Safety, Tolerability and Plasmodium Falciparum Transmission-reducing Activity of R0.6C Vaccine Adjuvanted With Alhydrogel Alone or Combined With Matrix-M in Healthy Malaria-naïve Adults in the Netherlands
Phase: PHASE1
Conditions: Malaria, Malaria,Falciparum
Sponsor: Radboud University Medical Center

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Clinical trial protocol 
Safety, Tolerability and Plasmodium falciparum transmission-
reducing activity of R0.6C vaccine adjuvanted with Alhydrogel 
alone or combined with Matrix-M in healthy malaria-naïve adults in 
the Netherlands  
(STOP-TRANS) 
Version 3.0 
Date: 14 June 2021 
 
 
 
 
 
 
 
 
CONFIDENTIAL 
 
 
 
 

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PROTOCOL TITLE: Safety, Tolerability and Plasmodium falciparum transmission-reducing 
activity of R0.6C vaccine adjuvanted with Alhydrogel alone or combined with Matrix-M in 
healthy malaria-naïve adults in the Netherlands 
 
Protocol ID 
STOP-TRANS 
NL76664.000.21 
Short title 
Safety and efficacy of R0.6C 
EudraCT number  
2021-000017-17 
Version 
3.0 
Date 
14Jun2021 
Protocol Author 
 
Matthew McCall MD PhD 
Manon Alkema MD 
Principal investigator  
Matthew McCall MD PhD 
Radboudumc, Department of Medical Microbiology 
Tel: +31 (0)24 3615363 
Email: matthew.mccall@radboudumc.nl 
Coördinating investigator 
Manon Alkema MD 
Radboudumc, Department of Medical Microbiology 
Tel: +31 (0)24 3619515 
Email: manonalkema@radboudumc.nl 
Sponsor  
Stichting Katholieke Universiteit Nijmegen 
Department of Medical Microbiology  
Geert Grooteplein-Zuid 10 
6525 GA, Nijmegen, The Netherlands 
Subsidising party 
EU project 733273 - OptiMalVax 

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Independent expert (s) 
Arjan van Laarhoven MD PhD 
Radboudumc Department of Internal Medicine 
Tel: +31 24 361 69 80. 
Email: Arjan.vanLaarhoven@radboudumc.nl 
Laboratory sites  
Eric Aaldring 
Radboudumc Clinical Chemical Laboratory  
Tel: +31 (0)24 3616997 
Fax: +31 (0)24 3568408 
e-mail: Eric.Aaldring@radboudumc.nl 
 
Heiman Wertheim MD PhD 
Clinical Microbiology Laboratory  
Tel: +31 (0)24 36 14281 
email: Heiman.Wertheim@radboudumc.nl 
Pharmacy  
Dr. Rob ter Heine 
Radboudumc Clinical Pharmacy - Clinical Trial Unit  
Tel: +31 (0)24 3616405 
Fax: +31 (0)24 3668755 
email: R.terHeine@radboudumc.nl 

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PROTOCOL SIGNATURE SHEET 
The signature below constitutes approval of this protocol and the attachments and provides 
required assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements, applicable to ICH E6 [R2] guidelines. 
 
 

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TABLE OF CONTENTS 
 
1. 
INTRODUCTION AND RATIONALE ................................................................................ 13 
2. 
1.2 Rationale ..................................................................................................................... 14 
3. 
OBJECTIVES .................................................................................................................... 15 
4. 
STUDY DESIGN ............................................................................................................... 16 
5. 
STUDY POPULATION ...................................................................................................... 17 
5.1 
Population (base)....................................................................................................... 17 
5.2 
Inclusion criteria ......................................................................................................... 17 
5.3 
Exclusion criteria........................................................................................................ 18 
5.4 
Sample size justification ............................................................................................ 19 
5.4.1 
Estimation of empirical power for an analysis of transmission reducing activity 
under different assumptions.............................................................................................. 19 
6. 
TREATMENT OF SUBJECTS .......................................................................................... 22 
6.1 
Investigational product/treatment .............................................................................. 22 
6.2 
Escape medication .................................................................................................... 22 
7. 
INVESTIGATIONAL PRODUCT ....................................................................................... 23 
7.1 
Name and description of investigational  product(s) ................................................. 23 
7.2 
Summary of findings from non-clinical studies.......................................................... 24 
7.3 
Summary of findings from clinical studies ................................................................. 25 
7.4 
Summary of known and potential risks and benefits ................................................ 30 
7.4.1 
Risk Assessment ................................................................................................ 30 
7.4.2 
Benefit assessment ............................................................................................ 32 
7.5 
Description and justification of route of administration and dosage ......................... 32 
7.6 
Dose preparation and administration ........................................................................ 32 
7.7 
Preparation and labelling of Investigational Medicinal Product ................................ 33 
7.8 
Drug accountability .................................................................................................... 33 
8. 
METHODS......................................................................................................................... 33 
8.1 
Study endpoints ......................................................................................................... 33 
8.2 
Randomisation, blinding and treatment allocation .................................................... 34 
8.3 
Study procedures....................................................................................................... 35 
8.3.1 
Screening visit .................................................................................................... 35 
8.3.2 
Medical history.................................................................................................... 36 
8.3.3 
Physical examination.......................................................................................... 36 
8.3.4 
Vital signs ........................................................................................................... 37 
8.3.5 
Inclusion visit....................................................................................................... 37 
8.3.6 
Administration of R0.6C TBV candidate ............................................................ 38 
8.3.7 
Follow up after R0.6C administration ................................................................. 38 
8.3.8 
Unscheduled visits.............................................................................................. 38 
8.3.9 
Patient reported outcomes (study diary) ............................................................ 38 
8.3.10 
Blood sampling and safety laboratory evaluations ............................................ 39 
8.3.11 
Urine Toxicology analysis................................................................................... 39 
8.3.12 
Pregnancy Test .................................................................................................. 39 

