First-in-human Trial of the Coronavirus Virus-like Particle Subunit Vaccine ABNCoV2 in SARS-CoV-2-naïve Adult Volunteers in Good Health
Phase: PHASE1
Conditions: Covid19, Severe Acute Respiratory Syndrome, SARS-CoV-2 Infection
Sponsor: Radboud University Medical Center

NL76192.000.20 / COUGH-1 
Safety and tolerability of ABNCoV2 
 
 
 
 
 
 
 
 
 
 
 
CLINICAL TRIAL PROTOCOL 
 
First-in-human trial of the Coronavirus virus-like particle subunit vaccine 
ABNCoV2 in SARS-CoV-2-naïve adult volunteers in good health 
 (COUGH-1) 
 
Version 1.6 
Date: 31 March 2021 
 
 
 
 
Confidentiality Statement 
 
This document is confidential and is to be distributed for review only to investigators, potential 
investigators, consultants, study staff, and applicable Independent Ethics Committees or Institutional 
Review Boards. The contents of this document shall not be disclosed to others without written 
authorization from CVIA (or others, as applicable). 
 
 
 

NL76192.000.20 / COUGH-1 
Safety and tolerability of ABNCoV2 
 
 
 
 
PROTOCOL TITLE ‘First-in-human trial of the Coronavirus virus-like particle subunit vaccine 
ABNCoV2 in SARS-CoV-2-naïve adult volunteers in good health’ 
 
Protocol ID 
COUGH-1 
Short title 
Safety and tolerability of ABNCoV2 
EudraCT number  
2020-004621-22 
Version 
1.6 
Date 
31 March 2021 
Protocol authors 
Benjamin Mordmüller, MD 
Jessica Brosnahan 
Meral Esen, MD 
Diane Egger-Adam, PhD 
Matthew McCall, MD, PhD 
Merel J. Smit, MD 
Morten Agertoug Nielsen, PhD 
Principal investigator(s)  
Benjamin Mordmüller, MD  
Radboudumc, Department of Medical Microbiology  
Tel: +31 (0)24 3619499 
Email: benjamin.mordmueller@radboudumc.nl 
Coordinating investigator 
Matthew McCall, MD, PhD 
Radboudumc, Department of Medical Microbiology 
Tel: +31 (0)24 3615363 
Email: matthew.mccall@radboudumc.nl 
Clinical investigator(s) 
Merel J. Smit, MD 
Radboudumc, Department of Medical Microbiology 
Tel: +31 (0)24 3619515 
Email: merel.smit@radboudumc.nl 
Sponsor 
Stichting Radboud universitair medisch centrum 
Department of Medical Microbiology  
Geert Grooteplein-Zuid 10 

NL76192.000.20 / COUGH-1 
Safety and tolerability of ABNCoV2 
 
 
 
 
6525 GA, Nijmegen, The Netherlands  
Sponsor representative:  
Heiman Wertheim, MD, PhD 
Clinical Microbiology Laboratory  
Tel: +31 (0)24 3614281 
Email: Heiman.Wertheim@radboudumc.nl 
Subsidising party 
European Union Horizon 2020 research and innovation 
action (RIA) project 101003608, PREVENT-nCoV 
Independent expert (s) 
Arjan van Laarhoven, MD, PhD 
Radboudumc Department of Internal Medicine 
Tel: 
24 361 69 80
(0)
+31 
. 
Email: Arjan.vanLaarhoven@radboudumc.nl 
Laboratory sites 
Eric Aaldring 
Radboudumc Clinical Chemical Laboratory  
Tel: +31 (0)24 3616997 
Email: Eric.Aaldring@radboudumc.nl 
Heiman Wertheim, MD, PhD 
Clinical Microbiology Laboratory  
Tel: +31 (0)24 3614281 
Email: Heiman.Wertheim@radboudumc.nl 
Pharmacy 
Rob ter Heine, PharmD, PhD 
Radboudumc Clinical Pharmacy – Clinical Trial Unit  
Tel: +31 (0)24 3616405 
Email: R.terHeine@radboudumc.nl 
Antibody Assays 
Morten Nielsen, PhD 
University of Copenhagen 
Faculty of Health Science 
Institute for Immunology and Microbiology 
Centre for Medical Parasitology 
Tel: +45 35326803 
Email: mortenn@sund.ku.dk 

NL76192.000.20 / COUGH-1 
Safety and tolerability of ABNCoV2 
 
 
 
 
PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Principal Investigator: 
Benjamin Mordmüller, MD 
Department of Medical Microbiology  
 
31 March 
2021 
 
 

NL76192.000.20 / COUGH-1 
Safety and tolerability of ABNCoV2 
 
 
 
 
TABLE OF CONTENTS 
1. 
INTRODUCTION AND RATIONALE ................................................................................ 15 
1.1 
Introduction ................................................................................................................ 15 
1.2 
Rationale .................................................................................................................... 17 
2. 
OBJECTIVES .................................................................................................................... 18 
2.1 
Primary Objectives..................................................................................................... 18 
3. 
STUDY DESIGN ............................................................................................................... 18 
3.1 
Visit intervals .............................................................................................................. 21 
4. 
STUDY POPULATION ...................................................................................................... 22 
4.1 
Population .................................................................................................................. 22 
4.2 
Inclusion criteria ......................................................................................................... 22 
4.3 
Exclusion criteria........................................................................................................ 23 
4.4 
Sample size calculation ............................................................................................. 24 
5. 
TREATMENT OF SUBJECTS .......................................................................................... 26 
5.1 
Investigational product/treatment .............................................................................. 26 
5.2 
Use of co-intervention................................................................................................ 26 
5.3 
Escape medication .................................................................................................... 27 
6. 
INVESTIGATIONAL PRODUCT ....................................................................................... 27 
6.1 
Name and description of investigational product(s) .................................................. 27 
6.1.1 
ABNCoV2 vaccine product................................................................................. 27 
6.1.2 
MF59-adjuvant.................................................................................................... 29 
6.2 
Summary of findings from non-clinical studies.......................................................... 29 
6.3 
Summary of findings from clinical studies ................................................................. 31 
6.4 
Summary of known and potential risks and benefits ................................................ 32 
6.4.1 
Risk Assessment ................................................................................................ 32 
6.4.2 
Benefit assessment ............................................................................................ 36 
6.5 
Description and justification of route of administration and dosage ......................... 36 
6.6 
Dosages, dosage modifications and method of administration ................................ 37 

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6.7 
Preparation and labelling of Investigational Medicinal Product ................................ 37 
6.7.1 
Presentation and formulation ............................................................................. 37 
6.7.2 
Stability and storage ........................................................................................... 38 
6.7.3 
Dose preparation and administration ................................................................. 38 
6.7.4 
Labelling ............................................................................................................. 39 
6.8 
Drug accountability .................................................................................................... 39 
7. 
NON-INVESTIGATIONAL PRODUCT.............................................................................. 40 
8. 
METHODS......................................................................................................................... 40 
8.1 
Study endpoints ......................................................................................................... 40 
8.1.1 
Main study parameters/endpoints ...................................................................... 40 
8.1.2 
Secondary study endpoint.................................................................................. 40 
8.1.3 
Exploratory study parameters/endpoints ........................................................... 40 
8.2 
Randomisation, blinding and treatment allocation .................................................... 41 
8.3 
Study procedures....................................................................................................... 41 
8.3.1 
Recruitment of subjects ...................................................................................... 41 
8.3.2 
Screening visit (visit 1) ....................................................................................... 42 
8.3.3 
Inclusion visit (visit 2) ......................................................................................... 43 
8.3.4 
ABNCoV2 inoculation visits (visits 3 + 9) ........................................................... 44 
8.3.5 
Follow-up visits after ABNCoV2 administration (visit 4-8 and 10-16) ............... 45 
8.3.6 
Unscheduled visits.............................................................................................. 45 
8.3.7 
Visits regarding vaccination with licensed SARS-CoV-2 vaccine ..................... 46 
8.3.8 
Medical history.................................................................................................... 46 
8.3.9 
Physical examination.......................................................................................... 47 
8.3.10 
Height and weight ............................................................................................... 47 
8.3.11 
Vital signs ........................................................................................................... 47 
8.3.12 
Laboratory evaluations ....................................................................................... 47 
8.3.13 
Urine toxicology analysis .................................................................................... 48 
8.3.14 
Pregnancy test.................................................................................................... 48 

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8.3.15 
Discontinuation of study procedures .................................................................. 48 
8.3.16 
Assay to determine concentration SARS-CoV-2-specific antibodies ................ 49 
8.3.17 
Virus neutralization assay .................................................................................. 49 
8.3.18 
Safety assessments ........................................................................................... 49 
8.3.19 
Time and event procedures ............................................................................... 51 
8.4 
Withdrawal of individual subjects .............................................................................. 53 
8.4.1 
Handling of withdrawals ..................................................................................... 53 
8.4.2 
Withdrawals at specific time-points .................................................................... 54 
8.5 
Replacement of individual subjects after withdrawal ................................................ 55 
8.6 
Follow-up of subjects withdrawn from treatment....................................................... 55 
8.7 
Premature termination of the study ........................................................................... 55 
9. 
SAFETY REPORTING ...................................................................................................... 56 
9.1 
Temporary halt for reasons of subject safety ............................................................ 56 
9.2 
AEs, SAEs and SUSARs ........................................................................................... 56 
9.2.1 
Adverse events (AEs)......................................................................................... 56 
9.2.2 
Serious adverse events (SAEs) ......................................................................... 56 
9.2.3 
Suspected unexpected serious adverse reactions (SUSARs) .......................... 57 
9.3 
Annual safety report................................................................................................... 57 
9.4 
Follow-up of adverse events ..................................................................................... 58 
9.5 
Safety Monitoring Committee .................................................................................... 60 
9.5.1 
Local safety monitor ........................................................................................... 61 
9.5.2 
Safety Meetings .................................................................................................. 61 
9.5.3 
Safety Reports .................................................................................................... 61 
9.5.4 
Dose escalation and adjuvant use in group 4 and above.................................. 62 
9.5.5 
Holding rules for dose escalation to groups 4-7: ................................................ 62 
9.5.6 
Decision on further development of ABNCoV2 ................................................. 64 
10. 
STATISTICAL ANALYSIS ............................................................................................. 64 
10.1 
Primary study endpoints ............................................................................................ 64 

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10.2 
Secondary study endpoint ......................................................................................... 65 
10.3 
Other study endpoints ............................................................................................... 65 
10.4 
Interim analysis (if applicable) ................................................................................... 65 
11. 
ETHICAL CONSIDERATIONS...................................................................................... 66 
11.1 
Regulation statement................................................................................................. 66 
11.2 
Recruitment and consent........................................................................................... 66 
11.3 
Benefits and risks assessment, group relatedness .................................................. 67 
11.4 
Compensation for injury............................................................................................. 67 
11.5 
Compensation............................................................................................................ 67 
12. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .......................... 68 
12.1 
Handling and storage of data and documents .......................................................... 68 
12.1.1 
Source data ........................................................................................................ 68 
12.1.2 
Confidentiality ..................................................................................................... 69 
12.2 
Monitoring and Quality Assurance ............................................................................ 70 
12.3 
Amendments .............................................................................................................. 71 
12.4 
Annual progress report .............................................................................................. 72 
12.5 
Temporary halt and (prematurely) end of study report ............................................. 72 
12.6 
Public disclosure and publication policy .................................................................... 72 
13. 
STRUCTURED RISK ANALYSIS ................................................................................. 72 
13.1 
Potential issues of concern........................................................................................ 72 
13.2 
Synthesis ................................................................................................................... 77 
14. 
REFERENCES .............................................................................................................. 77 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
General Assessment and Registration form (ABR form), the 
application form that is required for submission to the accredited 
Ethics Committee; in Dutch: Algemeen Beoordelings- en 
Registratieformulier (ABR-formulier) 
AE 
Adverse Event 
AEFI 
Adverse Event Following Immunization 
ALT 
Alanine aminotransferase 
AR 
Adverse Reaction 
AS 
Adjuvant System 
AST 
Aspartate aminotransferase (AST) 
AV 
AdaptVac 
BEVS 
Baculovirus Expression Vector System 
BMI 
Body Mass Index 
BP 
Bloodpressure 
CA 
Competent Authority 
CBC 
Complete blood count 
CCMO 
Central Committee on Research Involving Human Subjects; in 
Dutch: Centrale Commissie Mensgebonden Onderzoek 
CDT 
Carbohydrate-deficient transferrin 
cGMP 
current Good Manufacturing Practice  
CoV 
Coronavirus 
COVID-19 
Coronavirus disease 2019 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CV 
Curriculum Vitae 
DSMB 
Data Safety Monitoring Board 

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ELISA 
Enzyme-Linked immunosorbent assay 
EU 
European Union 
EudraCT 
European drug regulatory affairs Clinical Trials  
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene 
Verordening Gegevensbescherming (AVG) 
GLP 
Good Laboratory Practice 
GMP 
Good Manufacturing Practice 
HBV 
Hepatitis B virus 
HCV 
Hepatitis C virus 
HIV 
Human immunodeficiency virus 
HPV 
Human papillomavirus 
IB 
Investigator’s Brochure 
IC 
Informed Consent 
ID 
Identifier 
IEC 
Independent Ethics Committee 
IgG 
Immunoglobulin G 
IM 
Intramuscular 
IMP 
Investigational Medicinal Product  
IMPD 
Investigational Medicinal Product Dossier  
IRB 
Institutional Review Board 
ITT 
Intention-To-Treat 
IU 
International Unit 
LSM 
Local safety monitor 
MERS 
Middle East respiratory syndrome 
METC  
Medical research ethics committee (MREC); in Dutch: medisch-
ethische toetsingscommissie (METC) 

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NFU 
Nederlandse Federatie van Universitair Medische Centra 
NHP 
Non-human primate 
NPC 
Non-pharmaceutical interventions 
NZW 
New Zealand White 
PBMC 
Peripheral Blood Mononuclear Cell 
PCR 
Polymerase chain reaction 
PI 
Principal investigator 
PP 
Per protocol 
RBC 
Red blood cell 
RBD 
Receptor Binding Domain 
RNA 
Ribonucleic Acid 
RTCCS 
Radboudumc Technology Center Clinical Studies 
SAE 
Serious Adverse Event  
SARS 
Severe Acute Respiratory Syndrome 
SARS-CoV 
Severe Acute Respiratory Syndrome Coronavirus 
SARS-CoV-2 
Severe Acute Respiratory Syndrome Coronavirus 2 
SEM 
Standard error of the mean 
SOP 
Standard Operating Procedure 
SMC 
Safety Monitoring Committee 
SPC 
Summary of Product Characteristics; in Dutch: officiële 
productinformatie IB1-tekst 
S-Protein 
SARS-CoV-2 Spike Protein 
Sponsor 
The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. 
A party that provides funding for a study but does not commission it 
is not regarded as the sponsor, but referred to as a subsidising 
party. 
SUSAR  
Suspected Unexpected Serious Adverse Reaction 

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TLR 
Toll Like Receptor 
UAVG 
Dutch Act on Implementation of the General Data Protection 
Regulation; in Dutch: Uitvoeringswet AVG 
VE 
Vaccine efficacy 
VLP 
Virus Like Particle 
cVLP 
Capsid Virus Like Particle 
WHO 
World Health Organization 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet 
Medisch-wetenschappelijk Onderzoek met Mensen 
 
 

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SUMMARY 
Rationale: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic 
virus, primarily causing respiratory symptoms in humans, ranging from very mild to life 
threatening. The current outbreak of SARS-CoV-2 was first reported in late 2019 and has 
spread rapidly around the world, leading the World Health Organization (WHO) to declare a 
pandemic. A vaccine could complement non-pharmaceutical interventions (NPC), in order to 
protect vulnerable populations by reducing virus-spread, decrease the load on health care 
systems and reduce the social and economic impact of NPC. The ABNCoV2 vaccine is 
intended to protect against coronavirus disease 2019 (COVID-19) and limit spread of SARS-
CoV-2.  
Objective: The main objectives of the trial are to assess the safety and tolerability of two doses 
of ABNCoV2, formulated with and without the adjuvant MF59, in healthy adult volunteers and 
to identify the dosage and formulation that optimizes the immunogenicity-tolerability ratio 14 
days following first vaccination with ABNCoV2. 
Study design: COUGH-1 is a phase 1, single centre, open labelled trial in healthy, adult, 
SARS-CoV-2-naïve volunteers. The trial involves first-in-human administration, pre-defined, 
sequential dose escalation of ABNCoV2, and adjuvant selection. It intends to inform dosage 
and formulation for subsequent clinical development. 
Study population: Healthy, SARS-CoV-2-naïve, adult female and male volunteers, 18-55 
years old. 
Intervention: ABNCoV2 is a virus-like particle vaccine. It will be administered as two 
intramuscular injections in groups of up to 9 volunteers. The pre-defined escalation schedule 
will start with 6 μg ABNCoV2, followed by 12, 25 and 50 µg with a maximum dose of 70 µg. 
MF59-adjuvanted and non-adjuvanted formulations will be tested in parallel to detect 
superiority or futility of the MF59-adjuvanted against the non-adjuvanted formulation at the 6, 
12 and 25 µg dosage. Approval for further dose escalation and choice of adjuvant use will be 
provided by a safety monitoring committee (SMC), supported by pre-defined analyses of 
safety, tolerability and immunogenicity data at day 14 post-first-vaccination. Recruitment for 
the two best (safe, tolerable and most immunogenic) regimens will continue until 12 volunteers 
per regimen have been immunized. 
Main study parameters/endpoints: Safety – number of at least possibly related Grade 3 
adverse events (AE) and serious adverse events (SAE) from time of first administration of 
ABNCoV2 until the end of the follow-up period and number and severity of at least possibly 
related solicited AEs within one week following administration of ABNCoV2. Immunogenicity – 
concentration of ABNCoV2-specific antibodies 14 days following first vaccination.  

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Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: There is no direct benefit from participation in this trial. Information about 
their own general health status may be a potential indirect benefit for participants. It will be 
made clear that the vaccine is experimental and may not protect against COVID-19. 
Participating in this trial includes risks associated with intramuscular ABNCoV2 administration, 
immune-response against the vaccine and blood sampling. Volunteers will experience frequent 
follow-up visits, physical examinations, screening for HIV, hepatitis B and hepatitis C, a 
pregnancy test (for females) and COVID-19 diagnostics.  
 
