; 
- 
Directive 98/44/EC of the European Parliament and of the Council of 6 July 1998 
on the legal protection of biotechnological inventions;  
 
15.3 Description of Ethical Considerations Relating to the Trial  
The trial is based entirely on voluntary participation.  
 For each study visit, the subject will receive a travel compensation of 
approximately €15.00 and will only be given when the subject presents at each of 
the scheduled visits and when the corresponding travelling receipts are 
presented. 
 No other compensation will be given. 
 The vaccines will be provided by the sponsor, SEEK. All vaccines have been 
produced to GMP standard.  

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
79
Procedural sampling i.e. nasal and tonsil swabs, blood sampling, will be performed 
according to standard clinical practice.  
Over the course of the study, a total of 3 additional blood samples will be taken from all 
subjects.  
All study procedures will be performed by experienced health care personnel and to 
standard clinical practice.  
No serious risks are anticipated for the subjects.  
 
15.4 Information and Consent 
Prior to screening for the study each subject will be informed in detail about the study 
vaccine to be administered, and the nature of the clinical investigation with its risks and 
discomforts to be expected. The basic elements of informed consent as specified by the 
FDA (21 CFR 50.25) and ICH-GCP will be followed. The subjects will also be instructed 
that they are free to withdraw their consent and discontinue their participation in the 
study at any time without prejudice. Written consent will be obtained from each subject 
to be involved in the clinical trial by using the CCMO-approved Informed consent form 
(ICF) prior to the conduct of any study-related activity. A copy of the ICF will be 
submitted together with this protocol and must be approved by the CCMO prior to study 
commencement. Each subject will be given a copy of the written ICF, and each subject’s 
chart will include the signed ICF for study participation. The original subject signed and 
dated ICFs will be maintained by the site for 15 years.  Regulatory authorities may check 
the existence of the signed ICF in this central study folder if not having done so during 
the study.   
Information regarding the inclusion lab tests, pregnancy test (for females), nasal and 
tonsil swabs and details of the medical register data/records usage will be provided in 
the SIS and ICF.  Each Investigator involved will be responsible for the adequate storage 
of informed consents according to good clinical practice and national regulations.  
Prior 
to 
the 
commencement, 
and 
where 
applicable, 
copies 
of 
ethical 
approvals/opinions/notifications by the CCMO and the Minister of Health, Welfare and 
Sport will be submitted to the European Commission. 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
80
15.5 Compensation for Injury 
Appropriate liability insurance to cover against injury and damages arising from 
participation in the study will be provided by the Sponsor. 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
81
16. Provisional Timelines for the Study Phases 
The duration of the trial, from visit 1 (first subject first visit) to study conclusion will be 
a maximum of 1 year. 
The recruitment is estimated to take 1-4 months.  
 
Prof. Eelko Hak is the working package leader on the clinical trial program (WP6) within 
the UNISEC Consortium and scientific coordinator of the trials conducted within this 
working package. Other members of his team include Marcy Heng Liu, postdoc in 
vaccinology, Eva van Doorn, PhD research fellow, and Denise Mailly, senior data 
manager at TCC.  
The main tasks of this steering committee are to supervise the protocol for scientific 
content as well as the data management, statistics and report of the trial.   
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
82
17. Administrative Aspects, Monitoring and Publication 
17.1 Monitoring and Quality Assurance 
(1) Study Monitoring 
GCP stipulates that all clinical studies are adequately monitored. SEEK is responsible for 
ensuring the proper conduct of the study with regards to protocol adherence and the 
validity of the data recorded on the eCRFs. Subject confidentiality will be maintained 
throughout. 
In accordance with applicable regulations, GCP, and SEEK procedures, SEEK study 
monitors or monitors appointed by SEEK (e.g. monitors employed by a Contract 
Research Organization) will contact the site prior to the subject enrolment to review the 
protocol and data collection procedures with site staff. In addition, the monitor will 
periodically contact the site, including conducting on-site visits. The extent, nature and 
frequency of on-site visits will be based on such considerations as the study objective 
and/or endpoints, the purpose of the study, study design complexity, and enrolment rate. 
During these contacts, the monitor will: 
 