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8.3.13 
Analysis of transmission reducing activity ......................................................... 39 
8.3.14 
Immunological assays: ELISAs/ PBMCs  collection .......................................... 40 
8.3.15 
Case report forms and data collection ............................................................... 40 
8.3.16 
COVID-19 related measures .............................................................................. 40 
8.3.17 
Schedule of trial visits and measurements ........................................................ 41 
8.4 
Withdrawal of individual subjects .............................................................................. 44 
8.5 
Replacement of individual subjects after withdrawal ................................................ 45 
8.6 
Follow-up of subjects withdrawn from treatment....................................................... 45 
8.7 
Premature termination of the study ........................................................................... 45 
9. 
SAFETY REPORTING ...................................................................................................... 46 
9.1 
Temporary halt for reasons of subject safety ............................................................ 46 
9.2 
AEs, SAEs and SUSARs ........................................................................................... 46 
9.2.1 
Adverse events (AEs)......................................................................................... 46 
9.2.2 
Adverse event data collection and recording..................................................... 46 
9.2.3 
Assessment of causality ..................................................................................... 47 
9.2.4 
Serious adverse events (SAEs) ......................................................................... 48 
9.2.5 
Suspected unexpected serious adverse reactions (SUSARs) .......................... 49 
9.3 
Annual safety report................................................................................................... 49 
9.4 
Follow-up of adverse events ..................................................................................... 50 
9.5 
Safety Monitoring Committee .................................................................................... 50 
9.5.1 
Local safety monitor ........................................................................................... 50 
9.5.2 
Safety Meetings .................................................................................................. 50 
9.5.3 
Safety Reports .................................................................................................... 51 
9.5.4 
Inter-group progression ...................................................................................... 51 
9.5.5 
Safety holding rules ............................................................................................ 52 
10. 
STATISTICAL ANALYSIS ............................................................................................. 54 
10.1 
Primary study parameter(s) ....................................................................................... 54 
10.2 
Interim analysis .......................................................................................................... 55 
11. 
ETHICAL CONSIDERATIONS...................................................................................... 56 
11.1 
Regulation statement................................................................................................. 56 
11.2 
Recruitment and consent........................................................................................... 56 
11.3 
Benefits and risks assessment .................................................................................. 57 
11.4 
Compensation for injury............................................................................................. 57 
11.5 
Incentives ................................................................................................................... 58 
12. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .......................... 59 
12.1 
Handling and storage of data and documents .......................................................... 59 
12.2 
Monitoring and Quality Assurance ............................................................................ 59 
12.3 
Amendments .............................................................................................................. 60 
12.4 
Annual progress report .............................................................................................. 61 
12.5 
Temporary halt and (prematurely) end of study report ............................................. 61 
12.6 
Public disclosure and publication policy .................................................................... 61 
13. 
STRUCTURED RISK ANALYSIS ................................................................................. 62 
13.1 
Potential issues of concern........................................................................................ 62 

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13.2 
Synthesis ................................................................................................................... 64 
14. 
REFERENCES .............................................................................................................. 65 

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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 
AR 
Adverse Reaction 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
CV 
Curriculum Vitae 
DSMB 
Data Safety Monitoring Board 
ELISA 
Enzyme-linked immunosorbent assay 
EPD 
Electronic Patient Dossier 
EU 
European Union 
EudraCT European drug regulatory affairs Clinical Trials  
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
GP 
General Practicioner 
IB 
Investigator’s Brochure 
IC 
Informed Consent 
IM 
Intramuscular(ly) 
IMP 
Investigational Medicinal Product  
IMPD 
Investigational Medicinal Product Dossier  
METC  
Medical research ethics committee (MREC); in Dutch: medisch-ethische 
toetsingscommissie (METC) 
PBMC 
Peripheral Blood Mononuclear Cells 
(S)AE 
(Serious) Adverse Event  
SMFA 
Standard Membrane Feeding Assay 
SMC 
Safety Monitoring Committee 
SPC 
Summary of Product Characteristics; in Dutch: officiële 
productinformatie IB1-tekst 
Sponsor The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical 

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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 
TBV 
Transmission blocking vaccine 
TBA 
Transmission blocking activity 
TRA 
Transmission reducing activity 
UAVG 
Dutch Act on Implementation of the General Data Protection Regulation; 
in Dutch: Uitvoeringswet AVG 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 

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SUMMARY 
 
Rationale: Malaria, a disease caused by Plasmodium parasites, is one of the most important 
infectious diseases worldwide. After a period of success in global malaria control, progress 
has stalled in 2015-2018. The availability of a transmission blocking vaccine would be a 
critical step to move towards malaria elimination[1, 2]. The R0.6C fusion protein, consisting 
of the N-terminal region of Glutamate Rich Protein GLURP (R0) and the 6-cysteine C-
terminal fragment of the well-established Pfs48/45 antigen (6C), is a lead candidate for a 
transmission blocking vaccine.  
 
Objectives 
Primary safety  objective:  
1) To evaluate safety of R0.6C immunizations in healthy malaria-naïve volunteers in four 
dose-adjuvant combinations. 
Primary efficacy objective: 
1) To assess transmission reducing activity (TRA) to mosquitoes in the standard 
membrane feeding assay of sera after full course of R0.6C immunizations in each of 
the four dose-adjuvant combinations. 
 
Secondary safety objective; 
1) To evaluate tolerability of R0.6C immunizations in healthy malaria-naïve volunteers in 
four dose-adjuvant combinations. 
Secondary efficacy objectives: 
1) To assess the dynamics of TRA in the standard membrane feeding assay of sera 
collected during and after R0.6C immunizations in each of the four dose-adjuvant 
combinations. 
2) To assess the dynamics of anti-6C antibody quantities during and after R0.6C 
immunizations in each of the four dose-adjuvant combinations. 
 
Exploratory objectives:  
1) To assess transmission blocking activity (TBA) in the standard membrane feeding 
assay in each of the four dose-adjuvant combinations.  
2) To estimate antibody decay rate against the 6C fraction of R0.6C following R0.6C 
immunizations for each of the four dose-adjuvant combinations.   
3) To quantify antibody quantities against R0.6C and the R0 fraction of R0.6C after 
immunization with R0.6C in each of the four dose-adjuvant combinations.   
4) To analyse cellular immune responses after R0.6C immunization. 

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5) To compare TRA between the two dose groups (30 or 100µg R0.6C) and/or between 
adjuvant groups (alhydrogel and alhydrogel+Matrix-M). 
6) To compare anti-6C antibody quantities between the two dose groups (30 or 100µg 
R0.6C) and/or between adjuvant groups (alhydrogel and alhydrogel+Matrix-M). 
 
Study design: R0.6C is a first-in-human phase I, open-label, single-site, dose escalation 
study to determine the safety, tolerability and transmission reducing activity of the R0.6C 
vaccine in two different adjuvant combinations. 
Study population: 32 healthy, malaria naïve adults (males and females), aged 18 – 55 
years. 
Intervention: Each of the study arms will receive four intramuscular vaccinations on days 0, 
28, 56 and 168 with R0.6C adsorbed to Alhydrogel alone (Groups 1-4A), or combined with 
an additional adjuvant Matrix-M (groups 1-4B). Dose escalation with the two adjuvant arms 
will take place in parallel: first, a sentinel group (1A and 1B, n=3 per arm) will receive the low 
dose of 30µg R0.6C; subsequently the additional subjects (groups 2A and 2B, n=5 per arm) 
will receive the low dose of 30 µg R0.6C; if considered safe, a sentinel group (3A and 3B, 
n=3 per arm) will receive the high dose of 100µg R0.6C; and finally, the remainder of 
subjects will receive the high dose of 100µg R0.6C (groups 4A and 4B, n=5 per arm).  
 