 

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1. INTRODUCTION AND RATIONALE 
1.1 Introduction 
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified in late 2019 
after a number of pneumonia cases with unknown cause were reported in Wuhan City, China. 
Coronavirus disease 2019 (COVID-19), the disease caused by SARS-CoV-2, spread rapidly 
through China and other countries worldwide. In March 2020, the World Health Organisation 
(WHO) declared a global pandemic [1].  
SARS-CoV-2 is a Betacoronavirus (CoV), causing respiratory and systemic symptoms in 
humans, ranging from very mild to life threatening. Coronaviruses are a family of viruses that 
usually cause mild to moderate upper-respiratory tract illnesses in humans. Four human 
coronaviruses (HCov-229E, HCov-HKU1, HCov-NL63 and HCov-OC43) are endemic, causing 
disease with a peak in winter months [2]. However, in the past two decades three new 
coronaviruses have emerged from animal reservoirs and have caused serious illness, 
complications and death. SARS coronavirus (SARS-CoV) emerged in November 2002 and 
caused severe acute respiratory syndrome (SARS), until it disappeared in 2004 [3, 4]. 
Transmitted from an animal reservoir in camels, the Middle East respiratory syndrome (MERS) 
caused by the MERS coronavirus (MERS-CoV) was identified in 2012 [5]. MERS continues to 
cause sporadic and localized outbreaks. The third coronavirus to emerge in this century is 
SARS-CoV-2. SARS-CoV-2 is a zoonotic virus, likely originating from bats and transmitted to 
humans via an intermediary mammalian host. 
Whilst the genome of all three novel CoVs is similar, the S gene of SARS-CoV-2 is divergent 
to the other CoVs and information about protective immune responses to the virus is scarce, 
so far. All CoVs cause primarily respiratory illnesses – from common cold to severe respiratory 
distress, but other complication may occur, particularly with SARS-CoV-2 infections. 
The SARS-CoV-2 virus is transmitted through respiratory droplets, with a mean incubation 
period of 4-6 days. Most patients display symptoms within 14 days of infection [6]. The most 
prominent symptoms are fever, cough, and fatigue. However, a number of other symptoms 
have been identified as being associated with COVID-19, including loss of smell and taste, 
sore throat, diarrhoea, fatigue, neurological symptoms, cardiac symptoms, coagulopathies, 
and renal damage amongst others [7-9].  
Many patients experience only mild symptoms, and some are asymptomatic, leading to 
increased transmission due to social contact whilst feeling well. COVID-19 is associated with 
significant mortality in at-risk groups. The main risk factor for complications and death is old 
age, typically associated with a number of pre-existing conditions. Geographic variations may 
also exist, although different testing strategies bias mortality estimates.  

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Although a worldwide concerted effort was made to contain the virus by way of non-
pharmaceutical interventions (NPC) such as local “lockdowns”, social contact restrictions, 
travel restrictions and contact tracing with resultant quarantine, it continues to run rampant in 
countries such as the US and the UK. In the Netherlands and other European countries 
incidence decreased over the summer but since September infections resurge, and incidence 
remains high (in March 2021 ~30,000 new cases/week in the Netherlands) despite re-
implementation of NPCs. As of March 2021, there had been nearly 120 million cases world-
wide, with more than 2.5 million deaths [10].  
The ABNCoV2 vaccine is a sub-unit vaccine using a non-infectious virus-like particle (VLP) 
platform to optimize immunogenicity. A stabilized version of the receptor binding domain (RBD) 
of SARS-CoV-2 Spike glycoprotein (S) is bound to an VLP, and through vaccination is 
presented to the immune system in order to evoke a response resulting in protection from 
infection. Most vaccine candidates targeting SARS-CoV-2 and other members of the family 
Coronaviridae consist of S antigens and are optimized to achieve high invasion inhibition 
activities. Proof-of-concept studies of S antigen-based vaccine candidates have shown high-
level vaccine efficacy and transmission-blocking activity in dromedaries and other Camelidae 
[11-13], the main host of the Middle East respiratory syndrome coronavirus (MERS).  
Besides ABNCoV2, more than 200 vaccines against SARS-CoV-2 are under development 
[14]. First candidates have reached Phase 3 clinical trials and results have been published in 
the form of press releases [15-17], two providing more detail in form of an interim analyses [18, 
19]. Three studies have reached approximately 100 cases, which allows a reasonably estimate 
of a clinically relevant vaccine efficacy (VE). Observed VE was 60-90%. All three trials used 
candidates based on the S antigen as the immunogen and have observed significant 
immunogenicity following two vaccinations [20-22]. Two use an RNA-vaccine-platform and one 
a recombinant adenovirus. Follow up times are still short (median of ~3 months) but 
vaccination campaigns based on emergency-use authorization have already started in some 
countries. Platform technologies that enable such a fast development (RNA, viral vector) are 
relatively new and have only been used in the framework of clinical trials – many without an 
infectious disease prevention indication. RNA vaccines have mostly been used as proof-of-
concept vaccines for cancer. In the few studies against infectious diseases, they have shown 
satisfactory but not particularly long-lasting 
or broad responses 
[23, 24]. The 
Oxford/AstraZeneca platform has been used extensively but without success in malaria and 
tuberculosis vaccine development. In addition, systemic reactogenicity was significant in all 
three studies.  

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Virus like particles (VLPs) that display antigen on their surface are a technology to increase 
immunogenicity of soluble protein subunit vaccines and have an extensive safety record; e.g. 
as human papilloma virus (HPV) and hepatis B vaccines. Besides induction of better immune 
responses, memory and specificity of the response may also be improved. Since VLPs cannot 
replicate they can be safely used in immunocompromised and elderly populations, two 
populations at particular risk for severe COVID-19. In COUGH-1 a novel VLP technology that 
allows covalent, directional, high density binding of different proteins on the VLP surface will 
be used. Besides the immunological advantages, this decreases development time 
significantly, although not to levels as fast as RNA vaccines because proteins must still be 
expressed. The VLP itself consists of the coat protein of AP205, a structural protein of a phage 
(a virus infecting bacteria) that does not infect eukaryotes (including humans). AP205 VLPs 
have never been used in humans but the closely related Qβ phage-based VLP system has 
been tested in clinical trials [25-27].  
In pre-clinical models ABNCoV2 immunogenicity has been superior to the other platform 
technologies. In addition, it is optimized to comply with the WHO target product profile for 
SARS-CoV-2 vaccines [28]. If successful, it will be developed as a second-generation SARS-
CoV-2 vaccine.  
1.2 Rationale 
There is currently no cure for COVID-19. The disease itself, as well as the essential measures 
put in place to reduce its spread, are wreaking havoc on communities, economies and health 
systems worldwide. Not only are economies suffering as a result of lockdowns and restrictions, 
with recessions imminent, but healthcare systems are overwhelmed, resulting in increased 
morbidity and mortality due to other diseases and routine immunization programmes are being 
disrupted leaving millions of children worldwide at risk of preventable diseases. An effective 
vaccine is essential and urgently required, in order to protect populations all over the world 
from COVID-19. Even though first vaccines have been registered, the need for new products 
remains high. Since COVID-19 is a worldwide pandemic, no single supplier will be able to 
produce enough vaccine to cover the world’s population and not all vaccines in the pipeline 
are effective, safe and easy to deploy. A larger portfolio of vaccines will also increase resilience 
of the public health response including faster reaction to control spread of new virus strains 
and potential vaccine-escape mutants. In addition, the target product profile will differ between 
populations as will vaccine safety and acceptability. Hence, the use of different technologies 
will improve safety, coverage, effectivity, efficiency [29] and together with NPCs, control of the 
epidemic. The ABNCoV2 sub-unit vaccine also has the potential to prevent COVID-19 in health 
care workers, as well as limit spread of the disease in epidemic settings.  

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2. OBJECTIVES 
2.1 Primary Objectives 
Safety: 
- 
To assess safety and tolerability of two doses of ABNCoV2, formulated with and without 
MF59 in healthy adult volunteers. 
Immunogenicity: 
- 
To identify a dosage that optimizes the tolerability-immunogenicity ratio 14 days 
following first vaccination with ABNCoV2. 
3. STUDY DESIGN 
This first-in-human phase 1 trial of ABNCoV2 is a single center, sequential dose-escalation, 
open labelled trial to establish the safety and tolerability of two doses of ABNCoV2, formulated 
with and without MF59 in healthy, adult, SARS-CoV-2-naïve volunteers. The immunological 
objective of this trial is to identify a dosage that optimizes the immunogenicity-tolerability ratio 
14 days following first vaccination with ABNCoV2. The trial will be carried out by the Radboud 
University Medical Center (Radboudumc). 
The trial involves first-in-human administration, dose escalation of ABNCoV2 and adjuvant 
selection. Volunteers will be screened for eligibility and receive two vaccinations by 
intramuscular injection. While we cannot predict with certainty the safety in human subjects, 
we have adopted a safety-orientated staggered trial design with ascending doses of 
ABNCoV2. Seven groups of volunteers (n=6) will receive a given dose of ABNCoV2, either 
with or without MF59, followed by a booster with the same dose and formulation four weeks 
after the first vaccination. All vaccinations will be given as intramuscular injection. The pre-
defined escalation schedule will start with 6 μg ABNCoV2, with a maximum dose of 70 µg. 
Dose-escalation will proceed only in absence of protocol-defined safety signals. MF59-
adjuvanted and non-adjuvanted formulations will be tested in parallel at the first three 
escalation steps (Group 1-3) to detect superiority or futility of the MF59-adjuvanted against the 
non-adjuvanted formulation. Group 1A (n=3) will receive two doses of 6 μg non-adjuvanted 
ABNCoV2, Group 1B (n=3) will receive two doses of 6 μg adjuvanted ABNCoV2, Group 2A 
(n=3) will receive two doses of 12 μg non-adjuvanted ABNCoV2, Group 2B (n=3) will receive 
two doses of 12 μg adjuvanted ABNCoV2, Group 3A (n=3) will receive two doses of 25 μg 
non-adjuvanted ABNCoV2 and Group 3B (n=3) will receive two doses of 25 μg adjuvanted 
ABNCoV2. After the first vaccinations in group 3 subjects, a decision will be taken as to 
whether to proceed with or without adjuvant in the subsequent groups. Group 4 (n=6) will 
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receive two doses of 70 μg adjuvanted or non-adjuvanted ABNCoV2, Group 6 (n=6-9)1 will 
receive the optimal or second-highest adjuvanted or non-adjuvanted ABNCoV2 dose, and 
Group 7 (n=6-9) will receive the highest adjuvanted or non-adjuvanted ABNCoV2 dose 
achieved (Table 1). Up to forty-two (n=42) subjects will be enrolled, as well as one reserve 
subject per group. 
ABNCoV2 administration will be staggered at each dose-escalation and addition of adjuvant 
such that the first subject in each group will be administered ABNCoV2 and observed for the 
occurrence of any AEs. The second subject in each dose group will not receive their dose of 
ABNCoV2 sooner than 6 days after the first subject has received their first dose of ABNCoV2 
(Figure 1). Escalation to the next higher dosage group will be dependent upon no safety 
signals arising as described below and will follow the same staggered algorithm as shown in 
Figure 1.  
Decision on further dose escalation and adjuvant use will be made by the investigator and 
sponsor and will be discussed and approved by a safety monitoring committee (SMC), 
supported by pre-defined analysis (see below) of safety, tolerability and immunogenicity data 
up to day 14 post-vaccination. The SMC will review the safety data of Group 1-3 up to day 
14 after each participant’s respective first vaccination before proceeding to Group 4. The 
data to be reviewed includes the safety data (solicited AEs through day 7 and unsolicited 
AEs through day 14 post ABNCoV2 administration with and without adjuvant; see below) 
and clinically significant laboratory tests collected at 24 hours and 7 days following first 
ABNCoV2 administration for all subjects in the specified dose group. 
The ABNCoV2 dose will be escalated until: 
• 
The maximal dose of 70 µg is reached, or 
• 
The vaccine is too reactogenic and/or not tolerated, or 
• 
Immunogenicity-dose response curve is saturated (no further increase or decrease in 
antibody concentration with increasing dose) 
The highest safe/tolerable and the next lower dosage will be re-tested (in groups 7 and 6, 
respectively) to increase power to detect adverse reactions at the optimal dose and to inform 
dosing studies in the elderly population. If the immunogenicity dose-response is saturated 
before reaching a safety/tolerability limit, then this dose and the next lower dose will be re-
 
1 Group 6 and 7 ensure that at least 12 volunteers have received the highest and second -highest 
dosage. In case that dose-escalation is completed before 50 µg is reached, n=9 volunteers will be 
recruited, otherwise n=6 (Figure 1 for examples).  

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tested. At completion of the trial, 12 volunteers will have received the highest and second-
highest dosage.   
Table 1. Study groups 
 
* Whether Groups 4-7 will receive a MF59-adjuvanted formulation is dependent on the superiority or 
futility of the MF59-adjuvanted against the non-adjuvanted formulation in Groups 1-3. 
** In principle, the highest and second-highest safe/tolerable doses will be re-tested to increase power to 
detect adverse reactions at the optimal dose and to inform dosing studies in the elderly population. If 
the immunogenicity-dose response is saturated before reaching a safety/tolerability limit, then the 
highest achieved dose and the next lower dosage will be re-tested. 
 
 
 
Study 
group 
Number of 
subjects 
ABNCoV2 dosage 
 
MF59-adjuvant 
Administration Route 
Group 1A 
3 
6 μg 
no 
intramuscular 
Group 1B 
3 
6 μg 
yes 
intramuscular 
Group 2A 
3 
12 μg 
no 
intramuscular 
Group 2B 
3 
12 μg 
yes 
intramuscular 
Group 3A 
3 
25 μg 
no 
intramuscular 
Group 3B 
3 
25 μg 
yes 
intramuscular 
Group 4 
6 
50 μg 
t.b.d.* 
intramuscular 
Group 5 
6 
70 μg 
t.b.d.* 
intramuscular 
Group 6 
6 
Second-highest/optimal dose** 
t.b.d.* 
intramuscular 
Group 7 
6 
Highest dose** 
t.b.d.* 
intramuscular 

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A 
  
 
 
 
 
 
 
B 
  
Figure 1. Staggered dosing trial design for ABNCoV2 administration. A) Example algorithm 
in case that escalation up to 70 µg (Group 5) is required; B) example algorithm for an 
escalation up to 25 µg (Group 3). For both examples the date of first vaccination is arbitrarily 
set at 15 March 2021; coloured numbers indicate n, the vertical blue lines are the decision 
points for further use of adjuvant and dosage in groups 6 and 7, respectively (in example B 
both decisions will be made at the same time) 
3.1 Visit intervals 
The permissible study visit intervals are indicated in Table 2. 

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Table 2. Permissible study visit intervals 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4. STUDY POPULATION 
4.1 Population 
The study population will be comprised of adult male and female healthy subjects aged 18-55 
at time of first ABNCoV2 administration. A maximum of 42 subjects will be enrolled to 
participate in the study, in addition to up to 7 reserve subjects (1 reserve subject per dosing 
group). The investigator will ensure that all subjects being considered for the study meet 
the eligibility criteria described in section 4.2 and 4.3. Subject eligibility is to be established 
and confirmed by checking all inclusion/exclusion criteria at both screening and inclusion 
(baseline). A relevant record of the eligibility criteria will be stored with the source 
documentation at the study site. 
4.2 Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following criteria: 
1. Subject must sign written informed consent to participate in the trial. 
2. Subject is able to understand 
planned study procedures and demonstrate 
Visit 
Type of visit 
Day of visit 
Allowed interval 
Visit 1 
Screening 
I1-28 to I1-1 
- 28 to -1 days 
Visit 2 
Inclusion 
I1-3 
± 2 days 
Visit 3 
Administration 1st ABNCoV2 dose 
I1 
± 1 day 
Visit 4 
Follow-up 
I1+1 
0 day 
Visit 5 
Follow-up 
I1+2 
0 day 
Visit 6 
Follow-up 
I1+7 
± 1 day 
Visit 7 
Follow-up 
I1+14 
± 1 day 
Visit 8 
Inclusion 
I2-3 
± 2 days 
Visit 9 
Administration 2nd ABNCoV2 dose 
I2 
± 1 day 
Visit 10 
Follow-up 
I2+1 
0 day 
Visit 11 
Follow-up 
I2+2 
0 day 
Visit 12 
Follow-up 
I2+7 
± 1 day 
Visit 13 
Follow-up 
I2+14 
± 1 day 
Visit 14 
Follow-up 
I2+42 
± 2 days 
Visit 15 
Follow-up 
I2+91 
± 2 days 
Visit 16 
Follow-up (end-of-study visit) 
I2+168 
± 3 days 

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comprehension of the protocol procedures and knowledge of the study by passing a 
quiz (assessment of understanding). Subjects must score at least 80% correct on a 
multiple-choice quiz. If they do not score 80% on the initial quiz, the protocol 
information will be reviewed with them, and they will have the opportunity to retest. 
3. In the opinion of the investigator, the subject can and will comply with the requirements 
of the protocol. 
4. Subjects are available to attend all study visits and are reachable by phone throughout 
the entire study period from day -1 until 24 weeks following last vaccination (end of study). 
5. Subject is a male or non-pregnant and non-lactating female age ≥ 18 and ≤ 55 years and 
in good health at time of ABNCoV2 administration. 
6. Subject agrees to their general practitioner (GP) being informed about participation in the 
study and agrees to sign a form to request the release by their GP, and medical specialist 
when necessary, of any relevant medical information concerning possible contra-
indications for participation in the study to the investigator(s).  
7. The subject agrees to refrain from blood donation to Sanquin or for other purposes 
throughout the study period according to current Sanquin guidelines. 
8. Female subjects of non-childbearing potential may be enrolled in the study. Non-
childbearing potential is defined as pre-menarche, current bilateral tubal ligation or 
occlusion, hysterectomy, bilateral ovariectomy or post-menopause. All other female 
subjects must agree to use continuous adequate contraception2 for the duration of the 
study. Female subjects must have a negative pregnancy test at the inclusion visit. 
4.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from participation 
in this study: 
1. Any clinically significant abnormal finding on clinical examination or laboratory screening 
tests according to the US Food and Drug Administration (FDA) Toxicity Grading Scale 
for Healthy Adult and Adolescent Subjects Enrolled in Preventative Vaccine Clinical Trials 
[30]. 
2. History of COVID-19 infection. 
3. Chronic use of immunosuppressive drugs or other immune modifying drugs within six 
 
2 Acceptable forms of female contraception include: established use of oral, injected or implanted hormonal 
contraceptives; intrauterine device or intrauterine system; barrier methods (condoms or diaphragm with 
additional spermicide); male partner’s sterilization (with appropriate post-vasectomy documentation of absence 
of sperm in the ejaculate); true abstinence when this is in line with the preferred and usual lifestyle of the 
subject. Periodic abstinence (e.g., calendar, ovulation, symptom-thermal, post-ovulation methods) and 
withdrawal are not acceptable methods of contraception. Adequate contraception does not apply to subjects of 
childbearing potential with partners of the same sex. 