Check the progress of the study 
 
Review study data collected 
 
Conduct source document verification 
 
Identify any issues and address their resolution. 
This will be done to verify that the: 
 
Data are authentic, accurate, and complete 
 
Safety and rights of subjects are being protected 
 
Study is conducted in accordance with the currently approved protocol (and 
any amendments), ICH-GCP guidelines and all applicable regulatory 
requirements. 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
83
The Investigator will agree to allow the monitor direct access to all relevant documents 
and to allocate his/her time and the time of his/her staff to the monitor to discuss 
findings and any relevant issues. 
At study closure, monitors will also conduct the following activities in conjunction with 
the Investigator or site staff as appropriate: 
 Return of all study data to SEEK 
 Ensure timely resolution of outstanding data queries 
 Accountability, reconciliation and arrangements for unused investigational 
product 
 Review of site study records for completeness. 
(2) Quality Assurance 
To ensure compliance with ICH-GCP and all applicable regulatory requirements, SEEK 
may conduct a quality assurance audit. Regulatory agencies may also conduct a 
regulatory inspection of this study. Such audits/inspections can occur at any time during 
or after completion of the study. If an audit or inspection occurs, the Investigator and 
institution agree to allow the auditor/inspector direct access to all relevant documents 
and allocate his/her time and the time of his/her staff to the auditor/inspector to 
discuss findings and any relevant issues. 
(3) Records Retention 
SEEK will retain all original eCRFs, while the Investigator will retain a copy. Following 
closure of the study, the Investigator must maintain all site study records in a safe and 
secure location. The records must be maintained to allow easy and timely retrieval, 
when needed (e.g. for an audit or inspection) and where feasible, to allow any 
subsequent review of data in conjunction with assessment of the facility, supporting 
systems and staff. When permitted by local laws/regulations or institutional policy, 
some or all of these records can be maintained in a format other than hard copy (e.g. 
microfiche, scanned, electronic); however, caution needs to be exercised before such 
action is taken. The Investigator must assure that all reproductions are legible and are a 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
84
true and accurate copy of the original and meet accessibility and retrieval standards, 
including re-generating a hard copy, if required. Furthermore, the Investigator must 
ensure there is an acceptable back-up of these reproductions and that an acceptable 
quality control process exists for making these reproductions. 
SEEK will inform the Investigator of the time period for retaining these records to 
comply with all applicable regulatory requirements. The minimum retention time will 
meet the strictest standard applicable to that site for the study, as dictated by any 
institutional requirements or local laws or regulations, or SEEK standards/procedures; 
otherwise, the retention period will default to 15 years. 
The material to be stored shall include the following: 
 
Signed and dated copy of the final study protocol and any amendments 
 
Signed and dated Confidentiality Agreements 
 
Signed and dated letter of IEC approval, letter of constitution of the IEC and 
copies of any other correspondence relevant to the study with the IEC or 
regulatory authorities 
 
The IEC-approved subject information (SIS) and ICF 
 
Current curriculum vitae (signed and dated) of the Investigator and co-
workers with major responsibilities in the trial 
 
Sample eCRF 
 
Signed subject ICF 
 
Subject screening, identification and enrolment logs 
 
Complete authorised drug accountability and reconciliation record 
 
Authorised signature and delegation list of investigator site staff 
 
Laboratory certificates and reference ranges (signed and dated) 
 
The End of Study Report 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
85
 
Clinical raw data including the Medical Source Data Forms, all clinical 
laboratory report forms, subject eCRFs, drug accountability forms, and 
dispensing records, etc. 
 