Main study endpoints:  
Primary safety endpoints: 
1) The number of serious adverse events and solicited and unsolicited grade 3 adverse 
events possibly, probably or definitely related to the vaccine in the period from first 
R0.6C administration up to 84 days after the last immunization.  
Primary efficacy endpoints: 
1) The functional TRA in the standard membrane feeding assay of volunteer sera 
collected two weeks after the fourth R0.6C immunization (I4+14), compared to 
baseline (I1-1)  within each of the four dose-adjuvant groups.  
 
Secondary safety endpoints: 
1) The number of solicited and unsolicited grade 1 and 2  adverse events possibly, 
probably or definitely related to the vaccine in the period from first R0.6C 
administration up to 84 days after the last immunization.  
Secondary efficacy endpoints: 
1) The TRA at other timepoints (I1+14, I2+14, I3+14, I3+111 [I4-1], and I4+84) 
compared to baseline (I1-1) in each of the four dose-adjuvant groups. 

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2) The anti-6C antibody quantity in volunteer sera collected two weeks after fourth 
R0.6C immunization (I4+14) and at other time points (I1+14, I2+14, I3+14, I3+111 [I4-
1], and I4+84) compared to baseline (I1-1) in each of the four dose-adjuvant 
combinations, as determined by ELISA. 
 
Exploratory endpoints: 
1) The functional TBA in the SMFA of volunteer sera collected at different time points 
compared to baseline (I1-1) in each of the four dose-adjuvant combinations.  
2) The anti-6C antibody decay rate following R0.6C immunization for each of the four 
dose-adjuvant combinations. 
3) The anti R0.6C and anti-R0 antibody quantity in volunteer sera collected at different 
time points in each of the four dose-adjuvant combinations. 
4) The cellular immune responses in volunteers samples collected at different time 
points in each of the four dose-adjuvant combinations. 
5) The difference in TRA between the two dose groups (30 or 100µg R0.6C) and/or 
between adjuvant groups (alhydrogel and alhydrogel+Matrix-M). 
6) The difference in peak anti-6C antibody quantity between the two dose groups (30 or 
100µg R0.6C) and/or between adjuvant groups (alhydrogel and alhydrogel+Matrix-M). 
 
Nature and extent of discomfort and risks associated with participation, benefit and 
group relatedness: There are no individual benefits to participating in this study. In case 
that the vaccine is effective, vaccination campaigns will reduce the risk of transmission in a 
population. Subjects will not be protected against malaria after participating. The vaccine 
platform (Lactococcus lactis expressed recombinant protein vaccine) and both adjuvants 
(Alhydrogel, Matrix M) have been used extensively before. However, R0.6C has not been 
administered in humans before, therefore there is a theoretic risk of novel side effects to 
occur. Participating in this trial includes the discomfort of R0.6C vaccine administrations with 
potential adverse reactions, multiple blood sampling tests, follow-up visits, physical 
examinations, screening for HIV, Hepatitis B and Hepatitis C, pregnancy tests (for females), 
filling out a diary and abiding to all study rules. 

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1. INTRODUCTION AND RATIONALE 
Malaria is one of the most devastating infectious diseases worldwide. After a period of 
success in global malaria control, progress has stalled. Data from 2015-2018 highlight that 
no significant progress in reducing global malaria cases was made in this period. There were 
an estimated 228 million cases and 405 000 related deaths in 2018, mainly in children below 
five years of age[3]. In addition to the this burden of morbidity and mortality, this disease 
forms a profound economic burden for the affected countries, many of which are in any case 
characterised as Low and Middle Income Countries (LMIC)[4]. The urgency of the situation is 
further emphasized by the waning effectiveness of currently registered anti-malarials due to 
fast emergence and spread of resistance and the absence of a highly effective vaccine[1, 5]. 
 
Human malaria is caused by protozoa of the genus Plasmodium: P. falciparum, P. vivax, P. 
ovale, P. malariae, and P. knowlesi, of which P. falciparum is responsible for the greatest 
global burden of morbidity and mortality. Parasites (sporozoite stage) are injected into the 
skin by an infected female Anopheles mosquito when taking a blood meal. After penetration 
of the skin capillaries, sporozoites migrate to the liver, where they develop and multiply in 
liver cells before release into the blood (merozoite stage) and invading red blood cells for 
further maturation and multiplication. The cyclical proliferation of asexual stages within the 
human red blood cells is responsible for the occurrence of clinical symptoms. A small fraction 
of asexual stages commits to enter sexual development and ultimately form mature male and 
female gametocytes. These male and female gametocytes do not cause clinical pathology or 
symptoms, but can be taken up by a new Anopheline vector, in whose midgut they transform 
into male and female gametes, which fuse to form a zygote that in turn becomes motile and 
elongated (ookinete). This ookinete invades the mosquito midgut wall, developing into an 
oocyst which in turn releases new sporozoites. This ultimately renders the mosquito 
infectious upon its next bite, leading to onward transmission of malaria infections. 
Malaria transmission blocking vaccines (TBVs) and transmission-blocking drugs aim to 
interrupt transmission to or the development of parasites in the mosquito vector by an 
intervention applied to the human intermediate host[5]. Deployment of transmission-blocking 
drugs and TBVs will be an efficient complementary element in an integrated program of anti-
malarial interventions. With the implementation of TBVs we will be able to reduce the malaria 
burden, contain drug resistance and to move towards malaria elimination[2, 6].  
 
The sexual stage Pfs48/45 antigen is a well-established lead candidate for a P. falciparum 
transmission blocking vaccine because of its critical role in parasite fertilisation. Male 
gametes lacking Pfs48/45 are unable to bind female gametes in the mosquito midgut, thus 
preventing ookinete, oocyst and ultimately sporozoite development. The R0.6C fusion protein 

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is a chimera consisting of the 6-cysteine C-terminal fragment of PfS48/45 (6C) coupled to the 
N-terminal region of asexual stage Glutamate Rich Protein GLURP (R0) produced in 
Lactococcus lactis. Immunisation with R0.6C in rodents induces functional antibodies against 
the 6C subunit[7]. Anti-6C antibodies (Abs) are ingested during the blood meal and can bind 
male sexual forms in the mosquito gut, preventing their fertilisation of female gametes and 
thus ookinete and oocyst development. Sera of vaccinated animals were able to reduce 
transmission in the standard membrane feeding assay (SMFA) with cultured gametocytes. 
Anti-6C antibody titres were further increased by immunising with R0.6C adjuvanted with 
Alhydrogel or Alhydrogel and Matrix-M[8].   
 