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months prior to study onset (inhaled and topical corticosteroids and oral anti-histamines 
exempted) or expected use of such during the study period. 
4. Positive urine toxicology test for cannabis, cocaine or amphetamines at inclusion. 
5. Screening tests positive for SARS-CoV-2, SARS-CoV-2 antibodies, 
Human 
Immunodeficiency Virus (HIV), active Hepatitis B Virus (HBV), or Hepatitis C Virus (HCV). 
6. Receipt of any investigational or non-registered product (drug or vaccine) other than 
the study product in the 30 days preceding enrolment or during the study period. 
7. Participation in any other clinical study in the 30 days prior to the start of the study or 
during the study period. 
8. Immunization with any vaccines within the past four weeks or planned receipt of a vaccine 
during the study period with the exception of a licensed SARS-CoV-2 vaccine, given 
within the framework of the national SARS-CoV-2 vaccination campaign. The time 
between last vaccination with ABNCoV2 and a SARS-CoV-2 vaccine provided by the 
campaign shall be at least 4 weeks. 
9. Known hypersensitivity to any of the vaccine components (adjuvant or protein). 
10. Administration of immunoglobulins and/or any blood products within the three months prior 
to the first dose of ABNCoV2 or planned administration during the study period. 
11. Previous participation in a COVID-19 vaccine study. 
12. Body Mass Index (BMI) >35 kg/m2. 
13. Pregnancy, lactation or intention to become pregnant during the study period. 
14. History of drug or alcohol abuse interfering with normal functioning in the five years 
preceding enrolment. 
15. Being an employee or student of the department of Medical Microbiology of the 
Radboudumc, or a person otherwise related to the investigator other than a professional 
relationship for clinical trial purpose only. 
16. Any other condition or situation that would, in the opinion of the investigator, place the 
subject at an unacceptable risk of injury or render the subject unable to meet the 
requirements of the protocol. 
 
4.4 Sample size calculation 
COUGH-1 is an exploratory, Phase 1, first-in-human trial to evaluate the safety and tolerability 
of increasing doses of ABNCoV2 vaccine antigen with and without adjuvant, in healthy, 
COVID-19-naïve adults. Its outcome will inform dose and schedule for further clinical 
development. The overall objective is to assess safety and tolerability and identify an optimal 
immunogenicity-tolerability ratio. In case that the maximal dose of 70 µg is reached, a 
maximum number of 42 volunteers will be included.  

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The higher the probability of an adverse reaction (AR) in the vaccinated population, the smaller 
the sample of volunteers with at least one occurrence of the AR is. Table 3 shows probability 
of AR in vaccinees that can be detected with a given sample size at 90% power. A sample size 
of 6 can detect adverse reactions that occur in 32% of the participants with 90% power (with 
n=12, a prevalence of 17% can be detected). Assuming 42 volunteers are immunized, adverse 
reactions that occur in 4.7% of the volunteers (within the tested dose-range), can be detected 
with 90% probability.  
Table 3. Probability of adverse reaction (AR) 
Sample size (n) 
Probability of AR (%) 
Power 
6 
32 
0.9 
12 
17 
0.9 
18 
12 
0.9 
24 
9.1 
0.9 
30 
7.4 
0.9 
36 
6.2 
0.9 
42 
5.3 
0.9 
48 
4.7 
0.9 
54 
4.2 
0.9 
60 
3.8 
0.9 
 
The names used to describe probability groupings of events (e.g. AR) customarily follow the 
convention: Very common (≥1/10); common (≥1/100 to <1/10); uncommon (≥1/1,000 to 
<1/100); rare (≥1/10,000 to <1/1,000); very rare (<1/10,000). Consequently, COUGH-1, as a 
Phase 1 first-in-human trial, is powered to detect very common and common events. 
Immunogenicity 
Dose escalation will be done in groups of 6 volunteers, starting with split groups, where half 
(n=3) of the volunteers receive the non-adjuvanted and half (n=3) the MF59-adjuvanted 
vaccine formulation. 14 days after administration of the first vaccination in Group 1-3, it will be 
decided whether the non-adjuvanted or the MF59-adjuvanted vaccine formulation will be used 
in the remainder based on superiority upon immunogenicity endpoints. The decision will be 
taken by the sponsor and the investigators and approved by a safety monitoring board based 
on all immunogenicity, safety and reactogenicity data that have been accrued until this time. 
Assuming a sigmoid dose-immunogenicity relation, the optimal dose will be selected close to 
the upper asymptote, given it is still safe and well tolerated. To increase power at this threshold, 
the number of volunteers receiving the optimal dose ABNCoV2 will be doubled (total n=12 
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A statistical analysis plan will be developed during preparation of the trial and updated based 
on pre-clinical findings as well as published data from other vaccine candidates. Immunological 
analyses will be developed using discovery and pre-clinical data generated during the project. 
Up to 7 reserve subjects will be recruited (1 reserve subject per dosage group). 
5. TREATMENT OF SUBJECTS 
5.1 Investigational product/treatment 
Volunteers will sequentially receive an administration of non-adjuvanted or MF59-adjuvanted 
ABNCoV2 vaccination. All subjects will receive a second vaccination with the same dose and 
formulation 4 weeks following the first vaccination. In Group 1 (n=6), subjects will receive 6 µg 
ABNCoV2 intramuscularly, half of whom (n=3) will receive the non-adjuvanted vaccine 
formulation and the other half (n=3) will receive the MF59-adjuvanted vaccine formulation. In 
Group 2 (n=6), subjects will receive 12 µg ABNCoV2 intramuscularly, half of whom (n=3) will 
receive the non-adjuvanted vaccine formulation and the other half (n=3) will receive the MF59-
adjuvanted vaccine formulation. In Group 3 (n=6), subjects will receive 25 µg ABNCoV2 
intramuscularly, half of whom (n=3) will receive the non-adjuvanted vaccine formulation and 
the other half (n=3) will receive the MF59-adjuvanted vaccine formulation. In Group 4 (n=6), 
subjects will receive 50 µg non-adjuvanted or MF59-adjuvanted ABNCoV2 intramuscularly. In 
Group 5 (n=6), subjects will receive 70 µg non-adjuvanted or MF59-adjuvanted ABNCoV2 
intramuscularly. The subjects in Group 6 (n=6) will receive the next lower dosage of the highest 
non-adjuvanted or MF59-adjuvanted ABNCoV2 dose achieved intramuscularly, and the 
subjects in Group 7 (n=6) will receive the highest non-adjuvanted or MF59-adjuvanted 
ABNCoV2 dose achieved intramuscularly. There will be no placebo group. 
5.2 Use of co-intervention 
Systematic use of a co-intervention is not planned (e.g. preventive use of non-steroidal anti-
inflammatory drugs [NSAID]). In order to be eligible for the study, subjects must abide to the 
inclusion and exclusion criteria as described in section 4.2 and 4.3 of this protocol. Therefore: 
- 
Subjects must pass a quiz (assessment of understanding) by scoring at least 80% 
correct on the multiple-choice quiz; 
- 
Subjects may not chronically use immunosuppressive drugs or other immune 
modifying drugs within six months prior to study onset (inhaled and topical 
corticosteroids and oral anti-histamines exempted) or expect to use such during the 
study period; 
- 
Subjects must attend all study visits and must be reachable by phone throughout the 
entire study period from day -1 until 24 weeks following last vaccination (end of study); 
- 
All subjects must use continuous adequate contraception for the duration of the study. 

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Female subject must undergo a pregnancy test at the inclusion visit. Females may 
not breastfeed during the study period; 
- 
Subjects must refrain from blood donation to Sanquin or for other purposes 
throughout the study period; 
- 
Subjects may not receive any investigational or non-registered product (drug or 
vaccine) other than the study product in the 30 days preceding enrolment or during 
the study period; 
- 
Subjects may not receive any immunoglobulins and/or any blood products within three 
months preceding the first dose of ABNCoV2 or expect administration thereof during 
the study period; 
- 
Subjects may not participate in any other clinical trial in the 30 days prior to start of 
the study or during the study period; 
- 
Subjects must contact the trial physician if they experience any medical complaint 
throughout the entire study period; 
- 
Subjects may not get immunization with any vaccines within the past four weeks or 
during the study period, with the exception of a licensed SARS-CoV-2 vaccine, given 
within the framework of the national SARS-CoV-2 vaccination campaign following 
ABNCoV2 administration. The time between last vaccination with ABNCoV2 and a 
SARS-CoV-2 vaccine provided by the campaign shall be at least 4 weeks. 
 
5.3 Escape medication 
No ABNCoV2-inactivating escape medication is available. The volunteers will not receive any 
premedication prior to the intramuscular injection of ABNCoV2 (e.g. nonsteroidal anti-
inflammatory drugs – NSAIDs). In case of an anaphylactic reaction, the current version of the 
RUMC anaphylaxis Standard Operating Procedure (SOP) will be followed. The volunteers are 
allowed to take pain reliefers such as NSAIDs if required. They will be asked to document and 
report any use of concomitant medication during the trial.  
6. INVESTIGATIONAL PRODUCT  
6.1 Name and description of investigational product(s) 
6.1.1 
ABNCoV2 vaccine product 
The ABNCoV2 vaccine is a sub-unit vaccine using a non-infectious virus-like particle (VLP) 
platform. A stabilized version of the receptor binding domain (RBD) of SARS-CoV-2 Spike 

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glycoprotein (S) is bound to a VLP, and through vaccination will be presented to the immune 
system in order to evoke a response resulting in protection from infection. Almost all vaccine 
candidates targeting SARS-CoV-2 and other members of the family Coronaviridae are based 
on S antigens and are optimized to achieve high invasion inhibition activities [14]. Proof-of-
concept studies of S antigen-based vaccine candidates have shown high-level vaccine efficacy 
and transmission-blocking activity in dromedaries and other Camelidae [11-13], the main host 
of the Middle East respiratory syndrome coronavirus (MERS).  
VLPs that display antigen on their surface are a technology to increase immunogenicity of 
soluble protein subunit vaccines [31]. Besides induction of better immune responses, memory 
and specificity of the response may also be improved. Since VLPs cannot replicate, they can 
be safely used in immunocompromised and senescent populations, two populations at risk for 
severe COVID-19. VLP-based vaccines have an extensive and excellent safety record. 
Examples of licensed and marketed VLP-based vaccines are hepatitis B (e.g. Engerix, 
HBVaxPro), and HPV vaccines (e.g. Gardasil, Gardasil 9, Cervarix). 
In ABNCoV2 a novel VLP technology that allows covalent, directional, high density binding of 
different proteins on the VLP surface is used. The VLP consist of the Acinetobacter Phage 205 
(AP205) capsid VLP (cVLP). AP205 does not infect eukaryotes (including humans). AP205 
VLP was never used in humans so far but the closely related Phage Qβ phage VLP has been 
tested in clinical trials [25-27]. In model systems, AP205 VLPs increase and improve antibody 
responses when compared to soluble protein antigens [32-34].  
The self-assembling AP205 VLP of the ABNCoV2 vaccine has modified capsid proteins that 
contain the split-protein Tag/Catcher system for antigen coupling [35]. Those binding sites 
distribute evenly across the outer surface of the particle and allow for fusion of large antigen 
proteins. The RBD of SARS-CoV-2 S is genetically engineered to bind to AP205 VLP through 
the split-protein Tag/Catcher system. Following coupling RBD antigens are presented on the 
VLP display platform at high density (72 spike RBD antigens per VLP). The RBD antigens are 
also unidirectionally oriented so that the ACE2 binding epitopes are positioned on the 
outermost surface of the complex, making it highly accessible for immune recognition.  

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Figure 2. Structural Elements of ABNCoV2: Engineered AP205 VLP and Spike RBD 
Antigen. AP205: Acinetobacter Phage 205; cVLP: capsid virus-like particle; RBD: receptor 
binding domain; S2 cells: drosophila S2 cells; VLP: virus-like particle 
6.1.2 
MF59-adjuvant 
Adjuvants are used to modulate type, strength and longevity of immune responses following 
vaccination. Adjuvants are particularly important for recombinant subunit vaccines that often 
do not lead to strong immune responses following intramuscular injection. VLP vaccines 
typically have better immunogenicity than soluble protein subunit vaccines. Therefore 
COUGH-1 will assess ABNCoV2 with and without MF59-adjuvant. MF59 is an oil-in-water 
emulsion containing squalene, polysorbate 80 and sorbitan trioleate. It is marketed in the 
Netherlands as part of the influenza vaccine Fluad/Fluad Tetra. It is approved for use in the 
elderly (>65 years) and has a favourable safety record. In influenza vaccines, MF59 can be 
used to reduce vaccine dose (dose-sparing), mainly acts through enhanced recruitment of 
immune cells to the injection site and has immune-stimulatory effects even in T helper cell 
deficient conditions [36].  
6.2 Summary of findings from non-clinical studies 
Preclinical data is described in detail in the investigator’s brochure (IB) and Investigational 
Medicinal Product Dossier (IMPD). The immunogenicity of adjuvanted ABNCoV2 has been 
demonstrated in mice when formulated in the AddaVax, a bio-similar of GMP-produced MF59. 
BALB/c mice were vaccinated IM with soluble RBD, RBDn-CLP (ABNCoV2) or RBDc CLP (a 
VLP with the RBD bound to the c-terminus of the AP205 capsid protein instead of the n-
terminal Tag/Catcher AP205). The ABNCoV2 vaccine and RBDc-CLP induced CoV-2 spike 
protein-specific IgG in all mice after a single vaccination and a booster vaccination increased 
the magnitude of the response. In contrast, soluble RBD required 2 administrations to induce 

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a detectable increase in vaccine-specific antibody concentration. In this study, doses (6.5 μg 
ABNCoV2) that corresponds to a human equivalent dose 10 to 20 times higher than 70 μg 
were used, which indicates a broad safety margin. Doses as low as 1 µg were also 
immunogenic. 
Together with an increase in antibody concentration, a single vaccination with ABNCoV2 
resulted in 100% neutralization of SARS-CoV-2 at a serum dilution of 1:80. The titre post 
booster immunization increased to 1:10,240. A correlation analysis between the ELISA 
antibody titres and neutralizing antibodies showed that there was a positive correlation 
between these measurements (Ks = 0.7152, p = 0.0461) in mice immunized with ABNCoV2 or 
RBDc-CLP, but not in the mice vaccinated with soluble RBD (Ks = 0.316, p = 0.4679). When 
compared with virus neutralization data generated in small animal models with other COVID-
19 vaccine candidates, ABNCoV2 induced neutralizing antibody where highest (Figure 3). 
 
Figure 3. Neutralizing Antibody Levels Induced by Different COVID-19 Vaccines in Rodents. 
DNA: deoxyribonucleic acid; ID50: infectious dose 50; mRNA: messenger RNA; Pseudo V: 
pseudovirus; RBD: receptor binding domain; Repli V: replicating virus; RNA:  ribonucleic 
acid; saRNA: small activating RNA 
 
A Good Laboratory Practice (GLP) compliant repeat dose toxicity study in New Zealand White 
(NZW) rabbits showed no abnormalities following two and three injections of 100 µg MF59-

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adjuvanted ABNCoV2 that were considered a safety concern for progression in human trials. 
Compared to normal saline placebo, mild to moderate systemic and local reactogenicity was 
observed. All changes were reversible. In NZW rabbits, 100 µg translate to a human equivalent 
dose of >700 µg.  
An ongoing nonhuman primate (NHP) challenge study in rhesus macaques has shown a robust 
increase in functional antibody titres following a single ABNCoV2 vaccination (15 and 100 µg 
ABNCoV2 with and without MF59) with a peak beyond 4 weeks. As in COUGH-1, a 1:1 
formulation with MF59 was used. This study will go on to provide longer term immunogenicity 
and protection data, with a challenge ~5 months post vaccination. Immunogenicity and first 
safety results of the NHP study will be available before start of the trial, NHP challenge 
infections are scheduled later. Most likely, data will be available during the dose-escalation 
phase of COUGH-1.  
6.3 Summary of findings from clinical studies 
COUGH-1 is a first-in-human study. Preliminary early phase data as well as Phase 3 results 
of all published trials do not indicate a major risk related to the antigen [15-17, 20-22], e.g. 
vaccine-enhanced disease [37]. There are no past human experiences with the non-
adjuvanted or MF59-adjuvanted ABNCoV2 vaccine. To date, two mRNA SARS-CoV-2 
vaccines have been marketed in the Netherlands: Comirnaty and Moderna, see below in Table 
4.  
Table 4. Marketed SARS-CoV2 vaccines 
Product number 
(EMA) 
Name 
Characteristics 
Population 
Very common adverse 
events (≥1/10) 
EMEA/H/C/005735  
 
Comirnaty 
(Pfizer/BioNTech) 
mRNA vaccine  
≥16 years 
old 
Pain and swelling at the 
injection site, fatigue, 
headache, myalgia, 
arthralgia, chills, fever. 
EMEA/H/C/005791 
COVID-19 
Vaccine Moderna 
mRNA vaccine 
≥18 years 
old 
Lymphadenopathy, 
headache, 
nausea/vomiting, 
myalgia, arthralgia, pain 
and/or swelling at 
injection site, fatigue, 
chills, fever. 
 