17.2 Amendments 
All substantial amendments will be notified to the national regulatory and local medical 
ethical review committees in the country conducting the trial. Written approval of any 
substantial amendments will be obtained from such authorities before implementation. 
A ‘substantial amendment’ is defined as an amendment to the terms of the medical 
ethical review committee application, or to the protocol or any other supporting 
documentation, that is likely to affect to a significant degree: 
- 
The safety or physical or mental integrity of the subjects of the trial; 
- 
The scientific value of the trial; 
- 
The conduct or management of the trial; or 
- 
The quality or safety of any intervention used in the trial. 
Non-substantial amendments will not be notified to the authorities but will be recorded 
in the Trial Master File by the sponsor.  
 
17.3 Annual Progress Report 
The current study will last 1 year approximately (from first subject first visit to study 
conclusion). A summary of the progress of the trial in line with ICH Topic E2F will be 
submitted by SEEK to the relevant ethical review committees.  
 
17.4 End of Study Report 
SEEK will notify the relevant regulatory authorities and ethical review committees of the 
end of the study within the time frame requested. An End of Study Report will be 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
86
submitted to the relevant regulatory authorities within one year of the end of the trial. In 
case the study is ended prematurely, SEEK will notify the relevant regulatory authorities 
within the time frame requested, including the reasons for the premature termination.  
Exploratory endpoints may be reported separately from the main clinical study report. 
 
17.5 Public Disclosure and Publication Policy 
In the grant proposal for the FP7-UNISEC project, the following statements are stated: (1) 
“The research results will be made suitable for (further) publications in international 
scientific journals. The partners in UNISEC will actively pursue publications, as these are 
read across Europe by the target groups of the dissemination of foreground.” (2) “A 
“Plan for the use and dissemination of foreground intellectual property” will be 
prepared and maintained throughout the project duration and finalized at the end of the 
project. Prior to entering the European Commission contract, appropriate actions 
regarding knowledge management will be undertaken concerning the identification of -, 
access to - and exclusion of background intellectual property (IP), resulting in separate 
agreements. The coordinator will be responsible for the coordination of these actions 
and the development and maintenance of the “Plan for the use and dissemination of 
knowledge” and for ensuring compliance with articles II.29-30 of Part C of Annex II of 
the FP7 Contract. The policy of open access to scientific findings and publications 
resulting from publicly funded research, and the need to take into account the challenge 
of intellectual property (see Commission Recommendation C(2012) 4890), will be 
considered by the beneficiaries according to clause 39(on open access) of the FP7 model 
grant agreement.” 
 
 

 
 
FLUv 003 Protocol v4.1 -Dated 28 April 2017            |Evaluation of FLU-v vaccine-RCT 
87
18. References 
1. Pleguezuelos O, Robinson S, Stoloff GA, Caparrós-Wanderley W. Synthetic 
Influenza vaccine (FLU-v) stimulates cell mediated immunity in a double-blind, 
randomised, placebo-controlled Phase I trial. Vaccine. 2012;30(31):4655-60.  
2. WHO. Influenza Fact Sheet 2009.  
http://www.who.int/mediacentre/factsheets/fs211/en  
3. http://ec.europa.eu/health/ph_threats/com/Influenza/docs/seasonflu_rec2009_
en.pdf 
4. Tan PT, Khan AM, August JT. Highly conserved influenza A sequences as T cell 
epitopes-based vaccine targets to address the viral variability. Hum Vaccine. 
2011;7(4):402-9. 
5. Boon AC, de Mutsert G, van Baarle D, Smith DJ, Lapedes AS, Fouchier RA et al. 
Recognition of homo- and heterosubtypic variants of influenza A viruses by 
human CD8+ T lymphocytes. J Immunol. 2004;172(4):2453-60. 
6. Chua JV, Chen WH. Bench-to-bedside review: vaccine protection strategies during 
pandemic flu outbreaks. Crit Care. 2010;14(2):218.  
7. Evans D, Cauchemez S, Hayden FG. "Prepandemic" immunization for novel 
influenza viruses, "swine flu" vaccine, Guillain-Barré syndrome, and the detection 
of rare severe adverse events. J Infect Dis. 2009; Aug 1;200(3):321-8.