The SMFA is the most widely used assay to determine the TRA and an accepted 
surrogate of TRA for public health interventions [5]. Laboratory reared Anopheles mosquitoes 
are fed through membrane feeders with in vitro cultured gametocytes to which serum or 
purified anti-R0.6C antibodies in various concentrations are added. Subsequently, the 
oocyst densities in mosquitoes are compared between the experimental and control group 
by means of microscopy of the mosquito gut, parasite DNA detection or immune-assays [5, 
9]. The SMFA can also be used to determine the transmission blocking activity (TBA) by 
comparing the number of infected mosquitoes between the experimental and control group. 
 
Because the mode of action of TBVs is so explicit, there is a highly informative endpoint in 
the SMFA that is a clear indicator of later public health impact. For other vaccines, there is no 
such clear in vitro assay. The TRA measured in the SMFA assay around which there is 
confidence that blocking activity is present was set by consensus at 80% reduction in 
oocyst intensity, however even TBVs with a TRA of less than 80% could eliminate 
plasmodium at low transmission levels over consecutive transmission seasons[10]. 
 
2. 1.2 Rationale 
The renewed focus on malaria elimination has increased the priority of research towards 
development of interventions to block malaria transmission [10, 11]. By interrupting 
transmission of malaria parasites in mosquito vectors, a reduction in the number of 
secondary infections in the community is expected with an overall reduction in disease and 
mortality[12].  
The lead TBV candidate R0.6C has shown good transmission reducing efficacy in rodent 
models. Also, a monoclonal antibody TB31F against the same Pfs48/46 6C region has 
shown excellent safety and transmission reducing activity in humans in a recent clinical trial 
(van der Boor et al, manuscript in preparation). Efficacious TBVs would be a critical step on 

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the path towards malaria elimination and their safety and preliminary efficacy data can be 
acquired in malaria-naïve volunteers.  
 
3. OBJECTIVES 
 
Primary safety  Objective:  
1) To evaluate safety of R0.6C immunizations in healthy malaria-naïve volunteers in four 
dose-adjuvant combinations. 
Primary efficacy objective: 
1) To assess TRA in the standard membrane feeding assay of sera after full course of 
R0.6C immunizations in each of the four dose-adjuvant combinations. 
 
Secondary safety objective; 
1) To evaluate tolerability of R0.6C immunizations in healthy malaria-naïve volunteers in 
four dose-adjuvant combinations. 
Secondary efficacy objectives: 
1) To assess the dynamics of TRA in the standard membrane feeding assay of sera 
collected during and after R0.6C immunizations in each of the four dose-adjuvant 
combinations. 
2) To assess the dynamics of anti-6C antibody quantities during and after R0.6C 
immunizations in each of the four dose-adjuvant combinations. 
 
Exploratory Objectives:  
1) To assess TBA in the standard membrane feeding assay in each of the four dose-
adjuvant combinations.  
2) To estimate antibody decay rate against the 6C fraction of R0.6C following R0.6C 
immunizations for each of the four dose-adjuvant combinations.   
3) To quantify antibody quantities against R0.6C and the R0 fraction of R0.6C after 
immunization with R0.6C in each of the four dose-adjuvant combinations.   
4) To analyse cellular immune responses after R0.6C immunization. 
5) To compare TRA between the two dose groups (30 or 100µg R0.6C) and/or between 
adjuvant groups (alhydrogel and alhydrogel+Matrix-M). 
6) To compare anti-6C antibody quantities between the two dose groups (30 or 100µg 
R0.6C) and/or between adjuvant groups (alhydrogel and alhydrogel+Matrix-M). 

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4. STUDY DESIGN 
This is a first-in-human, phase I, open-label, single-site, dose escalation study to determine 
the safety tolerability and immunogenicity of the R0.6C vaccine in two different adjuvant 
combinations. Administration of R0.6C to volunteers, follow up and clinical chemical, 
microbiological analysis and storage of the samples will take place in the Radboudumc in 
Nijmegen.  
Thirty-two healthy adult volunteers will be recruited and divided over the study arms that will 
receive four vaccinations on days 0, 28, 56 and 168 with either 30µg or 100µg of R0.6C 
adjuvanted with Alhydrogel alone, or combined with Matrix-M (Table 1).  
Three volunteers (Group 1A, n=3) will receive four vaccinations with the lower dose of 30µg 
R0.6C with Alhydrogel, and, in parallel, three volunteers (Group 1B, n=3) will receive four 
vaccinations with the lower dose of 30µg R0.6C with Alhydrogel and Matrix-M. Volunteers 
will be closely monitored for adverse events for a period of minimally 14 days after the first 
vaccination. If safe, an additional 5 volunteers per adjuvant arm (groups 2A and 2B) will then 
receive four vaccinations with the lower dose (30µg R0.6C). If considered safe following a 
minimum of 14 days of follow-up after the first R0.6C administration of groups 2A and 2B, 
three volunteers per adjuvant arm (groups 3A and 3B) will start the vaccination regimen with 
the higher dose of 100µg R0.6C. Finally, a minimum of 14 days after administration of the 
first vaccination in groups 3A and 3B, if considered safe, an additional 5 volunteers per 
adjuvant arm (groups 4A and 4B) will initiate the vaccination regimen with the higher dose of 
100µg R0.6C. There will be no placebo group; TRA endpoints will be compared to pre-
vaccination values. All volunteers will be followed up for adverse events until 84 days after 
the last immunisation. Total trial duration is approximately 8 months for each subject. Blood 
will be collected to assess the functional TRA, TBA and immunogenicity at pre-specified time 
points after R0.6C vaccinations (also see section 8.3.17). If one of the volunteers is 
considered not eligible for participation on day 0, an different (‘back-up’) volunteer will 
replace him/her. For this purpose up to two additional volunteers may be screened and kept 
as back up in each group. 
Table 1: study arms. 
Study arm 
Number of 
volunteers 
Candidate vaccine dose and adjuvant combination 
1A 
n=3 
4x 30µg R0.6C Alhydrogel 
1B 
n=3 
4x 30µg R0.6C Alhydrogel + Matrix M 
2A 
n=5  
4x 30µg R0.6C Alhydrogel 
2B 
n=5  
4x 30µg R0.6C Alhydrogel + Matrix M 
3A 
n=3 
4x 100µg R0.6C Alhydrogel 

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3B 
n=3 
4x 100µg R0.6C Alhydrogel + Matrix M 
4A 
n=5  
4x 100µg R0.6C Alhydrogel 
4B 
n=5  
4x 100µg R0.6C Alhydrogel + Matrix M 
 
5. STUDY POPULATION 
5.1 Population (base)  
The study population will be comprised of adult male and female healthy subjects aged 18-
55 at time of first R0.6C administration. A total of 32 subjects will be enrolled to participate 
in the study as well as up to 8 reserve subjects (up to 2 reserve subject per dosing group). 
The investigator will ensure that all subjects being considered for the study meet the 
eligibility criteria described below. 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. 
 