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Although an AP205 cVLP vaccine like ABNCoV2 has not been previously tested in humans, 
there is extensive clinical experience with licensed VLP vaccines that protect against human 
papillomavirus (HPV) in addition to some early clinical experience with closely related Phage 
Qβ cVLP vaccines. cVLPs based on Phage Qβ (an RNA bacteriophage similar to AP205) have 
been examined in phase 1/2 clinical trials for indications of hypertension, asthma and smoking 
addiction, see Table 5. There is also a wealth of clinical data on influenza vaccines that are 
similarly adjuvanted with MF59. To date 270 million doses of recombinant VLP vaccines have 
been administered over the past 14 years. Overall VLP treatments have been shown to be 
generally safe and well tolerated, with reports of vaccine-related injection site and systemic 
reactions being most common. 
Table 5. Clinical experience of Phage Qβ cVLP vaccines 
NCT number 
Indication 
Intervention 
Characteristics 
Adverse events 
NCT00710372 
NCT00500786 
NCT00701649 
Hypertension 
CYT006-AngQb 
Phage Qβ cVLP 
vaccine targeting 
angiotensin 2 
Phase 1-2 
Transient flu-like 
symptoms, local 
injection site 
reactions. 
NCT00369616 
NCT01280968 
Nicotine 
(smoking 
cessation) 
Phage Qβ cVLP 
vaccine 
Phase 2 
Transient flu-like 
symptoms, 
pyrexia, 
headache, 
nasopharyngitis, 
chills, myalgia 
and local injection 
site pain. 
6.4 Summary of known and potential risks and benefits 
The investigational study product, ABNCoV2, to be used in this study has not been 
administered previously to humans. All known risks and precautions described here are 
explained in detail in the informed consent documents.  
6.4.1 
Risk Assessment 
The following section (Table 6) outlines the initial risk assessment and mitigation strategy 
for this study protocol. 
Table 6. Risk Assessment 

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Important 
potential/identified 
risk 
Data/rationale for risk 
Mitigation strategy 
Investigational study product (ABNCoV2 vaccine) 
Important potential 
risk: First-in-
human unknown 
risk 
There is no prior experience of 
ABNCoV2 vaccination in human 
subjects. 
A dose escalating design will be used for 
Group 1 up till Group 5, starting with the 
lowest dose of 6 µg, which is lower than in 
all pre-clinical studies where no toxicity was 
observed (absolutely and when calculated as 
human equivalent dose – preclinical toxicity 
was assessed at a human equivalent dose of 
>700 µg). Subjects will be observed closely 
by qualified clinicians, and emergency care 
will be immediately available to subjects. A 
minimum of 6 days is required between 
product administration to the sentinel 
subjects of each group. In case that related 
serious related adverse events occur dose-
escalation will be stopped.  
Important potential 
risk: 
Hypersensitivity 
(including 
anaphylaxis) 
As with other vaccines, 
hypersensitivity and anaphylaxis to 
one or several components of the 
vaccine can rarely occur. 
Subjects who report allergic reaction against 
constituents of the vaccine in the past will not 
be recruited. All vaccinated volunteers will be 
observed closely for at least 60 minutes 
following administration of the product 
ABNCoV2 with appropriate medical 
treatment readily available in case of severe 
adverse events. 
Local and 
systemic 
reactogenicity to 
intramuscular 
administration of 
ABNCoV2 
ABNCoV2 vaccine has never 
previously been administered 
intramuscularly in humans. The 
risks associated with vaccination 
include local inflammatory reactions 
to the injected product, such as pain 
Experienced and qualified medical 
personnel will administer the vaccination 
intramuscularly. Subjects will be informed 
about the local and systemic side effects that 
may occur and will be monitored closely for 
local and systemic adverse events. Subjects 

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and swelling at the injection site. 
Systemic effects generally 
associated with vaccination may 
include flu-like symptoms, fever, 
chills, nausea, gastrointestinal 
symptoms, headache, malaise, 
myalgia and arthralgia. 
will be advised to inform or call the study 
doctor immediately if they have any side 
effects.  
Study procedures 
Pain when taking 
blood samples 
Because of the necessity to obtain 
frequent blood sampling for 
laboratory safety analysis, 
determining the concentration of 
ABNCoV2-specific antibodies, and 
measuring cellular immune 
responses, there is a risk of feeling 
faint, or experience mild pain, 
bruising, irritation or redness at the 
site where blood was taken. In rare 
cases arteries or nervous tissue 
may be injured or a punctured 
vessel may occlude and induce 
inflammation of the surrounding 
tissue. 
Experienced and qualified medical personnel 
will draw blood. The amount of blood to be 
taken for sampling will not be harmful to the 
subject’s health. Total blood volume over the 
whole study period will be below <500 mL, 
less than the amount of a single whole blood 
donation. 
Pregnancy risks 
Pregnancy and 
lactating females 
Risks of the ABNCoV2 vaccine to 
unborn babies are unknown at this 
time; pregnant females will be 
excluded from this study. 
Females are only eligible for participation if 
they agree to use efficient contraception 
during the study period and the pregnancy 
test during the inclusion visit is negative. In 
case effective contraceptives are not 
available the clinical team may provide them. 
Volunteer who becomes pregnant before the 
second immunization will not receive 
subsequent trial immunizations and will be 
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administration. Pregnant volunteers will not 
be withdrawn from the trial. Conditional on 
their agreement, they will be followed-up until 
the end of the pregnancy. Immunological and 
other exploratory blood samplings will be 
reduced to a minimum. Lactating females 
will be excluded from this study. 
Risks to study personnel 
Needle-stick 
injuries 
The principal risk in the clinical 
setting is the handling of needles 
that may be contaminated with 
blood or body fluids and the 
associated risk of acquiring a 
blood-borne pathogen (including 
hepatitis B and C viruses and 
human immunodeficiency virus 
(HIV)). 
Adherence to standard operating procedures 
(SOP) for working with infectious agents 
and universal precautions will reduce the 
risk of exposure. Subjects will be screened 
for hepatitis B and C and HIV prior to 
inclusion. Individuals positive for HIV, 
hepatitis B and C are excluded from the 
study. 
Receiving another, authorized, SARS-CoV-2 vaccine 
Important potential 
risk: Unknown 
risk(s) associated 
with 
administration of 
a different SARS-
CoV-2 vaccine 
The risks associated with 
administration of a different SARS-
CoV-2 vaccine after having received 
ABNCoV2, remain unknown. 
Participants in the COUGH-1 trial offered an 
authorized SARS-CoV-2 vaccine as part of 
the official vaccination campaign during 
follow-up will be advised to receive it, 
although we will recommend participants not 
to do so earlier than 4 weeks after their 
second ABNCoV2 vaccination. This is to be 
able to attribute a majority of AE during follow 
up to ABNCoV2 administration and to 
decrease the risk of potential interaction 
between immune responses against 
ABNCoV2 and another SARS-CoV-2 
vaccine. The risk of SARS-CoV-2 infection 
and, if infected, of severe COVID-19 is very 
low in the study population. Particularly, as it 
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due to a combination of social distancing 
measures, mass vaccination and seasonal 
environmental factors. Volunteers who are 
offered an authorized SARS-CoV-2 vaccine 
during follow-up will be asked to attend 
additional study visits (before and after the 
vaccination), in order to document 
occurrences of (novel) adverse events, as 
well as changes in immunogenicity. 
 
6.4.2 
Benefit assessment 
There is no direct benefit for study subjects from participation in the trial. It will be made clear 
that the vaccine is experimental and may not protect against COVID-19. Subjects may 
indirectly benefit from general medical evaluation and health screening procedures including 
testing for HIV, hepatitis B, and hepatitis C. Subjects will be informed about the results of the 
screening and if necessary, they will be referred to their primary physician where they will 
receive counselling and further medical attention earlier than if they did not know of their 
disease status. Society as a whole may benefit from the development of an effective COVID-
19 vaccine, as this has the potential to improve the health of the worldwide population, as well 
as have extremely positive impacts on economic, social and health systems. Subjects will 
receive a financial compensation which is reasonable and in line with Dutch common practice 
(see 11.5). 
6.5 Description and justification of route of administration and dosage 
Enrolled participants will receive two intra-muscular injections of the ABNCoV2 vaccine in the 
deltoid muscle, preferably starting with the non-dominant arm at first vaccination, followed by 
the other arm at second vaccination. The administration site minimizes the risk that the 
injection goes into a blood vessel. In case of suspicion that a blood vessel is hit, the vaccine 
will not be administered. In case of trauma or anatomical abnormalities the same arm may be 
used for subsequent injections.  
The vaccine will be administered on days 0 and 28. The trial is designed to evaluate the 
safety and tolerability in a staggered dose escalation of five dosages (6 µg, 12 µg, 25 µg, 50 
µg and 70 µg). The initial starting dose proposed in this protocol is extrapolated from other 
VLP-type vaccines and pre-clinical data (see above). Four weeks after the first vaccination, a 
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6.6 Dosages, dosage modifications and method of administration 
Preparation of the syringe will be described in a separate pharmacy manual. All vaccinations 
will consist of the intramuscular injection with a syringe preferably starting with the non-
dominant deltoid muscle. The syringe will be prepared by the pharmacy and delivered with the 
appropriate documentation. ABNCoV2 vaccine will be tested for the first time in humans. 
One objective of the study is to identify a dosage that optimizes the immunogenicity-tolerability 
ratio 14 days following first vaccination. MF59-adjuvanted and non-adjuvanted formulations 
will be tested in parallel in Group 1 up till Group 3 to detect superiority or futility of the MF59-
adjuvanted against the non-adjuvanted formulation. If the adjuvanted vaccine formulation is 
superior based upon immunogenicity endpoints, whilst still remaining safe and tolerable 
(primary endpoints), subsequent Group 4 up till Group 7 will receive the adjuvanted vaccine. If 
not, Group 4 up till Group 7 will receive the non-adjuvanted vaccine. Dose-escalation will be 
performed until an optimal immunogenicity-tolerability ratio is reached with a ceiling of 70 µg. 
When immunogenicity dose-response is saturated (no further increase in immunogenicity and 
an acceptable tolerability profile) the highest reached and the next lower dosage will be 
repeatedly tested to increase power to detect adverse reactions at the optimal dose. Decision 
on adjuvant use and optimal dose will be made by the investigator and sponsor teams followed 
by a request of approval by a safety monitoring board (SMC), supported by analysis of 
tolerability, safety and immunogenicity data accrued up to the date of analysis. 
6.7 Preparation and labelling of Investigational Medicinal Product 
6.7.1 
Presentation and formulation 
ABNCoV2 (without adjuvant) will be supplied frozen in multidose glass vials closed with 
bromobutyl rubber stoppers, crimped with flip-off caps. The vial contents will be combined with 
MF59 adjuvant at the site pharmacy and distributed within the indicated shelf life of the 
formulated product.  
The MF59 adjuvant will be supplied in glass-lined prefilled syringes that protect against light. 
Prior to mixing ABNCoV2 with MF59 at the clinical trial site, the formulation of ABNCoV2 is 
supplied as 0.5 mg/mL +/- 0.1 mg/mL ABNCoV2, 1.2 mM KH2PO4, 8.1 mM Na2HPO4, 136.9 
mM NaCl, 2.7 mM KCl, 10.0 mM tris, 200 mM sucrose, pH 8.6. The drug product ABNCoV2 is 
a clear to opalescent liquid. Formulation will be done by the pharmacy at Radboudumc 
according to a study specific pharmacy manual. The procedure will be fully documented. The 
trial is not blinded. Hence, investigators and volunteers are aware of the content of the syringe 
used for injection.  

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6.7.2 
Stability and storage 
ABNCoV2 must be stored frozen at -20°C ± 5°C. MF59-adjuvant must be stored between 2°C 
and 8°C. It should not be frozen. ABNCoV2 drug product and MF59-adjuvant will be stored in 
a secure area with access limited to authorized study personnel, and will not be used for any 
other purpose other than the trial. ABNCoV2 formulated vaccines are stored at 2-8 °C and will 
be used within 24 hours. The storage conditions will be assessed during pre-trial monitoring 
visits. The storage temperature will be continuously monitored and recorded with calibrated 
temperature monitoring device(s). In case of temperature excursions, they will be reported 
in degrees Celsius. Any deviation from the recommended storage conditions will be 
immediately reported to the clinical trial monitor and the sponsor. The use of the impacted 
study vaccine must be interrupted until the sponsor has given authorization for its continued 
use. 
6.7.3 
Dose preparation and administration 
Dose preparation, as described in the pharmacy manual, will be carried out by qualified 
personnel under controlled conditions. The ABNCoV2 vaccine product and adjuvant will be 
mixed prior to dosing at the clinical trial site pharmacy under the responsibility of a pharmacist 
according to a standard operating procedure (SOP). Syringes will be prepared under sterile 
conditions. Syringes contain either: 
• 
6 µg ABNCoV2 
• 
6 µg ABNCoV2 + MF59-adjuvant 
• 
12 µg ABNCoV2 
• 
12 µg ABNCoV2 + MF59-adjuvant 
• 
25 µg ABNCoV2 
• 
25 µg ABNCoV2 + MF59-adjuvant 
• 
50 µg ABNCoV2 +/- MF59-adjuvant 
• 
70 µg ABNCoV2 +/- MF59-adjuvant 
From 50 to 6 µg two-fold serial dilutions will be used during the preparation of the vaccine 
product. Reported doses are rounded to the next lower integer for the two lowest doses. 
All preparations will be diluted to give an injection volume of 0.5 mL. A pharmacy manual 
containing instructions for storage, preparation and handling of the vaccine will be provided to 
the pharmacist. Furthermore, trainings on the procedure will be performed before the first 
injection. Individual sterile single use 1 mL syringes for injection will be delivered to the person 
in charge of the injection. A separate sterile vanish point syringe will be used for each individual 
trial participant to prevent transmission of infectious agents. Before injection, the site of 
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In case that a blood vessel is accidentally punctured, the vaccine is not injected and a novel 
syringe needs to be prepared. As for any injectable vaccine, appropriate equipment will be 
available in case of immediate allergic reactions. Enrolled participants will receive two 
intramuscular injections of the vaccine. The first will be given preferably in the non-dominant 
deltoid muscle, the second in contralateral muscle. The vaccine will be administered on day 0 
and day 28. Caution will be taken to ensure that the injection does not go into a blood vessel 
(see above). In case of trauma or anatomical abnormalities a single arm or may be used. Side 
and site of injection will be documented.  
6.7.4 
Labelling 
ABNCoV2 vaccine product and the MF59-adjuvant will be labelled and packaged according to 
applicable regulatory requirements. The labels will mention that the products are for 
investigational use only. The pharmacy at Radboudumc has a manufacturing authorization and 
may label the ABNCoV2 drug product and MF59 to fully comply with regulatory requirements. 
Details are given in the pharmacy manual.  
6.8 Drug accountability 
The drug product and adjuvants will be shipped to Radboudumc according to the relevant 
national and international regulations. The site pharmacist will maintain complete records of all 
study products received from the sponsor, an inventory, and an accountability record of the 
drug product and adjuvants for this study. The site pharmacist will also ensure the security of 
these documents. Vials will only be used for human administration within COUGH-1 and not 
for other in vitro or in vivo experimental studies. At the end of the study, the site will receive 
instruction from the manufacturer regarding the final disposition of any remaining study 
products. The person in charge of the product management will return a completed dispatch 
note to the sponsor, which will be attached to the package, as acknowledgement of receipt. 
The person in charge of product receipt will check that the cold chain was maintained during 
shipment. In case of a problem, the clinical trial monitor and the Sponsor must be alerted 
immediately. The acknowledgement of receipt will be dated and signed by the person in charge 
of product management. One copy will be kept archived; the other copy will be returned to the 
trial sponsor. 
In accordance with all applicable regulatory requirements, the person in charge of the product 
management at the site must keep up to date the following inventory records: 
• 
Receipt of product at the clinical trial site  
• 
Treatment administered to each subject (including number of injection) 
• 
Inventory of product at the clinical trial site 
• 
Log of any unused or expired investigational product 

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These records should include dates, quantities, batch numbers, period of use or expiration 
dates (if applicable), and any unique code numbers assigned to the product subjects.  
These records should document adequately that: 
• 
The subjects were provided the doses specified by the protocol 
• 
All products provided are fully reconciled 
These records will be monitored and verified by the clinical trial monitor regularly, as part of 
the routine monitoring procedure. 
Unused or open products will be either returned to the sponsor or designee at the end of the 
vaccination period together with the form "Return of unused or open products" or destroyed 
according to the sponsor’s instructions. 
7. NON-INVESTIGATIONAL PRODUCT 
Not applicable. 
8. METHODS 
8.1 Study endpoints 
8.1.1 Main study parameters/endpoints 
- 
Primary safety endpoint: Number of at least possibly related Grade 3 adverse events 
(AE) and serious adverse events (SAE) from time of first administration of ABNCoV2 
until the end of the follow-up period. 
- 
Primary immunogenicity endpoint: Concentration of ABNCoV2-specific antibodies 14 
days following first vaccination. 
8.1.2 Secondary study endpoint 
- 
Number and severity of at least possibly related solicited AEs within one week following 
administration of ABNCoV2. 
8.1.3 Exploratory study parameters/endpoints 
- 
Concentration of ABNCoV2-specific antibodies at baseline and during immunization 
and follow up. 
- 
Inhibitory titre in invasion inhibition assay at baseline and during immunization and 
follow up. 
- 
Cellular immune responses (T and B cell) at baseline and during immunization and 
follow up. 
- 
Correlation of response vaccine to habitual sleep using the Pittsburgh Sleep Quality 
Index (PSQI) [38, 39].  

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8.2 Randomisation, blinding and treatment allocation 
This will be an open-label trial to enable appropriate safety evaluation prior to proceeding to 
the next dose group. If the enrolment criteria are met, eligible subjects will be entered into the 
study. Randomization will not be used in this study. Subjects will be allocated based on 
logistical considerations (e.g. availability during follow up visits). The date/time of allocation 
and corresponding allocation group will be documented in the electronic case report form 
(eCRF).  
8.3 Study procedures 
It is the responsibility of the investigator to ensure only medically qualified study staff perform 
medical assessments and procedures which require medical training. Results of 
assessments should be reviewed with the subject, as appropriate, in a timely manner. It 
is also the responsibility of the investigator to ensure strict observance of the intervals 
between visits/procedures. These intervals are precisely defined for subjects in accordance 
with the protocol and are reflected in the study schedule outlined in section 8.3.19. In the 
context of the ongoing COVID-19 outbreak, the clinical investigator will follow current local and 
national guidelines concerning the mitigation of SARS-CoV-2 transmission and dedicated to 
minimizing the risk of exposure of study volunteers, study personnel, hospital staff & patients 
and the general public. This applies to all study visits. 
8.3.1 
Recruitment of subjects 
The purpose of recruitment will be to obtain up to 42 eligible subjects for participation in 
this trial plus up to 7 reserve subjects (1 reserve subject per group). In order to recruit 
sufficient subjects, advertisements will be placed in prominent public places (e.g. as flyers), as 
well as on the University intranet. The study will be advertised on social media and local 
newspapers. The advertisement text will indicate a telephone number to call, an e-mail 
address, and a website to request further information. Interested subjects can register for the 
(digital) information meetings by filling in an online form which includes basic questions 
regarding their general state of health, use of medication, and participation in previous 
scientific studies. All subjects with an interest in participation will be advised to join one of 
the (digital) information meetings, where they will receive information on study participation 
and the rights and duties of subjects, as well as the corresponding documents (the study 
information sheet and study schedule, the application form, general information brochure 
on medical-scientific research from the Ministry of Health, Welfare and Sport, and the 
insurance text). If they are still interested after the information meeting and/or after reading 
the documents, an appointment for a screening visit will be made. The screening visit will be 
planned at least 24 hours after the subject receives the information sheet and informed 

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consent form. Since the information meeting is not mandatory, the documents can be e-
mailed to subjects instead. Applicants will receive a pseudonymised screening number of 
two letters and a corresponding volunteer number as soon as they return their application 
form. This number will be used for source documents. This volunteer code will not contain 
any directly identifiable information. 
8.3.2 
Screening visit (visit 1) 
The screening visit will take place within 28 days before the first vaccine administration. The 
screening appointment for individual subjects is planned at least 24 hours after the subject 
has received the information sheet and the informed consent form. The purpose of the 
screening visit is to provide and clarify study information to the volunteer and answer any 
questions the subjects may have, to obtain informed consent and to determine whether 
interested subjects are eligible for participation. The trial physician is responsible for providing 
the study information and performing the medical screening. A study nurse may draw 
venous blood, and collect vital parameters. 
Upon arrival for the screening visit, the volunteer is given a medical questionnaire and a short 
quiz about the study to fill out. Subsequently, and prior to review of the questionnaire and 
quiz or any other screening activities, study staff will review the informed consent process 
with the volunteer. The possibility of withdrawal from the study, at any time and without any 
declaration of the reason, will be pointed out to the subjects. The investigator, or a person 
designated by the investigator, will fully inform the volunteer of all pertinent aspects of the 
study and individual consent will be documented by a signature. Subjects may only participate 
in screening if they have signed the informed consent form. All subjects must consent to a 
SARS-CoV-2, HIV, hepatitis B and hepatitis C serological screening, urine toxicology and for 
females also a pregnancy test. Subjects who sign the informed consent will undergo complete 
screening. After subject consents, the below activities will occur: 
- 
The study quiz will be reviewed and if any incorrect answers were given, these will 
be given additional attention. If the score on the quiz was <80%, the subject will be 
given the opportunity to retake it. 
- 
Patient history: the answers to the medical questionnaire are discussed with the 
volunteer and clarified where needed. The subject will be further interviewed to 
collect demographic data, medical history including details of any chronic or 
recurrent medical and psychiatric conditions and use of adequate contraception;  
- 
Study information: the study schedule and study rules are discussed with the subject 
and any questions are answered; 
- 
A physical examination including vital signs, height and weight will be performed; 

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- 
Blood specimens will be collected for routine clinical laboratory testing of 
biochemical and hematological parameters, SARS-CoV-2 antibodies, as well as HIV, 
hepatitis B and hepatitis C serological screening; 
- 
A urine specimen will be collected for toxicology screening and for females a 
pregnancy test if the subject is of childbearing potential; 
- 
If necessary, consent to inform a medical specialist of study participation, will be 
signed by the subject and sent after screening. 
 
If physical examination, vital signs or laboratory values are out of the normal range, a repeat 
measure may be obtained as deemed necessary by the investigators. The medical history, 
physical examination, and laboratory findings for subjects will be recorded in the source 
screening data documents. All subjects will be asked to supply a phone number of a partner 
or roommate who may be contacted in case of emergency. Concomitant medications is 
recorded at all study visits. All results of the screening will be reviewed with the subject. 
Subjects are informed by phone if they have satisfied all the inclusion criteria. If clinically 
significant abnormalities are identified during screening, subjects will be referred to their 
primary health provider or appropriate medical center. If identified during the study, subjects 
may be asked to return to the study site for further evaluation, including clinical evaluation 
and repeat laboratory testing as warranted. If more than 28 days have passed since screening 
before the intended day of first vaccine administration, the subject will be rescreened.  
8.3.3 
Inclusion visit (visit 2) 
Subjects meeting the eligibility criteria during screening (section 4.2 and 4.3) will be invited 
back for enrolment into the study at the inclusion visit, which will occur prior to the 
planned administration day. Baseline assessments will be taken on the inclusion day. For 
each subject, study start (day 0) will be defined as the day of first ABNCoV2 administration. 
For subjects that do not show up for inclusion visit (visit 2) or first inoculation visit (visit 3), 
alternate subjects may be included. At study inclusion, the following activities will occur: 
- 
Patient history will be taken and all AE that have occurred since screening will be 
noted. Only subjects who still meet the inclusion criteria will be included to receive 
ABNCoV2; 
- 
A nasopharyngeal swab for SARS-CoV-2 PCR; 
- 
Blood specimens will be collected for routine clinical laboratory testing of 
biochemical and hematological parameters; 
- 
Non-invasive sampling of saliva and mucosal secretions of the respiratory organs will 
be done to compare presence of antibodies at the site of infection to serum.  

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- 
A urine specimen will be collected for toxicology screening and for females a 
pregnancy test if the subject is of childbearing potential; 
- 
Subject will be issued a thermometer and symptom diaries to record any local and 
systemic symptoms and medication use; 
- 
Subject will be issued an emergency notification card that details their participation 
in the study and provides contact phone numbers of the investigators. 
8.3.4 
ABNCoV2 inoculation visits (visits 3 + 9) 
The purpose of these visits is to inoculate enrolled subjects with ABNCoV2. On the day of the 
first ABNCoV2 administration, the subjects will be admitted for approximately 4 hours. On the 
day of the second ABNCoV2 administration, the subjects will be admitted for approximately 1 
hour. Subjects will be briefly assessed regarding any new medical events since the inclusion 
visit or most recent follow-up visit. Temperature, blood pressure, and pulse rate will be 
recorded. To confirm eligibility, results of all assessments will be reviewed. If the subject is 
still eligible for participation, the following activities will occur as detailed below: 
- 
ABNCoV2 product will be prepared as described in the pharmacy manual by 
pharmacy staff based on group assignment. 
- 
ABNCoV2 will be administered by intramuscular injection in the deltoid muscle 
(if possible into alternating sides, starting with the non-dominant arm). Subjects 
will be observed directly for at least 60 minutes after administration.  
- 
Subjects may leave the unit at the Radboud Technology Center for Clincial 
Studies (RTCCS, hereafter ‘study center’) after 4 hours on the day of the first 
ABNCoV2 administration and after 1 hour on the day of the second 
ABNCoV2 administration. Subjects return the next day to the clinical research 
center for the 24-hour blood sampling time point. 
- 
Vital signs, AE (solicited and unsolicited) and blood will be collected at each 
time point as indicated in section 8.3.19. 
- 
A list of solicited local and systemic symptoms will be reviewed with the subject 
prior to and after administration. If the occurrence of an AE or use of medication 
is confirmed by the study physician, it is recorded in the clinical trial database. 
- 
The PSQI questionnaire about sleep behaviour will be filled in by the subject.  
- 
After leaving the study center, subjects will be asked to examine the site of 
injection and record local signs and symptoms for seven days, including 
bruising, erythema, swelling or induration. Subjects will be asked to measure 
their oral temperature daily until day 7 after each administration. Subjects will 
be asked to record any AEs in the memory aid booklet until day 196 of the 

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study. At the end of the study, the memory aid will be collected and kept as 
source data with the subject’s study file. 
 
All inoculations are performed by a trained nurse under the supervision of one of the clinical 
investigators. Another clinical investigator will be on call in case of emergency. Local hospital 
procedures will be followed. Study subjects will be monitored at the RTCCS Unit on the days 
of ABNCoV2 administration. Follow-up visits will take place as described below. 
8.3.5 
Follow-up visits after ABNCoV2 administration (visit 4-8 and 10-16) 
Follow-up visits will be carried out on an outpatient basis at the study center. Subject will be 
asked to examine the site of injection and record local signs and symptoms in their 
memory aid booklet (including bruising, erythema, swelling or induration) for seven days after 
injection as well as systemic symptoms. Subjects will also be asked to measure their body 
temperature in the mornings for 6 consecutive days after ABNCoV2 administration. The 
subject diary will be reviewed at each study visit and used as a basis for discussion of possible 
local and systemic AE or medication use. If the occurrence of an AE or use of medication is 
confirmed by the study physician, it is recorded in the clinical trial database. At the end of the 
study, the diary will be collected and kept as source data with the subject’s study file. Follow-
up visits not requiring blood collection may be carried out by telephone instead of at the study 
center. During the study period subjects will be instructed to call the trial physicians at 
any time if they experience significant symptoms. If clinically significant abnormalities are 
identified during the study, subjects may be asked to return to the study center for further 
evaluation, including clinical evaluation and repeat laboratory testing as warranted. The trial 
physician can decide to initiate any additional diagnostics (including safety laboratory 
evaluations) at all times. For unexpected laboratory abnormalities, the laboratory test may 
be repeated. If there is any ambiguity regarding these results, a trial physician will discuss the 
case with the local safety monitor. 
8.3.6 
Unscheduled visits 
Subjects may need to present to the study center for an unscheduled visit should they 
experience any AE that requires evaluation by the trial clinician. Data for any examinations 
performed on the subject at an unscheduled visit must be recorded in the eCRF. If an 
unscheduled visit is performed, the procedures for the next following visit should not be made 
earlier than scheduled above. 

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8.3.7 
Visits regarding vaccination with licensed SARS-CoV-2 vaccine 
Subjects who are offered an authorized SARS-CoV-2 vaccine during the follow-up period will 
be allowed to receive it. Preferentially, late following their second ABNCoV2 vaccination (not 
earlier than 4 weeks). This is to be able to attribute a majority of AE during follow-up to 
ABNCoV2 administration and to decrease the risk of potential interaction between immune 
responses against ABNCoV2 and another SARS-CoV-2 vaccine. The risk of SARS-CoV-2 
infection and, if infected, of severe COVID-19 is very low in the study population. Particularly, 
as it is expected that incidence will have dropped due to a combination of social distancing 
measures, mass vaccination and seasonal environmental factors. Subjects who are offered an 
authorized SARS-CoV-2 vaccine during follow-up will be asked to attend additional voluntary 
study visits before and after the vaccination. Subjects will be compensated 25 euros per extra 
on-site follow-up visit. 
8.3.8 
Medical history 
The trial clinician will review the medical history of potential study subjects during the 
screening visit. The starting point will be a medical questionnaire the subjects have filled 
out. Particular attention will be paid to: 
- 
Current or recent (within the previous two weeks) acute respiratory illness with or 
without fever, including symptoms associated with COVID-19; 
- 
COVID-19 infection in the past; 
- 
Recent receipt of immune globulin or other blood products, or injected corticosteroids 
or other immune modulator therapy within the previous six months; 
- 
Hypersensitivity of any kind; 
- 
Clinically relevant history of cardiovascular, renal, gastrointestinal, hematological, 
dermatological, endocrine, neurological or immunological diseases; 
- 
Known or suspected immunologic function impairment of any kind and/or known HIV 
infection, hepatitis B or hepatitis C infection; 
- 
Mental illness; 
- 
Tobacco, alcohol, or drug use; 
- 
Medication use in the past 6 months; 
- 
For women, pregnancy and contraceptive use and/or history of surgical sterilisation. 
For males, use of contraception; 

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8.3.9 
Physical examination 
A physical examination will be performed during screening, including the examination of 
general appearance, skin, neck, eyes, throat, lungs, heart, abdomen, back and extremities, 
and a routine vascular and neurological examination. During follow-up visits a focused 
physical examination will be performed if deemed necessary by the trial physician. 
8.3.10 Height and weight 
Height (cm) and body weight (to the nearest kilogram [kg] in indoor clothing, but without shoes) 
will be measured at the screening visit. Body mass index (BMI) will be calculated using the 
following formula: BMI = Body weight (kg) / [height (m)]2 and converted to an integer. 
8.3.11 Vital signs 
Vital signs including body temperature (degrees Celsius), blood pressure (BP, millimeters of 
mercury) and pulse measurements (beats per minute) will be recorded at the screening visit. 
Additional measurements will take place at the discretion of the physician. Systolic and 
diastolic BP will be measured while the subject is sitting, with the back supported and both 
feet placed on the floor, using an validated automated device, with an appropriately sized cuff. 
In case the cuff sizes available are not large enough for the subject’s arm circumference, 
a sphygmomanometer with an appropriately sized cuff may be used. If vital signs are out-of-
range at screening or baseline, the investigator may obtain two additional readings, so that 
a total of up to three consecutive assessments are made, with the subject seated quietly 
for approximately five minutes preceding each repeat assessment. At least the last reading 
must be within the normal range in order for the subject to qualify. Additionally, subjects will be 
given a thermometer to measure their oral temperature on a daily basis for one week after 
ABNCoV2 administration, which they will collect in their study diaries. 
8.3.12 Laboratory evaluations 
During the study, blood samples will be drawn for screening, safety and research purposes. 
The blood sampling schedule is shown in the flowchart (section 8.3.19). Following universal 
precautions, blood will be collected by venipuncture into vacutainer tubes. Blood specimens 
will be affixed with coded labels that link the specimen to the subject, specimen type, specimen 
collection date, and time-point. The specimen collection tube must be appropriate for the type 
of specimen required. The cumulative blood draw for each subject over the entire course of 
study participation will be ≤500 mL (the approximate amount of one blood donation). Biological 
safety parameters will be measured in plasma or serum samples at the central laboratory of 
the Radboudumc. In the case a laboratory assessment is outside the reference range, a 
decision regarding whether the result is of clinical significance or not shall be made by the 

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investigator and shall be based, in part, upon the nature and degree of the observed 
abnormality. The assessment may first be repeated for confirmation. All abnormalities will be 
documented in the source documents, including clinical considerations. 
Complete Blood Count (CBC) 
Complete hematology tests, including hemoglobin, hematocrit, red blood cell count, white 
blood cell count with differential (including neutrophils, basophiles, eosinophils, monocytes 
and lymphocytes) and platelet count will be measured when appropriate. Complete 
hematology will be done at screening, at 24 hours and days 7, 27, 29, 35 and 196. HbA1C will 
only be measured at screening. 
Clinical Chemistry 
Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, urea, sodium, 
potassium, bilirubin, yGT and LDH will be measured at screening, at 24 hours and days 27, 29 
and 196. Glucose, cholesterol and triglyceride will only be measured at screening. 
In addition to blood samples, non-invasive sampling of saliva and mucosal secretions of the 
respiratory organs will be done to compare presence of antibodies at the site of infection to 
serum.  
8.3.13 Urine toxicology analysis 
A midstream urine sample (approx. 30ml) will be obtained, in order to avoid contamination 
with epithelial cells and sediments, to allow proper assessment. A toxicology screening will be 
performed at inclusion (visit 2). A positive test for amphetamines and cocaine is a reason for 
exclusion.  
8.3.14 Pregnancy test 
A midstream urine sample (approx. 30ml) will be obtained and assessed by commercially 
available hCG urine tests. This test will be done at screening (visit 1), inclusion (visit 2) and the 
day before the second vaccination (visit 8). 
8.3.15 Discontinuation of study procedures 
Participants have the right to decline study procedures for any reason and at any time 
during the study. If a subject declines study procedures, this will be recorded as a study 
deviation and the reason will be clearly documented in the source document. The subject 
will be encouraged to complete all remaining safety related follow-up visits and procedures 
through the entire study period, including 
unscheduled visits. The reason for all 
discontinuations will be documented in source documents and the eCRF. 

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8.3.16 Assay to determine concentration SARS-CoV-2-specific antibodies  
Antigen-specific total IgG titers will measured by enzyme-linked immune-sorbent assay 
(ELISA). Briefly, 96-well plates (Nunc MaxiSorp) will be coated overnight at 4°C with 0.1µg/well 
recombinant ExpreS2 produced SARS-CoV-2 Spike (35-1227) protein in PBS. After blocking 
for 1 hour at room temperature (RT) using 0.5% skimmed milk in PBS human serum prediluted 
1:100 in blocking buffer will be added to the plate in a 3-fold dilution, followed by incubation for 
1 hour at RT. Plates will be washed three times in PBS in between steps. In order to measure 
total serum IgG or Isotype specific IgGs, Horseradish peroxidase (HRP) conjugated goat anti-
human IgG, IgG1, IgG2, IgG3 or IgG4 diluted 1:1000 in blocking buffer will be added followed 
by 1 hour incubation at RT. Plates were developed with TMB X-tra substrate (Kem-En-Tec, 
4800A) and absorbance was measured at 450nM. Data were collected on Microsoft excel 
(Microsoft 360, excel 2016) and analysed using graphpad prism (San Diego, USA, version 
8.4.3). Data will be represented by Area under the curve, IC50 and Endpoint titres. 
8.3.17 Virus neutralization assay 
Invasion inhibition assays are based upon the method described previously [40]. Briefly, 
SARS-CoV2, Freiburg isolate, FR-4286 and Leiden-001, will be propagated in VeroE6 
expressing cells expressing human TMPRSS2 (VeroE6-hTMPRSS2) with a multiplicity of 
infection (MOI) of 0.05. Supernatant containing new virus progeny will be harvested 72h post 
infection, and concentrated on 100kDa Amicon ultrafiltration columns (Merck) by centrifugation 
for 30 minutes at 4000 g. Virus titer will be determined by TCID50% assay and calculated by 
Reed-Muench method [41]. Participant serum will be heat-inactivated (30 min, 56 °C), and 
prepared in a 2-fold serial dilutions in DMEM (Gibco) + 2% FCS (Sigma-Aldrich) + 1% 
Pen/Strep (Gibco) + L-Glutamine (Sigma-Aldrich). Sera will be mixed with SARS-CoV-2 at a 
final titer of 100 TCID50/well, and incubated at 4°C overnight. No serum and a no virus 
(uninfected) controls will also be established. The following day virus:serum mixtures will be 
added to 2 x 104 Vero E6 TMPRSS2 cells seeded in flat-bottom 96-well plates, and incubated 
for 72h in a humidified CO2 incubator at 37 ˚C, 5% CO2, before fixing with 5% formalin (Sigma-
Aldrich) and staining with crystal violet solution (Sigma-Aldrich). The plates will read using a 
light microscope (Leica DMi1) with camera (Leica MC170 HD) at 4x magnification, and 
cytopathic effect (CPE) scored. 
8.3.18 Safety assessments 
Planned safety assessments will provide the data for active monitoring of investigational 
product safety during conduct of the trial and for the primary reactogenicity and safety 
endpoints. Subjects will be observed directly for 60 minutes following product administration. 