5.2 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 a male or non-pregnant and non-lactating female age ≥ 18 and ≤ 55 years and 
in good health. 
3. Subject is able to understand planned study procedures and demonstrate 
comprehension of the protocol procedures and knowledge of study by passing a quiz 
(assessment of understanding).  
4. In the opinion of the investigator, the subject can and will comply with the requirements 
of the protocol. 
5. Subjects are available to attend all study visits and are reachable by phone throughout 
the entire study period from day -1 until day 224 (end of study). 
6. The subject will remain within reasonable travelling distance from the study center 
from day -1 until day 7 after each R0.6C administration and agrees not to travel to a 
malaria-endemic area during the study period 
7. 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). 
8. The subject agrees to refrain from blood donation to Sanquin or for other purposes 
throughout the study period according to current Sanquin guidelines. 

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9. 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 subjects of 
childbearing potential must agree to use continuous adequate contraception* until 2 
months after completion of the study. Female subjects must agree not to breastfeed 
from 30 days prior to R0.6C administration until 2 months after completion of the study. 
Female subjects must have a negative pregnancy test at the inclusion visit. 
 
*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, symptothermal, post-
ovulation methods) and withdrawal are not acceptable methods of contraception. 
Adequate contraception does not apply to subjects of child bearing potential with partners 
of the same sex. 
 
5.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from 
participation in this study: 
1. Acute or chronic disease at time of R0.6C administration, clinically significant 
pulmonary, cardiovascular, hepatic, renal, neurological or immunological functional 
abnormality, as determined by medical history, physical examination or laboratory 
screening tests: 
a. Acute disease is defined as the presence of a moderate or severe illness with 
or without fever. For subjects with an illness on the day of R0.6C administration, 
the vaccination may be postponed up to 7 days. 
b. Fever is defined as an oral, axillary or tympanic temperature ≥ 38.0°C. 
c. Any abnormal and clinically significant baseline laboratory screening tests of 
ALT, AST, creatinine, hemoglobin, platelet count or total white blood cell 
count, as defined in the protocol according to the FDA Toxicity Grading Scale 
for Healthy Adult and Adolescent Subjects Enrolled in Preventative Vaccine 
Clinical Trials (appendix 1). 
2. History of malignancy of any organ system (other than localized basal cell carcinoma of 
the skin), treated or untreated, within the past 5 years. 
3. Chronic use of i) immunosuppressive drugs, iii) or other immune modifying drugs 
within three months prior to study onset (inhaled and topical corticosteroids and oral 
anti-histamines exempted) or expected use of such during the study period. 
4. History of drug or alcohol abuse interfering with normal social function in the period of 
one year prior to study onset, positive urine toxicology test for cocaine or amphetamines 
at screening or at inclusion. 
5. Screening tests positive for Human Immunodeficiency Virus (HIV), active Hepatitis B 
Virus (HBV), Hepatitis C Virus (HCV). 

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6. Use of any other investigational or non-registered product (drug or vaccine) during 
the study period. 
7. Known hypersensitivity to macrolides.  
8. Participation in any other clinical study involving an investigational product in the 30 
days prior to the start of the study or during the study period. 
9. Receipt of any other vaccination within 30 days prior to or up to 14 days after any R0.6C 
vaccination. Exceptions are made for vaccination against influenza and the novel 
coronavirus SARS-CoV2. 
10. Any history of malaria, positive serology for P. falciparum, or previous participation in 
any malaria (vaccine) study or CHMI. 
11. Body weight > 115 kg 
12. Being an employee or student of the department of Medical Microbiology of the 
Radboudumc at the time of screening, or a person otherwise related to the investigator.  
13. 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, or affects the interpretability of the trial results. 
 
5.4 Sample size justification 
 
R0.6C is a phase 1 safety and efficacy trial in healthy volunteers, designed to identify safety 
concerns associated with R0.6C administration at different dosages and/or adjuvants. A total 
of 32 healthy adults (males and females) aged 18 – 55 will be divided over four groups. Each 
group will include eight subjects.  
Sample size for each group is appropriate based on similar trial designs in malaria protein 
vaccination studies. This is a small Phase 1 study; therefore, it is largely descriptive and is 
intended to provide primarily safety data of 2 doses of R0.6C combined with 2 adjuvants. 
Comparative statistics will be performed but will have low power to detect anything other than 
very large differences between the groups.  
 
Furthermore, if we assume an effect size of 75% TRA at 14 days after the fourth R0.6C 
vaccination (I4+14) compared to baseline (I1-1), with the subject size of n=8 per 
dose/adjuvant arm we will have an empirical power of >99%, based on calculations and 
assumptions as described below in paragraph 5.4.1.  
 
 
5.4.1 
Estimation of empirical power for an analysis of transmission 
reducing activity under different assumptions 
 
  The simulation algorithm 
1. We generate 𝑛0 subjects in the control (pre-treatment) group and 𝑛1 subjects in the 
treatment (post-treatment) group with a variable 𝑥𝑖 to indicate whether the subject is in 
the control group (𝑥𝑖= 0) or in the treatment group (𝑥𝑖= 1). 
2. We generate 𝑚 mosquito dissections for each subject. 

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3. We simulate subject-specific random intercepts (𝑧𝑖) from a normal distribution with a mean 
of 0 and a specified standard deviation 𝜎, i.e. 𝑍~𝑁(0,𝜎). 
4. Lastly we generate the oocyst counts (𝑦𝑖𝑗) for each subject 𝑖 and mosquito 𝑗 using a 
negative binomial distribution such that 𝑌~𝑁𝐵(𝜇,𝜃) where 𝜃 is the anticipated dispersion 
parameter and 𝜇𝑖𝑗= exp(𝛽0 + 𝛽1𝑥𝑖𝑗+ 𝑧𝑖) where 𝛽0 is the anticipated mean of the log 
oocyte counts in the control group and 𝛽1 is the anticipated regression coefficient used to 
estimate the TRA, such that 
𝛽1 = log (1 −𝑇𝑅𝐴
100). 
  The model 
To analyze the simulated data, we make the of the 𝑚𝑔𝑐𝑣 package in R. We run a Generalized 
Additive Mixed Model such that, 
log(𝑦) = 𝛽0 + 𝛽1𝑥+ 𝑧𝑖. 
From each simulated dataset, we estimate the 𝑇𝑅𝐴= (1 −exp(𝛽^
1)) × 100 and 𝑝 (the p-
value) of the test for whether 𝛽1 is significantly different from 0. We then report the average 
𝑇𝑅𝐴 across simulations, which should be close to the anticipated value, and the emperical 
power which we define as the percentage of 𝑝's less than 0.05. 
 