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AEs will be recorded prior to administration and at all follow-up visits. Clinical laboratory 
results assessed as AEs will be scored for severity using US FDA Toxicity Tables [30]. 
 

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8.3.19 Time and event procedures 
Type of contact 
Visit 1 
(Screening) 
Visit 
2 
Visit 
3 
Visit 
4 
Visit 
5 
Visit  
6 
Visit  
7 
Visit  
8 
Visit  
9 
Visit 
10 
Visit 
11 
Visit 
12 
Visit 
13 
Visit 
14 
Visit 
15 
Visit 
16 
Study day 
 
I1-3 
I1 
I1+1 
I1+2 
I1+7 
I1+14 
I2-3 
I2 
I2+1 
I2+2 
I2+7 
I2+14 
I2+42 
I2+91 
I2+168 
Time after first administration 
D-28 to D-1 
D-3 
D0 
D1 
D2 
D7 
D14 
D25 
D28 
D29 
D30 
D35 
D42 
D70 
D119 
D196 
Informed consent 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
● 
● 
 
 
 
 
 
● 
 
 
 
 
 
 
 
 
Demographic data and medical history 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sleep behaviour questionnaire 
 
 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination1 
● 
●1 
●1 
●1 
 
●1 
●1 
●1 
●1 
●1 
 
●1 
●1 
●1 
●1 
●1 
Blood pressure, temperature, pulse2 
● 
●2 
●2 
●2 
 
●2 
●2 
●2 
●2 
●2 
 
●2 
●2 
●2 
●2 
●2 
Record any concomitant medications 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
HIV, HBV, HCV 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Drugs screening (urine) 
 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test (urine) 
● 
● 
 
 
 
 
 
● 
 
 
 
 
 
 
 
 
Distribution of emergency notification card, 
memory card and thermometer 
 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SARS-CoV-2 PCR (swab) 
 
● 
 
 
 
 
 
● 
 
 
 
 
 
 
 
 
SARS-CoV-2 antibodies 
● 
 
 
 
 
 
● 
● 
 
 
 
 
● 
● 
● 
● 
ABNCoV2 inoculation3 
 
 
●3 
 
 
 
 
 
●3 
 
 
 
 
 
 
 

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Solicited AEs (days 0–7 and 28-35) 
 
 
● 
● 
● 
● 
 
 
● 
● 
● 
● 
 
 
 
 
Unsolicited AEs (days 0-196) 
 
 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
Complete blood count4  
● 
 
 
● 
 
● 
 
● 
 
● 
 
● 
 
 
 
● 
Biochemistry tests5  
● 
 
● 
 
 
 
● 
 
● 
 
 
 
 
 
● 
PBMC6 
 
● 
 
 
 
● 
 
● 
 
 
 
● 
 
● 
 
● 
Serum banking6  
● 
 
 
 
 
 
● 
 
 
 
 
 
 
 
● 
Safety Report7 
● 
 
 
 
 
● 
End of study visit 
 
 
● 
Cumulative blood volume (mL)8 
≤500 mL 
 
1. 
A physical examination including height and weight and vital parameters will be performed at the screening visit. Additional measurements will take place at the discretion 
of the physician. 
2. 
Vital signs including body temperature, blood pressure and pulse will be recorded at the screening visit. Additional measurem ents will take place at the discretion of the 
physician. 
3. 
Subjects will receive the first dose of ABNCoV2 on day 0, followed by a booster with the same dose and formulation on day 28. 
4. 
CBC test includes: hemoglobin, hematocrit, platelets, red blood cell count, MCV, MCH, white blood cell count + differentiation. Additional at screening: HbA1C. 
5. 
Biochemistry test includes: creatinine, urea, sodium, potassium, bilirubin, yGT, AST, ALT and LDH. Additional at screening: cholesterol, triglyceride, and glucose. 
6. 
Blood will be collected for PBMC’s and serum banking. Saliva will be collected in parallel.  
7. 
Safety report: A safety report for Groups 1-3 is prepared by the clinical investigator once the final subject in Group 3 has completed 14 days of follow-up. A safety report 
of all the safety data is made after the close-out visit. 
8. 
Sampled blood volume will be tracked over the whole study period to ensure that ≤500 mL (equivalent to one whole blood donation) will be sampled in total and all safety 
analyses can be done throughout.  

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8.4 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. Subjects can also be withdrawn from the study procedures at the discretion of 
the clinical investigator or the local safety monitor for urgent medical reasons or if exclusion 
criteria are met. The following reasons may lead to withdrawal of individual subjects: 
- 
Withdrawal of informed consent by volunteer; 
- 
Any SAE; 
- 
Discovery of a condition or medication that according to clinical judgment of the 
investigator is considered as a definite contraindication to proceeding with the study 
procedures; 
- 
Immunosuppressant or other immune-modifying drugs administered chronically 
(i.e., more than 14 days in total) during the study period. Inhaled and topical 
corticosteroids and oral anti-histamines exempted; 
- 
Immunoglobulin and/or any blood products administered during the study period; 
- 
Pregnancy; 
- 
Complete lost to follow-up; 
- 
Ineligibility (arising during the study or retrospectively, having been overlooked at 
screening); 
- 
If the investigator or safety monitor believes that continuation would be 
detrimental to the subject’s well-being; 
- 
Volunteer non-compliance with study requirements; 
- 
Any other protocol deviation that results in a significant risk to the subject’s safety. 
8.4.1 
Handling of withdrawals 
For subjects lost to follow-up (i.e. those subjects whose status is unclear because they fail to 
appear for study visits without stating an intention to withdraw), extensive effort (i.e. 
documented phone calls and e-mails) will be undertaken to locate or recall the volunteer 
or at least to determine his or her health status. The investigator should show “due diligence” 
by documenting in the source documents steps taken to contact the subject. In case of 
premature withdrawal for any reason, the investigator will exert his/her best effort to: 
• 
Update any ongoing AE/SAEs that remained ongoing at the time of the subject’s last 
visit prior to withdrawal. 
• 
Conduct an interview to determine if the subject has had any reaction or AE (serious 
or non- serious) since the last visit. Where possible, the investigator should visibly or 
physically assess any reported adverse reaction or AE and document whether it led to 
the withdrawal. 

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• 
Conduct a physical examination. 
• 
Collect blood for biochemical and hematological clinical laboratory parameters. 
• 
Document the reason for premature withdrawal on the CRF. 
• 
Update the subject's contact information. 
8.4.2 Withdrawals at specific time-points 
During screening 
If the investigator ascertains that the subject does not meet the inclusion/exclusion criteria 
after the subject signs the informed consent form, the investigator only needs to note the 
reason for subject exclusion on the screening log. 
As a result of erroneous inclusion 
If a subject who does not meet the inclusion/exclusion criteria is inadvertently included in the 
trial, the investigator will terminate the subject’s participation in the trial, the sponsor will be 
informed immediately prior to administration of study product and a protocol deviation will be 
completed. In the event that the study product has already been given, the subject will be 
asked to complete all remaining safety related follow-up visits and procedures through the 
last study visit. However, the subject’s data will be excluded from the per-protocol analysis. 
As a result of withdrawal of informed consent 
If a subject withdrawal occurs for any reason, the investigator must make every effort to 
determine the primary reason for a subject’s withdrawal from the study and record this 
information in the study file. However, in accordance with the principles of the current version 
of the Declaration of Helsinki, it is acknowledged that a subject does have the right to withdraw 
from the study at any time and for any reason and is not obliged to give her or his reasons for 
doing so. With permission of the subject, all data generated before withdrawal will be 
included in final study analysis, unless it is compromised or the subject objects. Blood 
samples collected before withdrawal will be used/stored unless the subject specifically requests 
otherwise. 
As a result of occurrence of an adverse event/serious adverse event 
If any subject develops an AE/SAE leading to premature withdrawal, the event will be fully 
followed-up. For premature withdrawal in connection with the emergence of an AE/SAE that 
is considered to be clinically relatively favourable (for example, the diagnosis is known, and 
it is expected to be resolved completely within a week), intermittent follow-up may be 
accepted as long as the plan for follow-up of the event is fully described in the notes section 
of the subject’s CRF. Follow-up visits will be carried out for all subjects who are withdrawn 
due to occurrence of an AE or in connection with a change in any other safety indicator (vital 
sign or clinical laboratory result). All data generated before withdrawal will be included in the 

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final study analysis, unless it is compromised or the subject objects. Blood samples collected 
before withdrawal will be used/stored unless the subject specifically requests otherwise. 
As a result of loss to follow-up 
To prevent loss to follow-up, subjects will be reminded by phone, email, or text message of 
their next study visit. In the event of a missed visit, subject will be contacted by phone within 
1 day. A subject who misses two consecutive visits and cannot be reached or located after 5 
attempts will be considered lost to follow-up. Efforts to contact the subject will be documented 
in source documents. Any subject who fails to attend the final study visit will also be classified 
as lost to follow-up. There will be no replacement for subjects who are lost to follow-up. All 
data generated before loss to follow-up will be included in final study analysis with subject’s 
permission, unless it is felt that the data is compromised. Blood samples collected before loss 
to follow-up will be used/stored unless the subject specifically requests otherwise. 
8.5 Replacement of individual subjects after withdrawal 
If an assigned subject does not present on the day of ABNCoV2 study product administration 
or elects to withdraw the consent on the day of study product administration, one of the reserve 
subjects will replace the withdrawn subject. Subjects who have received the first dose of  
ABNCoV2 cannot be replaced. 
8.6  Follow-up of subjects withdrawn from treatment 
A subject may end his or her participation in the study and still be followed up for safety, 
unless the subject chooses to have complete withdrawal of the consent for further 
participation in any trial procedures. If a subject chooses to withdraw from the study, the 
investigator will make a reasonable effort to determine the reason for the subject’s withdrawal 
and complete the study termination eCRF. 
8.7 Premature termination of the study 
The study may be discontinued by the sponsor: 
- 
On advice of the local safety monitor 
- 
On advice of the Safety Monitoring Committee (SMC) 
- 
On advice of the clinical investigator 
- 
On advice of the METC (CCMO) 
 
The investigators, local safety monitor, SMC, METC or sponsor may decide to put the study 
on hold based on AEs, pending discussion with the sponsor, SMC, METC, local safety 
monitor or investigators. Following discussion, it may be decided to terminate the study. 
 

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9. SAFETY REPORTING 
9.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the study if 
there is sufficient ground that continuation of the study will jeopardise subject health or safety.  
The sponsor will notify the accredited METC without undue delay of a temporary halt including 
the reason for such an action. The study will be suspended pending a further positive decision 
by the accredited METC. The investigator will take care that all subjects are kept informed.  
9.2 AEs, SAEs and SUSARs 
9.2.1 Adverse events (AEs) 
AE are defined as any undesirable experience occurring to a subject during the study, whether 
or not considered related to the investigational product or trial procedure. All AE reported 
spontaneously by the subject or asked about or observed by the investigator or his staff will be 
recorded. 
9.2.2 Serious adverse events (SAEs) 
A SAE is any untoward medical occurrence or effect that  
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; or 
- 
any other important medical event that did not result in any of the outcomes listed 
above due to medical or surgical intervention but could have, based upon appropriate 
judgement by the investigator. 
An elective hospital admission will not be considered as a SAE. 
The investigator will report all SAEs to the sponsor without undue delay after obtaining 
knowledge of the events. The sponsor will report the SAEs through the web portal 
ToetsingOnline to the accredited METC that approved the protocol, within 7 days of first 
knowledge for SAEs that result in death or are life threatening followed by a period of maximum 
of 8 days to complete the initial preliminary report. All other SAEs will be reported within a 
period of maximum 15 days after the sponsor has first knowledge of the SAE. 

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9.2.3 Suspected unexpected serious adverse reactions (SUSARs) 
Adverse reactions are all untoward and unintended responses to an investigational product 
related to any dose administered. 
Unexpected adverse reactions are SUSARs if the following three conditions are met: 
1. the event must be serious (see chapter 9.2.2); 
2. there must be a certain degree of probability that the event is a harmful and an 
undesirable reaction to the medicinal product under investigation, regardless of the 
administered dose; 
3. the adverse reaction must be unexpected, that is to say, the nature and severity of the 
adverse reaction are not in agreement with the product information as recorded in: 
a. Summary of Product Characteristics (SPC) for an authorised medicinal product; 
b. Investigator’s Brochure for an unauthorised medicinal product. 
The sponsor will report expedited the following SUSARs through the web portal ToetsingOnline  
to the METC: 
- 
SUSARs that have arisen in the clinical trial that was assessed by the METC; 
- 
SUSARs that have arisen in other clinical trials of the same sponsor and with the same 
medicinal product, and that could have consequences for the safety of the subjects 
involved in the clinical trial that was assessed by the METC 
The remaining SUSARs are recorded in an overview list (line-listing) that will be submitted 
once every half year to the METC. This line-listing provides an overview of all SUSARs from 
the study medicine, accompanied by a brief report highlighting the main points of concern.  
The expedited reporting of SUSARs through the web portal Eudravigilance or ToetsingOnline 
is sufficient as notification to the competent authority. The sponsor will report expedited all 
SUSARs to the competent authorities in other Member States, according to the requirements 
of the Member States.  
The expedited reporting will occur not later than 15 days after the sponsor has first knowledge 
of the adverse reactions. For fatal or life threatening cases the term will be maximal 7 days for 
a preliminary report with another 8 days for completion of the report.  
9.3 Annual safety report 
In addition to the expedited reporting of SUSARs, the sponsor will submit, once a year 
throughout the clinical trial, a safety report to the accredited METC, competent authority, and 
competent authorities of the concerned Member States. 
This safety report consists of: 

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− a list of all suspected (unexpected or expected) serious adverse reactions, along with 
an aggregated summary table of all reported serious adverse reactions, ordered by 
organ system, per study; 
− a report concerning the safety of the subjects, consisting of a complete safety analysis 
and an evaluation of the balance between the efficacy and the harmfulness of the 
medicine under investigation. 
9.4 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been reached. 
Depending on the event, follow up may require additional tests or medical procedures as 
indicated, and/or referral to the general physician or a medical specialist. SAEs need to be 
reported till end of study within the Netherlands, as defined in the protocol.  
9.4.1 
Adverse event data collection 
Signs and symptoms will be recorded in study diaries and reviewed during admission at the 
clinical trial research center, at all follow-up visits, and whenever a trial volunteer reports signs 
or symptoms to the trial physician between visits. 
Solicited adverse events 
After the administration of ABNCoV2, the following local and systemic AE will be solicited until 
7 days after administration: 
- 
Local AE: pain, tenderness, erythema and induration at injection site.  
- 
Systemic AE: headache, fatigue, fever, drowsiness, chills. 
The time points that these symptoms are solicited is indicated in section 8.3.19. 
Unsolicited adverse events 
Unsolicited AE occurring through 196 days following the first ABNCoV2 administration will be 
collected during the entire study period. 
9.4.2 
Recording of adverse event data collection 
The trial physician will train subjects on the use of memory aid booklets for self-assessment of 
solicited local and systemic AE through 7 days post- administration of ABNCoV2, and for self-
assessment of unsolicited AE and concomitant medications during the entire study period. The 
trial physician will review and discuss with the subject all recorded events to ensure appropriate 
documentation and scoring prior to transferring of the diary card record to the eCRF. 
If known, the trial clinician will record the diagnosis (i.e., disease or syndrome) rather than 
component signs, symptoms and laboratory values. If the signs and symptoms are considered 

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unrelated to an encountered syndrome or disease they should be recorded as individual AEs. 
If a primary AE is recorded, events occurring secondary to the primary event should be 
described in the narrative description of the case (e.g. primary AE = Orthostatic hypotension; 
secondary event may be fainting, head trauma, etc.). In case of hospitalizations for surgical or 
diagnostic procedures, the pre-existing condition should be recorded as the SAE, not the 
procedure itself. 
All adverse events/reactions (solicited and unsolicited), noted by the investigators will be 
accurately documented in the case report form by the investigators. For each event/reaction 
the following details will be recorded: 
1. Description of the event(s)/reactions(s) 
2. Date and time of occurrence 
3. Duration 
4. Intensity 
5. Relationship with the intervention 
6. Action taken, including treatment 
7. Outcome 
In addition, symptoms will be ranked as (1) mild, (2) moderate, or (3) severe, depending on 
their intensity according to the following scale: 
Mild (grade 1): 
awareness of symptoms that are easily tolerated and do not 
interfere with usual daily activity. 
Moderate (grade 2): 
discomfort that interferes with or limits usual daily activity. 
Severe (grade 3): 
disabling, with subsequent inability to perform usual daily 
activity, resulting in absence or required bed rest. 
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. 
If an AE changes in intensity during the specified reporting period, a new description of the AE 
will be added. Interrupted AEs are registered as one AE if the interruption is <24 hours. When 
an AE/SAE occurs, it is the responsibility of the investigators to review all documentation (e.g. 
hospital progress notes, laboratory, and diagnostics reports) related to the event. The 
investigators will then record all relevant information regarding an AE/SAE on the CRF or SAE 
Report Form. 
 
 

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9.4.3 
Assessment of causality 
The investigators are obliged to assess the relationship between study procedures and the 
occurrence of each AE/SAE. The investigators will use clinical judgment to determine the 
relationship. Alternative causes, such as natural history or the underlying diseases, 
concomitant therapy, other risk factors and the temporal relationship of the event will be 
considered and investigated. The relationship of the AE with the study procedures will be 
categorized as: 
Definitely: 
administration of the IMP is the cause, another etiology causing the 
AE is not known. 
Probable: 
administration of the IMP is the most likely cause: however, there are 
alternative reasonable explanations, even though less likely. 
Possible: 
there is a potential association between the event and administration 
of the IMP, however, there is an alternative etiology that is more 
likely. 
Unlikely: 
a relationship to the administration of IMP is unlikely, however, it 
cannot be ruled out. 
Not related: 
a relationship to the administration of the IMP cannot be reasonably 
established; another etiology is known to have caused the AE or is 
highly likely to have caused it. 
When a regulatory authority requests a binary classification (related vs. unrelated), definitely, 
probably and possibly related are considered to be “related”, while not related and unlikely 
related are considered to be “unrelated”. Thus, an intervention-related AE refers to an AE for 
which there is a possible, probable or definite relationship to the study intervention. The 
investigator will use clinical judgment to determine the relationship. 
9.5 Safety Monitoring Committee 
An independent SMC composed of three independent individuals will be appointed. The SMC 
will include a local safety monitor, and two experts nominated by the PI. The SMC will be 
established for the purpose of monitoring the study, to discuss and approve the investigator 
and sponsor’s decision on further dose escalation and adjuvant use in groups 4 and above, as 
well as to provide independent, non-binding advice on safety and ethics. The responsibilities 
and procedures of the SMC members are defined in the SMC Charter. 
The advice(s) of the SMC will only be sent to the sponsor of the study. Should the sponsor 
decide not to fully implement the advice of the SMC, the sponsor will send the advice to the 

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reviewing METC, including a note to substantiate why (part of) the advice of the SMC will not 
be followed. 
9.5.1 
Local safety monitor 
For this study, a local safety monitor will be appointed, who will be involved in the review of 
severe and serious AE and volunteer safety. She/he is independent of the sponsor and the 
investigator. The local safety monitor is notified of all SAEs, as well as at least all grade 3 AE 
probably or definitely related to ABNCoV2 administration and persisting at grade 3 for >48 
hours. 
9.5.2 
Safety Meetings 
The SMC will review safety data upon completion of ≥14 days follow-up after the first 
immunisation in all subjects up to and including group 3. The chair of the SMC will determine 
whether a (digital) meeting will be held, or whether a recommendation from all members may 
be formalized through e-mail. In addition, safety data for all participants will be assessed by 
the SMC at the end of the study. An ad hoc SMC meeting may be convened at any time or at 
the request of the PI or local safety monitor to review safety data from subjects and/or groups 
who meet any of the holding rules as specified in the protocol and SMC charter, or if otherwise 
deemed necessary. 
9.5.3 
Safety Reports 
A safety report will be prepared by the clinical investigator for review by the committee prior to 
each scheduled data review. These reports will provide at a minimum the following information: 
- 
Accrual data and subject status data with regard to completion of/discontinuation from 
the study. 
- 
Summaries of solicited AEs, classified by severity. 
- 
Unsolicited AEs (including SAEs), categorized by MedDRA coding, severity and 
relatedness to study vaccine. 
- 
Safety laboratory test results outside of normal institution reference ranges and 
considered clinically significant, classified by severity grading scale (irrespective of 
whether assessed as AEs). 
- 
Any new or updated AEs that have met the holding rules. 
The SMC will review the safety data within 2 working days. The SMC will summarize their 
recommendations to the study sponsor as to whether they approve of the investigator and 
sponsor’s decision on further dose escalation and adjuvant use in groups 4 and above, whether 
there are safety concerns and whether the study should continue without change, be modified, 

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or be terminated. If at any time a decision is made to permanently discontinue administration 
of ABNCoV2 administrations in all subjects, the sponsor will notify the CCMO expeditiously. 
9.5.4 
Dose escalation and adjuvant use in group 4 and above 
Further dose escalation and choice of adjuvant use in group 4 and above require approval by 
the SMC following review of all available safety, tolerability and immunogenicity data up to 14 
days after first immunisation in all subjects up to and including group 3. The decision will be 
taken by the sponsor and the investigators and approved by a safety monitoring board based 
on all immunogenicity, safety and reactogenicity data that have been accrued until this time. 
The data to be reviewed includes all of the following available safety data: 
- 
Solicited AEs through day 7 post first ABNCoV2 administration for all subjects in 
groups 1-3 
- 
Unsolicited AEs through day 14 post first ABNCoV2 administration for all subjects 
in groups 1-3 
- 
Clinically significant laboratory tests collected at 1 and 7 days following first 
ABNCoV2 administration for all subjects in groups 1-3 
- 
Solicited and unsolicited AEs through day 7 post second ABNCoV2 administration 
for subjects in group 1A and sentinel subjects in group 1B and 2A. 
The ABNCoV2 dose will be escalated until: 
- 
The maximal dose of 70 μg is reached, or  
- 
The vaccine is too reactogenic and/or not tolerated, or  
- 
Immunogenicity-dose response curve is saturated (no further increase or decrease in 
antibody concentration with increasing dose)  
 
The pre-defined criteria to decide on the optimal dose and formulation are: 
- 
Safety and tolerability concerns during initial dose escalation of groups 1-3. 
- 
Occurrence of related grade 3, serious and severe adverse reactions 
- 
Dose response and adjuvant-dependency of anti-SARS-CoV-2 antibody titre 
 
The SMC will review the AEs (local, systemic, and laboratory) and subsequently make their 
recommendation(s). 
9.5.5 
Holding rules for dose escalation to groups 4-7: 
Decision on proceeding to group 4 will depend on the positive review by the SMC of the 
safety data containing solicited AEs through day 7 and unsolicited AEs through day 14 post 
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days following first ABNCoV2 administration for all subjects in groups 1-3. If either of the 
subjects in group 1-3 meet a holding rule (see below), administration of ABNCoV2 will be 
held for all remaining subjects in subsequent dose groups. The following holding rules apply 
to subjects in groups 1-3: 
• 
One or more participants experience a SAE that is determined to be at least 
possibly related to the administration of ABNCoV2 
• 
Two or more subjects experience a grade 3 adverse event (local, systemic or 
laboratory) possibly, probably or definitely related to ABNCoV2 administration and 
persisting at grade 3 for >48 hours 
 
The study site member first aware of the event meeting the holding rule will notify the 
Principal Investigator and the Local Safety Monitor. The PI will alert the appropriate parties. 
The SMC will be notified within 24 hours. An ad hoc SMC review will be performed. The 
following considerations must be discussed: 
• 
Relationship of the AE or SAE to the study product 
• 
Relationship of the AE or SAE to ABNCoV2 dose 
• 
If appropriate, additional screening or laboratory testing is provided to other 
subjects to identify subjects who may develop similar symptoms 
• 
If any study related SAE is not listed on the current informed consent form 
(ICF), the PI will revise the ICF and subjects will be asked to provide consent on 
the new ICF 
ABNCoV2 administration to subjects within the affected group and to the next higher dosage 
group may resume only if the local safety monitor, PI, SMC and the sponsor agree it is 
safe to resume ABNCoV2 administration. If the accredited METC has recommended safety 
hold, re-initiation of the study will require METC concurrence. The accredited METC will be 
informed of a safety hold by the sponsor. Following discussion, it may be decided to terminate 
the study. 
All subjects who have received the study product will be followed for safety until 
resolution or stabilization (if determined to be chronic sequelae) of their AE(s). If at any time 
a decision is made to discontinue administration of study product in all subjects, expeditious 
notification will be provided by the Sponsor to the METC within 48 hours. The PI, local 
safety monitor, SMC or METC may stop or suspend the use of this product at any time. 

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9.5.6 
Decision on further development of ABNCoV2 
ABNCoV2 development will not be continued in case that no dosage and regimen shows 
acceptable safety and tolerability. Immunogenicity following first immunization (D14) of the 
optimized regimen shall be at least within the range of convalescent sera.  
10. STATISTICAL ANALYSIS 
COUGH-1 is an exploratory Phase 1 study without confirmatory aim. It is designed to inform 
dose, formulation and schedule for a larger Phase 2 clinical trial that will extent into higher age-
groups – the most important risk group. Safety and tolerability data will be listed, 
immunogenicity data will be presented graphically and as aggregated data. A statistical 
analysis plan will be developed and finalized before first vaccination of a study participant.  
Demographic and safety data will be presented as descriptive data without hypothesis testing. 
Immunogenicity data will be modelled using a Bayesian approach. This allows repeated 
estimation of the parameters following completion of each group and booster vaccination. 
Since the number of volunteers is small and the follow up is not complex, we do not expect 
much missing data. Nevertheless, we will perform sensitivity analyses to assess the effect of 
handling missing values, values below the lower limit of detection, different priors, models and 
distributional assumptions.  
All analyses will be coded using R and follow guidelines for reproducible research.  
10.1 Primary study endpoints 
# Number of at least possibly related Grade 3 AE and SAE from time of first administration of 
ABNCoV2 to the end of the follow-up period. 
The total number of at least possibly related Grade 3 and SAE will be tabulated together with 
their severity grade (3 or serious) and relation to the vaccine (possible, probable or definitely 
related). Separate tables will be given for single groups, dosage and as number of volunteers 
experiencing AE.  
All SAE will be described in from of short clinical summaries.  
# Concentration of ABNCoV2-specific antibodies 14 days following first vaccination. 
ABNCoV2-specific antibodies will be measured by enzyme-linked immunosorbent assay 
(ELISA). Data will be transformed logarithmically to the base of 10 before aggregation or 
modelling. Data from pre-clinical assessment as well as published data show that log-
transformation leads to a distribution for which the normal assumption can be assumed. Data 
below the lower limit of detection (i.e. most baseline data) will be imputed using half the mean 
of the lower limit of detection and the standard deviation.  

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10.2 Secondary study endpoint 
# Number and severity of at least possibly related solicited AEs within one week following 
administration of ABNCoV2 (day 0 to 7). 
Here, the full AE tables will be given using the same tabulation method as for the primary 
endpoint. Severity will be reported on a 4-level ordinal scale: Grade 1, 2, 3, serious, causal 
relationship on a 5-level ordinal scale: not, unlikely, possible, probable, definitely related.  
Since full AE tables are typically not easy to interpret (due to their size) and are a poor 
representation of AE patterns (e.g. interactions and co-occurrences) AE-networks will be 
displayed using igraph.  
10.3 Other study endpoints 
Other study endpoints are considered exploratory and hypothesis generating.  
# Concentration of ABNCoV2-specific antibodies at baseline and during immunization and 
follow up. 
The kinetics of ABNCoV2-specific will be described using area under the curve, peak 
concentration and decay kinetics following first and second vaccination.  
# Inhibitory titre in invasion inhibition assay at baseline and during immunization and follow up. 
The kinetics of functional activity of sera (by invasion inhibition assay) will be described using 
area under the curve, peak concentration and decay kinetics following first and second 
vaccination.  
# Cellular immune responses (T and B cell) at baseline and during immunization and follow 
up. 
Cellular responses will be analysed by cytometry and enzyme-linked absorbent spot (ELISpot) 
assay.  
10.4 Interim analysis (if applicable) 
No interim analysis as in a confirmatory study setting is performed since a Bayesian approach 
to statistical calculation is used. Following first vaccination of Group 3B the database will be 
cleaned, AE data codes (using the medical dictionary for regulatory activities: MedDRA) and 
the immunogenicity data will be completed and validated. An interim report will be generated 
to guide the decision of investigator and sponsor to use adjuvanted or non-adjuvanted 
ABNCoV2 and to further dose-escalate. The decision process will be documented and 
presented, together with the data, to the SMC for approval.  
The trial will be put on hold when a SAE at least possibly related to the vaccine occurs.   

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11.  ETHICAL CONSIDERATIONS 
11.1 Regulation statement 
This study will be conducted in accordance with the latest Fortaleza revision of the 
Declaration of Helsinki (2013), the Medical research Involving Human Subjects act (WMO), 
the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for 
Human Use (ICH) Good Clinical Practice (GCP), and local regulatory requirements. The 
investigators are responsible for obtaining all relevant ethical approvals of the protocol and any 
subsequent amendments in compliance with local law before the start of the study. 
11.2 Recruitment and consent 
As soon as the study is approved by the accredited METC and competent authority, healthy 
subjects will be recruited to participate in the study. Advertisements will be placed in 
prominent places on university campuses and other public places as well as on the intranet 
of the University and social media. The advertisement will indicate a telephone number to 
call and an e-mail address for contact to request further information. When seemingly 
suitable subjects contact investigators via e-mail, telephone or the online form, they will be 
invited to a (digital) information meeting during which the study will be explained to them by 
the study physician. Directly after the meeting they will be provided with documents to review 
at home (the test subject information, the informed consent form, the application form and 
the insurance text). During and after the meeting there will be time for questions. After this 
free discussion with the study physician, and any follow-up discussion if necessary, the 
volunteer will be given sufficient time to consider participation. Subjects who are interested 
in participating will be asked to fill in the application form and will be invited to come for a 
screening visit. Eligible subjects may only be included in the study after providing written, 
METC-approved informed consent. Informed consent must be obtained before conducting 
any study-specific procedures (i.e. all of the procedures described in the protocol, including 
screening procedures). The process of obtaining informed consent should be documented in 
the subject source documents. During the screening visit, the questionnaire answers will be 
discussed and inclusion and exclusion criteria will be checked. Again, the study physician 
will answer any questions the volunteer has. The possibility of withdrawal from the study, at 
any time, without penalty and without any declaration of the reason will be pointed out to the 
subjects. The investigators will be responsible for providing adequate verbal and written 
information regarding the objectives and procedures of the study, the potential risks involved 
and the obligations of the subjects. Subjects will be informed that they will not gain health 
benefits from this study. Trainees or other students who might be dependent on the 
investigators or the study group will not be included in the study. 

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11.3 Benefits and risks assessment, group relatedness 
The compelling need for COVID-19 vaccines needs to be balanced with the potential risks 
and discomforts for the subjects. Risks for subjects are related to administration with 
ABNCoV2. There are no direct benefits to participation in the trial for subjects. Subjects will 
be advised to follow all the current national guidelines to minimize the risk of SARS-CoV2 
transmission. All partners in this proposal are aware of and follow the relevant national and 
international rules and regulations as they pertain to access to material of human origin and 
clinical research. International agreements such as the Declaration of Helsinki will be observed 
and respected 
11.4 Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of the 
WMO. The sponsor (also) has an insurance which is in accordance with the legal requirements 
in the Netherlands (Article 7 WMO). The insurance offers a maximum coverage of: 
1. € 650.000,-- (i.e. six hundred and fifty thousand euro) for death or injury for each 
subject who participates in the research; 
2. € 5.000.000,-- (i.e. five million euro) for death or injury for all subjects who participate 
in the research; 
3. € 7.500.000,-- (i.e. seven million five hundred thousand euro) for the total damage 
incurred by the organisation for all damage disclosed by scientific research for the 
sponsor as ‘verrichter’ in the meaning of said Act in each year of insurance coverage. 
This insurance provides cover for damage to research subjects through injury or death 
caused by the study. 
11.5 Compensation 
Enrolled subjects will receive up 1000,- euros in compensation for their time and for the 
inconveniences of taking part in this study. These amounts are based on predefined criteria: 
• 
Screening and inclusion: 100,- euros 
• 
Inconvenience of blood tests and/or visits: 25,- euros per blood sampling 
• 
2 x 1 day admission to hospital: 125,- euros per admission day 
• 
ABNCoV2 administration: 300,- euros 
• 
Compensation length of study: 20,- euros per month 
Travel expenses will be additionally reimbursed by €0,19 per km, provided that the participant 
is travelling to the Radboudumc by car or public transport and lives more than 5 km from the 
Radboudumc. Compensation will not be provided to subjects who are not enrolled i.e. screen 
failures. Eligible subjects who are enrolled at the inclusion visit as back-ups, will be 
compensated 50,- euros for each inclusion visit. If a subject withdraws from the study prior to 

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completion, they will receive reimbursement proportional to number of visits they attended. 
These compensation amounts are reasonable and in line with Dutch common practice. In 
case of unexpected medical complications, there will be access to state-of-the-art medical 
treatment with full costs covered by the insurance of Radboudumc. 
12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1 Handling and storage of data and documents 
A data management plan will be developed prior to start of study describing data 
management activities from project set-up through data lock and transfer. Designated trial 
staff will enter the data required by the protocol into the eCRF. All data should be recorded, 
handled and stored in a way that allows its accurate reporting, interpretation and verification. 
An external monitor will review the data entered into the eCRFs by investigational staff for 
completeness and accuracy and will instruct the site personnel to make any required 
corrections or additions. Queries are made during each monitoring visit. Designated 
investigator site staff are required to respond to the queries and confirm or correct the 
data. Medical history/current medical conditions be coded using the ICD-10 terminology. AE 
will be coded using MedDRA.  
12.1.1 Source data 
Prior to the start of the trial it will be determined which documents completed by the 
investigative team will be considered source documents. Only authorized study staff and 
representatives of the sponsor and authorized regulatory agencies may have direct access 
to source documents containing study data. Data for this study will include biographical, 
medical history, clinical (signs, symptoms, prescription and non-prescription medical 
treatments), safety and laboratory data. All information in original records and certified 
copies of original records, clinical findings, or observations will be considered source data 
for this study. The memory aids, produced by the study subjects are also considered source 
data. They will be kept as source documents in the investigator’s clinical file. Since all subjects 
will be healthy, there is no medical file for the study subjects, with exception of the medical file 
in case of adverse events/reactions resulting in a medical consultation or hospitalization. In 
this case the medical file will also be considered as the source data. 
All data collected by the investigators is reported in the eCRF. All information in eCRFs 
must be traceable to the source documents in the subject's file. As with all parts of the eCRF, 
there is an audit trail in place to register every data entry. The investigator will also keep the 
original informed consent form signed by the subject (a signed copy is given to the 
subject). The investigator’s team will maintain the information collected from CRFs and in 

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the eCRFs and all source documents that support the data collected from each subject 
in a secure area and treated as confidential material. Documents and data pertaining to 
the study will be kept in a locked cabinet under the responsibility of the investigator. Periodic 
monitoring visits will ensure that the data is safe and stored in a secure place and that only 
those authorized study staff have access to the data. 
Source data from this study will be stored for 25 years, in line with the Nederlandse Federatie 
van Universitair Medische Centra (NFU) guideline. Biological samples from this study will be 
stored for maximum 15 years for research related to SARS-CoV2 immunology. New 
immunological or molecular tests may become available in the future that could strengthen or 
validate this research or help find important new findings. Should material be used for research 
not related to SARS-CoV2, permission from the METC must be granted. 
12.1.2 Confidentiality 
All parties agree to adhere to the principles of medical confidentiality in relation to clinical 
study subjects involved in this trial and shall not disclose the identity of subjects to third parties 
without prior written consent of the subject. 
All data will be pseudonymised; volunteer data will be identified by a unique study number 
in the eCRF. Separate confidential files containing identifiable information will be stored in 
secured locations. All plasma samples, or other biological samples, with exception of those 
taken for safety diagnostics, will be labelled with the volunteer study identification number. 
Samples taken for safety diagnostic (processed by the central clinical laboratory) will be 
labelled with part of the subject identification code, study identification name and a fictitious 
birth date (only using the subjects actual birth year). The samples will not be labelled with 
volunteer names or actual birth dates. The subject identification code will be kept by the 
investigator. 
The investigator will maintain and retain appropriate medical and research records and 
essential documents for this trial in compliance with ICH GCP Guidelines, any regulatory 
requirements, and institutional requirements for the length of storage and for the protection of 
confidentiality of subjects. The investigator will permit direct access to study records and 
source documents to authorized representatives of the sponsor, responsible ethics 
committee(s), regulatory agencies, and the external monitor(s), for the purposes of quality 
assurance reviews, audits / inspections, and evaluation of the study safety and progress. 
Direct access includes examination, analysis, verification, and reproduction of de-identified 
records and reports that are important to the evaluation of the trial. 