  Simulation results 
We chose 𝑚= 20 as the standard amount of mosquito dissections per subject. We used a 
previous study (NL69779.091.19) to extract anticipated values for the mean log oocyst count 
in the control group, the dispersion parameter 𝜃 and the standard deviation of the random 
effects 𝜎. i.e. 𝛽0 = 3.7037, 𝜃= 4.451 and 𝜎= 0.64. For the anticipated 𝑇𝑅𝐴 (and thus 
anticipated 𝛽1) we used a range of assumed values, i.e. 𝑇𝑅𝐴= (25%, 50%, 75%). We also 
varied the number of people in the control and treatment groups, i.e. 𝑛0 and 𝑛1. The tables 
below show the emperical power under these scenarios. 
𝑻𝑹𝑨
= 𝟐𝟓% 
𝒏𝟏 
𝒏𝟎 
5 
6 
7 
8 
9 
10 
10 
17.0 
18.4 
17.4 
18.4 
18.2 
19.0 
11 
17.2 
15.8 
16.6 
17.6 
19.6 
19.4 
12 
17.0 
17.6 
18.2 
18.4 
19.4 
19.2 
13 
17.2 
16.4 
17.6 
17.4 
20.6 
19.6 
14 
16.6 
17.8 
19.8 
20.2 
19.4 
20.2 
15 
15.4 
17.4 
18.8 
19.4 
20.8 
22.6 
16 
14.6 
15.2 
17.8 
19.8 
20.4 
21.0 
17 
14.4 
15.8 
18.6 
19.0 
20.6 
22.4 
18 
14.4 
16.2 
18.2 
18.8 
21.0 
22.6 
19 
17.4 
19.0 
18.6 
19.4 
22.2 
24.8 
20 
15.8 
17.8 
19.4 
22.2 
25.8 
26.8 
 

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𝑻𝑹𝑨=
𝑻𝑹𝑨
= 𝟓𝟎% 
𝒏𝟏 
𝒏𝟎 
5 
𝟔 
7 
8 
9 
10 
10 
52.0 
57.0 
60.4 
63.8 
64.4 
67.0 
11 
54.2 
58.8 
62.4 
63.8 
65.2 
69.6 
12 
55.0 
59.4 
62.8 
65.4 
69.2 
70.6 
13 
54.6 
58.2 
63.6 
67.0 
69.4 
71.8 
14 
58.4 
62.4 
64.8 
68.2 
70.4 
73.0 
15 
55.6 
59.2 
65.0 
66.6 
70.4 
74.0 
16 
55.2 
60.4 
64.0 
68.0 
71.6 
75.8 
17 
54.6 
60.6 
66.8 
69.8 
75.8 
78.2 
18 
54.2 
61.6 
67.8 
72.2 
75.6 
78.4 
19 
59.6 
66.8 
71.0 
76.8 
78.8 
81.6 
20 
61.8 
68.2 
72.8 
78.6 
80.6 
82.8 
  
𝑻𝑹𝑨
= 𝟕𝟓% 
𝒏𝟏 
𝒏𝟎 
5 
𝟔 
7 
8 
9 
10 
10 
97.2 
99.0 
99.6 
99.8 
99.8 
100.0 
11 
98.2 
99.6 
99.8 
99.6 
99.6 
100.0 
12 
98.4 
99.2 
99.2 
99.8 
100.0 
100.0 
13 
97.4 
99.0 
99.6 
99.8 
100.0 
100.0 
14 
96.6 
98.8 
99.2 
99.8 
100.0 
100.0 
15 
97.8 
99.2 
99.8 
100.0 
100.0 
100.0 
16 
98.4 
98.8 
99.6 
99.8 
100.0 
99.8 
17 
99.4 
99.8 
100.0 
100.0 
100.0 
100.0 
18 
99.0 
100.0 
100.0 
99.8 
100.0 
100.0 
19 
99.2 
99.6 
99.6 
100.0 
100.0 
100.0 
20 
99.0 
99.2 
99.8 
100.0 
100.0 
100.0 
 
 

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6. TREATMENT OF SUBJECTS 
6.1 Investigational product/treatment 
Volunteers will sequentially receive four administrations of R0.6C intramuscularly in the 
deltoid muscle on alternating sides on days 0, 28, 56 and 168. A total of 8 subjects will 
receive four times 30µg R0.6C adjuvated with 240 µg  Alhydrogel (Alhydrogel, groups 1A and 
2A), eight subjects will receive four times 30µg R0.6C adjuvated with 240 µg Alhydrogel and 
15 µg Matrix-M (‘Alhydrogel/MM’ groups 1B and 2B), eight subjects will receive four times 
100µg R0.6C adjuvated with 800 µg Alhydrogel (groups 3A and 4A) and eight subjects will 
receive four times 100µg R0.6C adjuvated with 800 µg Alhydrogel and 49 µg Matrix-M 
(groups 3B and 4B). There will be no placebo group. 
 
6.2 Escape medication  
Escape medication is not applicable, appropriate medication (e.g. paracetamol or 
antihistamines) for symptomatic relief of any untoward side effects will be advised as 
necessary.  

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7. INVESTIGATIONAL PRODUCT  
 
7.1 Name and description of investigational  product(s) 
The R0.6C fusion protein is a chimera consisting of the 6-cysteine C-terminal fragment of 
PfS48/45 (6C) coupled to the N-terminal region of asexual stage Glutamate Rich Protein 
GLURP (R0) produced in Lactococcus lactis (Singh et al. 2020, Singh et al. 2017; Singh et 
al. 2015). The carrier protein R0 helps to enhance the immune response against Pfs48/45 
(Theisen et al. 2014). R0.6C and the position of the Pfs48/45-6C domain is shown in Figure 
1.  
 
 
Figure 1: Schematic representation of Pfs45/48 and the fragment that is cloned in the R0.6C 
fusion protein 
 
The recombinant R0.6C protein is formulated in 10 mM HEPES, 2.5% glucose, 0.5 mM 
EDTA, 155 mM NaCl and absorbed to 1.6 mg/mL Alhydrogel [Al(OH)3], at a fill volume of 0.8 
mL in a 2 mL borosilicate glass vial and stored at 2-8°C.  
 
Alhydrogel [Al(OH)3] is an aluminium hydroxide adjuvant supplied by Croda (Denmark, USA.  
It has been used as a vaccine adjuvant to enhance immune responses to vaccines. The final 
aluminium composition in the R0.6C/Alhydrogel product is 0.8 mg/mL Aluminum. 
 
Matrix-M is a saponin based adjuvant manufactured by Novavax (Sweden). The saponin-
based Matrix-M improves immune responses and enables vaccine dose-sparing. Matrix-M is 
supplied ready-to-mix as a refrigerated solution (stored 2-8°C) at a concentration of 0.375 
mg/mL and a fill volume of 0.75 mL. Although it is not yet fully understood how the Matrix-M 
adjuvant achieves its stimulatory effects, this adjuvant is known to transiently enhance the 
number of activated immune cells in the draining lymph nodes which may in turn lead to 
increased uptake and presentation of vaccine antigens to elicit a competent immune 
response (Reimer et al. 2012). Specifically, it has been shown that there is an increase of 
CD169+ macrophages, as well as activated dendritic cells, to the draining lymph nodes after 
immunization with Matrix-M adjuvanted vaccines, which may help to increase antigen 
presentation (Magnusson et al. 2018). Hence, CD169+ macrophages has previously been 
shown to have a role in transporting antigens to B lymphocytes by trapping them in the 
draining lymph node  and to facilitate cross-presentation of the antigen to CD8+ T 
lymphocytes (Gray and Cyster 2012; Carrasco and Batista 2007). This may lead to increased 
humoral and cellular immune responses, manifested by cross-reactive antibodies and multi-

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functional CD4+ T lymphocytes (Bengtsson et al. 2016; Shinde et al. 2020). Consequently, 
Matrix-M has been shown to contribute to antigen dose-sparing and increased duration of 
humoral and cellular vaccine responses (42). 
 
7.2 Summary of findings from non-clinical studies 
Preclinical studies focused on safety, immunogenicity, and functional activity of the R0.6C 
vaccine candidate. Table 2 summarizes the preclinical studies conducted in various animal 
species. For more detailed information, please refer to D1. Investigator's Brochure 
R0.6C_AlOH, section 8.0 preclinical studies. 
 
Table 2:  Summary of Preclinical Animal Studies for R0.6C candidate Vaccines 
 
Animal 
Specie
s 
Study 
Protocol 
Material 
Purpose 
Dose, Route, & 
Regimen 
Summary 
 
Rat 
 
Radboud 
UMC under 
approval 
number 
2016-0020 
 
[13] 
 
Small scale 
lab-
produced 
R0.6C 
 
Perform dose-
titration studies 
within suspected 
dose range to 
demonstrate that 
R0.6C elicit TB 
antibodies and to 
provide data for 
potency 
evaluation. 
 
0.03, 1.2, 4.7, 9.4, 
18.9, and 37.7µg 
of R0.6C 
adjuvanted on 
Alhydrogel, 3 SC 
injections on D0, 
D14, and D28. 
Terminal bleed 
on SD43. 
 
Doses are within the 
suspected range for 
potency evaluation. 
Pfs48/45-6C 
antibody titers are 
associated with 
TRA. 
 
Rat 
 
MVI-01-
2015/2016 
under P 20-
204 
 
[7] 
 
Up-scaled 
lab-
produced 
R0.6C 
 
Perform dose-
titration studies to 
demonstrate that 
up-scaled R0.6C 
product elicit TB 
antibodies. 
 
2.5, 10, 25µg of 
R0.6C 
adjuvanted in 
Montanide 
ISA720, 3 SC 
injections on D0, 
D21, and D42. 
Terminal bleed 
on D63. 
 
Doses are within the 
suspected range for 
potency evaluation. 
Pfs48/45-6C 
antibody titers are 
associated with 
TRA. 
 
Mice 
 
Radboud 
UMC under 
approval 
number 
2016-0020 
 
Up-scaled 
R0.6C 
reference 
material 
 
Perform two 
studies within 
suspected dose 
ranges and at the 
intended route of 
administration to 
confirm 
immunogenicity of 
DP formulations. 
 
0.4, 2, 10µg of 
R0.6C 
adjuvanted on 
Alhydrogel or 
Alhydrogel + 
Matrix M, 2 IM 
injections on D0, 
and D28. 
Terminal bleed 
on D63. 
 
Both R0.6C/AlOH 
and R0.6C/AlOH + 
Matrix M elicit high 
levels of functional 
antibodies when 
injected twice by the 
intended route of 
immunization. 

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Rabbits 
 
CRL 
Study no. 
78813 
 
Tox Grade 
Material 
 
Toxicity of repeated 
dose of R0.6C/AlOH 
and R0.6C/AlOH + 
Matrix-M 
 
 
A total of 4 doses by 
IM, with 100 µg 
R0.6 with or without 
50 µg Matrix-M  
No systemic 
toxicological changes. 
Local test item related 
histopathology 
changes were 
observed at the 
injection sites and 
consisted of an up to 
marked focal 
inflammatory response. 
Partial recovery was 
recorded when 
comparing the site 
dosed 3 days prior to 
necropsy with the site 
dosed 46 days prior to 
necropsy. 
 
7.3 Summary of findings from clinical studies 
There is no past human experiences with the R0.6C/AlOH and R0.6C/AlOH + Matrix-M 
vaccines. However, the GLURP-R0 region, which constitute 76.7% of R0.6C, forms part of 
the malaria vaccine candidate, GMZ2, which has been tested in both European and African 
clinical trials listed in Table 3. Notably, GMZ2, adjuvanted with aluminum hydroxide 
(GMZ2/AlOH) was tested in 880 children 12-60 month of age in Burkina Faso (Banfora; 
n=580, Sapone; n=300). 
To date 994 individuals, of whom the majority are African children below 5 years of age, have 
been immunized with GMZ2/AlOH. The GMZ2/AlOH formulation has shown excellent safety 
and tolerability confirming the safety of the expression system and the safety of the GLURP-
R0 portion of R0.6C. 
 
Table 3: Clinical experience of GMZ2/AlOH.  
Trial 
registration 
number 
(Reference) 
Title 
Interventions 
Charact
eristics 
Population 
NCT00397449 
[14] 
Safety and 
immunogenicity of 
GMZ2 - a MSP3-
GLURP fusion protein
malaria vaccine 
candidate 
Biological: 
• 
10µ 
GMZ2/AlOH 
• 
30µ 
GMZ2/AlOH 
• 
100µg 
GMZ2/AlOH 
Phase 
1a 
30 healthy malaria-
naïve German adults; 
10 / group 
NCT00424944 
[14] 
Safety and 
immunogenicity of the 
malaria vaccine 
candidate GMZ2 in 
malaria-exposed, 
Biological: 
• 
100µg 
GMZ2/AlOH 
• 
VerorabTM 
Phase 
1b 
40 healthy malaria-
exposed Gabonese 
adults; 20 / group 

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adult individuals from 
Lambaréné, Gabon 
NCT00703066 
[15] 
A Randomized 
Controlled Phase Ib 
Trial of the Malaria 
Vaccine Candidate 
GMZ2 in African 
Children 
Biological: 
• 
30µ 
GMZ2/AlOH 
• 
100µg 
GMZ2/AlOH 
• 
VerorabTM 
Phase 
1b 
30 healthy malaria-
exposed Gabonese 
children one to five 
years of age; 10 / 
group 
PACTR201006
0002033537 
[16] 
A phase 2b 
randomized, 
controlled trial of the 
efficacy of the GMZ2 
malaria vaccine in 
African children 
Biological: 
• 
100µg 
GMZ2/AlOH 
• 
VerorabTM 
Phase 
2b 
1735 healthy malaria-
exposed Gabonese 
children received 
three doses of 
vaccine (868 GMZ2, 
867 control-vaccine). 
 