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12.2 Monitoring and Quality Assurance  
General considerations 
Before study initiation, the protocol and eCRFs together with relevant SOPs will be reviewed 
by the sponsor, the investigators and their staff. During and after completion of the study, 
the data monitor will visit the site to check the completeness of records, the accuracy of 
entries on the eCRFs, the adherence to the protocol and to Good Clinical Practice and the 
progress of enrolment. 
The investigator will maintain source documents for each subject in the study, consisting of 
case and visit notes containing demographic and medical information, laboratory data, 
subject’s diaries, and the results of any other tests or assessments. All information on eCRFs 
must be traceable to these source documents in the subject's file. As with all parts of the 
eCRF, there is an audit trail in place to register every data entry. The investigator will also 
keep the original informed consent form signed by the subject (a signed copy is given to the 
subject). 
The investigator will give the external monitor access to all relevant source documents to 
confirm their consistency with the eCRF entries. According to the NFU risk classification 
system, this clinical trial has been classified as high risk. The monitor will perform full 
verification for the presence of informed consent, adherence to the inclusion/exclusion criteria 
and documentation of SAEs. The recording of data that will be used for all primary and safety 
endpoints will be assessed for 100% of included subjects. 
External monitoring 
To ensure that the study is conducted in accordance with ICH GCP and regulatory 
requirements, 
monitoring responsibilities will be provided by a Contract Research 
Organization or equivalent (CRO). A site initiation visit will be conducted prior to beginning 
of the study, and monitoring will be conducted at initiation, during, and at closeout of the study. 
During the course of the study, monitors will verify compliance to the protocol; completeness, 
accuracy, and consistency of the data and study product accountability; and adherence to 
ICH GCP and applicable regulations. As needed and when appropriate, the monitors will also 
provide clarifications and additional training to help the site resolve issues identified during 
the monitoring visit. As appropriate and informed by risk assessment, remote centralized 
monitoring activities may be considered in place of or to supplement onsite monitoring. 
These may include analysis of data quality (e.g., missing or inconsistent data), identification 
of data trends not easily detected by onsite monitoring, and performance metrics (e.g. 
screening or withdrawal rates, eligibility 
violations, timeliness, and accuracy of data 
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The extent and frequencies of the monitoring visits will be described in a separate monitoring 
plan developed prior to study initiation. The investigator will be notified in advance of 
scheduled monitoring visits. The monitors should have access to all trial related sites, subject 
medical records, study product accountability, and other study-related records needed to 
conduct monitoring activities. The CRO will share the findings of the monitoring visit, including 
any corrective actions, with the site investigator. The site PI and the monitors must 
cooperate to ensure that any problems detected in the course of these monitoring visits are 
resolved in a predefined timeframe. All queries must be resolved prior to database lock. 
Essential documents must be filed in the site study file on an ongoing basis and be available 
for review by the CRO. 
Independent Auditing 
Sponsor representatives may audit the study to ensure that study procedures and data 
collected comply with the protocol and applicable SOPs at the clinical site and that data 
are correct and complete. The site PIs will permit auditors to verify source data validation 
of the regularly monitored clinical study. The auditors will compare the entries in the eCRFs 
with the source data and evaluate the study site for its adherence to the clinical study protocol 
and ICH GCP guidelines and applicable regulatory requirements. 
Regulatory agency auditing (inspection) 
The site PIs must be aware that regulatory authorities may wish to inspect the site records to 
verify the validity and integrity of the study data and protection of human research subjects. 
The site PIs must make the relevant records available for inspection and will be available to 
respond to reasonable requests and audit queries made by authorized representatives of 
regulatory agencies. 
12.3 Amendments  
A ‘substantial amendment’ is defined as an amendment to the terms of the METC 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. 
All substantial amendments will be notified to the METC and to the competent authority. Non-
substantial amendments (e.g. typing errors and administrative changes like changes in names, 
telephone numbers and other contact details of involved persons mentioned in the submitted 

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study documentation) will not be notified to the accredited METC and the competent authority, 
but will be recorded and filed by the sponsor. 
12.4 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the accredited 
METC once a year. Information will be provided on the date of inclusion of the first subject, 
numbers of subjects included and numbers of subjects that have completed the trial, serious 
adverse events/ serious adverse reactions, other problems, and amendments.  
12.5 Temporary halt and (prematurely) end of study report 
The sponsor will notify the accredited METC and the competent authority of the end of the 
study within a period of 90 days. The end of the study is defined as the last patient’s last visit.  
The sponsor will notify the METC immediately of a temporary halt of the study, including the 
reason of such an action. In case the study is ended prematurely, the sponsor will notify the 
accredited METC and the competent authority within 15 days, including the reasons for the 
premature termination. Within one year after the end of the study, the investigator/sponsor will 
submit a final study report with the results of the study, including any publications/abstracts of 
the study, to the accredited METC and the Competent Authority. 
12.6 Public disclosure and publication policy 
The study will be entered by the investigator into a primary registry of the International Clinical 
Trials Registry Platform (ICTRP) network of the WHO (e.g. clinicaltrials.gov). The final report 
will be prepared by the investigators at the Radboud university medical center. It will be 
signed by the project leader or the principal investigator. The investigators will publish the 
results of the study, preferably in a peer-reviewed journal. 
 
13. STRUCTURED RISK ANALYSIS  
13.1 Potential issues of concern 
a. Level of knowledge about mechanism of action 
The ABNCoV2 vaccine is a sub-unit vaccine using a non-infectious virus-like particle (VLP) 
platform. A stabilized version of the receptor binding domain (RBD) of SARS-CoV-2 Spike 
glycoprotein (S) is bound to an VLP, and through vaccination will be presented to the immune 
system in order to evoke a response that should result in protection from infection. 
Virus like particles (VLP) that display antigen on their surface are a technology to increase 
immunogenicity of soluble protein subunit vaccines. Their size (20-200nm) allows direct 

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drainage into lymph nodes and it is optimal for uptake by antigen-presenting cells and cross 
presentation. Their highly repetitive surface structures also facilitate complement fixation and 
B cell receptor clustering, leading to the activation of the innate immune system, greater B cell 
activation, and ultimately increased antibody production [31]. Heterologous antigens display 
on VLPs can assume a similar immunogenicity as the underlying particle, making VLPs good 
antigen-presenting platforms to increase immune responses against otherwise poorly 
immunogenic antigens [31, 42]. Besides induction of better immune responses, memory and 
specificity of the response may also be improved. Since VLPs cannot replicate they can be 
safely used in immunocompromised and senescent populations, two populations at risk for 
severe COVID-19.  
In ABNCoV2  a novel VLP technology that allows covalent, directional, high density binding of 
different proteins on the VLP surface is used. The VLP consist of the coat protein of AP205, a 
structural protein of a virus that does not infect eukaryotes (including humans). In pre-clinical 
models ABNCoV2 immunogenicity has been superior to the other platform technologies [43]. 
In addition, it is optimized to comply with the WHO target product profile for SARS-CoV-2 
vaccines [28]. 
MF59 is an oil-in-water emulsion adjuvant comprised of squalene, polysorbate 80, and sorbitan 
trioleate and is marketed in amongst others the Netherlands in seasonal influenza vaccines 
(e.g., Fluad/Fluad Tetra). MF59-adjuvanted vaccines spare vaccine doses and enhance 
hemagglutination inhibiting antibodies against homologous and heterologous influenza virus 
strains. The mechanisms of MF59 include rapid induction of chemokines and inflammatory 
cytokines, recruitment of multiple immune cells, increased uric acid (a post-apoptotic danger 
signal) and benign apoptosis of certain innate immune cells after IM injection [36]. The adjuvant 
effects of MF59 on generating vaccine-specific isotype-switched Immunoglobulin G (IgG) 
antibodies, effector CD8 T cells, and protective immunity were retained (even in a CD4-
deficient condition) by inducing an effective immune-competent microenvironment with various 
innate and APCs in a mouse model. CD4-independent adjuvant effects of MF59 might 
contribute to improving vaccine efficacy in children, the elderly, and immunocompromised 
patients as well as in healthy adults [36]. 
b. Previous exposure of human beings with the test product(s) and/or products with a similar 
biological mechanism 
Besides ABNCoV2, more than 200 vaccines against SARS-CoV-2 are under development 
[14]. First candidates have reached Phase 3 clinical trials and promising results have been 
published in the form of press releases [15-17] and interim analyses [18, 19]. Three studies 
have reached approximately 100 cases, which allows a reasonable estimate of clinical relevant 

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vaccine efficacy. Observed VE was 60-90%. All three trials used candidates based on S 
antigen as the immunogen and have observed significant immunogenicity following two 
vaccinations. Two use an RNA-vaccine-platform and one a recombinant adenovirus. Follow 
up times are still short (median of ~3 months) but vaccination campaigns based on emergency-
use authorization have started in several countries. In addition, systemic reactogenicity was 
significant in all three studies. 
A AP205 VLP has never yet been administered to humans, but the closely related Phage Qβ 
phage VLP has been tested in clinical trials against non-infectious conditions. The CYT006-
AngQb Phage Qβ cVLP vaccine targeting angiotensin 2 to reduce ambulatory blood pressure 
displayed an acceptable safety profile in phase 2 clinical trials (NCT00710372, NCT00500786, 
and NCT00701649). While transient flu-like symptoms were commonly observed, the most 
common AE were local injection site reactions. The vaccine caused a greater decrease in 
blood pressure when compared with a control group [25, 27]. 
A Phage Qβ cVLP has also been clinically evaluated as a vaccine against nicotine, intended 
for smoking cessation in phase 2 clinical trials (NCT00369616 and NCT01280968). For this 
vaccine, the most common AEs were transient flu-like symptoms followed by pyrexia, 
headache, nasopharyngitis, chills, and myalgia. For local reactions, injection site pain was the 
most frequently experienced AE, while injection site swelling, erythema, and edema were 
rarely experienced. The vaccine was found highly immunogenic and induced a mild attenuation 
of smoking behavior [26, 44].  
The Phage Qβ cVLP has also been utilized as a toll-like receptor 9 (TLR 9) agonist to improve 
clinical outcome parameters of patients with allergic conditions in phase 1/2 clinical trials (NCT 
00890734, NCT00800332, NCT00575003, NCT00574223, NCT00293904, NCT00574704, 
NCT00652223). In a trial examining patients with persistent allergic asthma, the most common 
AEs were injection site erythema, pruritus, and swelling and were primarily mild in intensity. 
Nasopharyngitis was also commonly reported, but not unexpected in this patient population. 
Of 33 patients who received cVLP treatment, 1 patient experienced moderate flu-like 
symptoms nearly a week after the third injection [45]. Rhino-conjunctivitis symptoms were 
significantly lower in patients treated with high dose of the cVLP TLR-9 agonist as compared 
with placebo [46]. 
Overall cVLP treatments have been shown to be generally safe and well tolerated in humans, 
with reports of mild flu-like symptoms and common vaccine-related injection site and systemic 
reactions being most common. 
MF59 adjuvant is an oil-in-water emulsion marketed in the Netherlands as part of the influenza 
vaccine Fluad/Fluad Tetra. It is approved for use in the elderly (>65 years). The most common 

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injection-site reactions with MF59-adjuvanted Fluad influenza vaccines are injection site pain 
and tenderness [47]. The most common systemic reactions are myalgia, headache, and 
fatigue. In a clinical trial assessing elderly subjects aged 65 years or older (NCT02587221), 
injection site pain and influenza-like illness were the only unsolicited adverse reactions 
reported in ≥ 1% of subjects (1.7% and 1.5%, respectively) following vaccination with Fluad 
Quadrivalent. In another study (NCT03314662) injection site bruising was the only related 
unsolicited adverse reaction reported in ≥ 1% of subjects (1.0%) [48]. There were no SAE, AE 
of special interest, or deaths in either study that were related to study vaccine. 
Overall MF59 adjuvanted influence vaccines have been shown to be generally safe and well 
tolerated in humans, with reports of common vaccine-related injection site and systemic 
reactions being most common. 
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo human 
cell material? 
The ABNCoV2 vaccine induced CoV-2 spike protein-specific IgG in BALB/c mice after a single 
IM vaccination and these were boosted following a second dose. There was moreover 
significant correlation between ELISA antibody titers and neutralizing antibodies (Ks = 0.7152, 
p = 0.0461) in mice immunized with ABNCoV2. A single vaccination with ABNCoV2 resulted 
in 100% neutralization of SARS-CoV-2 at a serum dilution of 1:80, and serum post booster 
immunization could be diluted up to 1:10,240. It has been suggested that a >50% virus 
neutralization at an endpoint titer dilution of 100 to 500 would be required to confer protection 
[49]. Indeed, serum from mice immunized only once with ABNCoV2 displayed comparable 
neutralizing activity to high titer convalescent samples from patients recovered from COVID-
19, whereas mice sera after multiple immunizations showed significantly greater virus 
neutralization activity than any human convalescent sera [43].  
d. Selectivity of the mechanism to target tissue in animals and/or human beings 
A repeat-dose toxicity study of ABNCoV2 with MF59 adjuvant in a 1:1 emulsion, vs. control 
(saline), administered IM 2 or 3 times (14 days apart) to New Zealand White (NZW) rabbits to 
assess toxicity and local tolerance showed no adverse risk profile.   
The AP205 VLP has previously also been tested in non-human primates as a platform for 
delivering the malaria antigen, VAR2CSA. Overall, these vaccines were well-tolerated by the 
40 aotus monkeys in this study, with no visible side effects, signs of systematic reactions, or 
behavioral changes. Additionally, the AP205 cVLP particle has been administered to hundreds 
of mice as well as rabbits, rats, and aotus monkeys in different vaccine formulations. In all 

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these studies, no issues were observed with weight loss, ulceration, or any other overt 
toxicities. 
e. Analysis of potential effect 
The ABNCoV2 dose administered in the rabbit toxicity study (0.1 mg/rabbit) translates to a 
human equivalent dose of >700 μg for a 60 kg human, providing, a safety margin of >7x higher 
than the highest dose envisaged to be administered in this FIH trial (70 μg). 
In the aforementioned BALB/c mice study [43], the higher dose of 6.5 μg ABNCoV2 
administered corresponds to a 10 to 20 times higher human equivalent dose (on a body surface 
area equivalence basis) than the highest dose to be tested in this trial. 
f. Pharmacokinetic considerations 
Biodistribution studies of ABNCoV2 have not been conducted because the vaccine does not 
include live virus and is not capable of replication or productive infection. The route of 
administration (IM) is well understood, and the vaccine will be formulated in MF59, a licensed 
adjuvant with previously defined characteristics. Consistent with International Council for 
Harmonisation (ICH) guidelines, formal pharmacokinetic studies were not conducted. 
g. Study population 
Subjects included in this trial are healthy adult volunteers, who have been extensively screened 
for any evidence of co-morbidity, including but not limited to: immune deficiency, 
hypersensitivity and cardiovascular risk factors. Female subjects of child-bearing potential are 
screened for pregnancy by urine test and are required to use contraception throughout the 
study period. 
h. Interaction with other products 
ABNCoV2 is not known to interact or be incompatible with any other products. 
i. Predictability of effect 
ABNCoV2-specific antibody concentrations will be measured by ELISA during immunisation 
and follow-up. Inhibitory titres will be measured in an in vitro SARS-CoV-2 invasion inhibition 
assay. In mice immunized with ABNCoV2 a significant correlation was observed between 
ELISA antibody titers and neutralizing antibodies (Ks = 0.7152, p = 0.0461). It has been 
predicted that a >50% virus neutralization at an endpoint titer dilution of 100 to 500 would be 
required to confer protection [49]. 
 
 

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j. Can effects be managed? 
Potential mild-moderate local or systemic adverse reactions to immunization will be managed 
symptomatically (e.g. paracetamol, ibuprofen, antihistamines). In the event of an anaphylactic 
reaction, this will be managed according to hospital-wide emergency protocols. 
13.2 Synthesis 
Preclinical studies show excellent immunogenicity of ABNCoV2, including high titres of 
neutralising antibodies, which are considered to represent a correlate of protection against 
SARS-CoV-2 infection and COVID-19. This is a first-in-human study and although pre-clinical 
toxicity results of ABNCoV2 are pending, substantive pre-clinical safety data exists on similar 
vaccines based on AP205 VLP, including in non-human primates. In addition, the closely 
related Phage Qβ phage VLP has been shown to be generally safe and well tolerated in 
humans, with reports of mild flu-like symptoms and common vaccine-related injection site and 
systemic reactions being most common. 
The trial incorporates a number of safety designs to help minimize these and other residual 
risks: 1) Cautious dose-escalation, starting at 6µg (equating to ~140-400x below the human 
equivalent dose used in pre-clinical animal studies) and escalating 2-fold; 2) Administration of 
a give dose of the vaccine to a groups of volunteers first without MF59 adjuvant, before 
proceeding to administration of that dose to a separate group in combination with adjuvant; 3) 
Use of sentinel subjects within each dose-adjuvant group; 4) Review of safety data up to and 
including group 3 (25 µg with and without adjuvant) and approval by SMC required prior to 
further dose-escalation and decision on whether to include adjuvant in subsequent groups; 5) 
Repeat of highest and second-highest safe and tolerable doses in two additional groups of 
volunteers in order to further support confidence in safety data before proceeding to phase 2 
trial(s). 
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