The Matrix-M adjuvant technology [17] is a promising technology which has been explored 
for various infectious diseases and has a good safety profile in humans [18].  To-date Matrix-
M has been utilized in more than 25+ clinical trials, including multiple Phase III trials (Table 
4). In these studies, Matrix-M has been combined with multiple malaria vaccine candidates, 
influenza, covid-19, and other disease indications. The most common dosage of Matrix-M 
has been 50 µg in adults, with a maximum dosage of 75 µg.  Further, Matrix-M is currently 
being evaluated with another malaria vaccine candidates (R21) In Burkina Faso under Phase 
I and Phase II clinical studies (NCT02925403) in children. There the tested dosages of 
Matrix-M are 25 and 50 µg. 
In one recent example (NCT01444482), Matrix-M adjuvant has also been tested in a 
randomized, observer-blinded, active-comparator controlled trial during the 2019-2020 
influenza season (doi: https://doi.org/10.1101/2020.08.07.20170514). In brief, 2654 clinically-
stable, community-dwelling adults ≥65 years of age were randomized to receive a single 
intramuscular dose of either Matrix-M-adjuvanted quadrivalent nanoparticle influenza vaccine 
(qNIV) or a licensed inactivated influenza vaccine (IIV4). Local reactogenicity, primarily mild 
to moderate and transient pain, was higher in the qNIV group. Interpretation: qNIV was well 
tolerated and produced a qualitatively and quantitatively enhanced humoral and cellular 
immune response in older adults.  
 
 

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Table 4: Key clinical experience of Matrix-M with various vaccine antigens. 
 
   NCT 
Number 
Title 
Conditions 
Interventions 
Characteristics 
Population 
NCT04201431 
Safety, Immunogenicity 
and Efficacy of the 
Blood-stage 
Plasmodium Vivax 
Malaria Vaccine 
Candidate PvDBPII in 
Matrix M1 
Malaria, 
Vivax 
• Biological: 
PvDBPII/Matrix M1 
Phase 1 
Phase 2 
18 Years to 45 
Years (Adult) 
NCT01669512 
Adjuvanting Viral 
Vectored Malaria 
Vaccines With Matrix M 
Malaria 
• Biological: Low Dose 
Matrix M Regimen 
• Biological: Standard 
Dose Matrix M Regimen 
Phase 1 
18 Years to 50 
Years (Adult) 
NCT04130282 
VAC077: Safety and 
Immunogenicity of the 
Pfs25-IMX313/Matrix-M 
Vaccine 
Malaria 
• Biological: Pfs25-
IMX313/ Matrix-M1 
Phase 1 
18 Years to 45 
Years (Adult) 
NCT04318002 
Safety and 
Immunogenicity of 
RH5.1/ Matrix-M in 
Adults and Infants 
Living in Tanzania 
Malaria 
• Biological: 
RH5.1/Matrix-M 
Phase 1 
6 Months to 45 
Years (Child, 
Adult) 
NCT03896724 
Safety, Immunogenicity 
and Efficacy of R21 
Matrix-M in 5-17 Month 
Old Children in Nanoro, 
Burkina Faso 
Malaria 
• Biological: R21 
adjuvanted with 25mcg 
Matrix-M 
• Biological: R21 
adjuvanted with 50mcg 
Matrix-M 
Phase 1 
Phase 2 
5 Months to 17 
Months (Child) 
NCT04271306 
Safety, Immunogenicity 
and ex Vivo Efficacy of 
Pfs25-IMX313/Matrix-M 
in Healthy Volunteers in 
Bagamoyo, Tanzania. 
Malaria 
• Biological: Pfs25-
IMX313 (10ug)/Matrix-M 
(50ug) 
• Biological: Pfs25-
IMX313 (50ug)/Matrix-M 
(50ug) 
• Biological: Pfs25-
IMX313 (50ug)/Matrix-M 
(50ug) 
& Pfs25-IMX313 (10ug)/ 
Matrix-M (50ug) 
Phase 1 
5 Years to 45 
Years (Child, 
Adult) 
NCT02572388 
A Study to Assess the 
Safety and 
Immunogenicity of the 
Malaria Vaccine, R21, 
Administered With and 
Without Matrix-M1 
Malaria 
• Biological: R21 
• Biological: Matrix-M1 
Phase 1 
18 Years to 50 
Years (Adult) 
NCT02925403 
A Study to Assess the 
Safety and 
Immunogenicity of the 
Malaria Vaccine, R21, 
With Matrix-M1 
Adjuvant 
Malaria 
• Biological: R21/Matrix-
M1 
• Other: Saline 
Phase 1 
Phase 2 
18 Years to 45 
Years (Adult) 
NCT01444482 
Study of Parenterally 
Administrated 
Adjuvanted Seasonal 
Influenza Vaccine in 
Healthy Elderly 
Volunteers 
Influenza 
• Biological: Matrix M 
• Biological: Seasonal 
influenza vaccine 
Phase 1 
65 Years to 75 
Years (Older 
Adult) 

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   NCT 
Number 
Title 
Conditions 
Interventions 
Characteristics 
Population 
NCT04611802 
A Study Looking at the 
Efficacy, Immune 
Response, and Safety 
of a COVID-19 Vaccine 
in Adults at Risk for 
SARS-CoV-2 
COVID-19 
• Biological: SARS-CoV-2 
rS/ Matrix-M1 Adjuvant 
• Other: Placebo 
Phase 3 
18 Years and 
older (Adult, 
Older Adult) 
NCT04368988 
Evaluation of the Safety 
and Immunogenicity of 
a SARS-CoV-2 rS 
Nanoparticle Vaccine 
With/Without Matrix-M 
Adjuvant 
COVID-19 
• Biological: SARS-CoV-2 
rS - Phase 1 
• Biological: SARS-CoV-2 
rS/ Matrix-M Adjuvant, 
Days 0 and 21 - Phase 2 
• And more 
Phase 1 
Phase 2 
18 Years to 84 
Years (Adult) 
NCT04533399 
A Study Looking at the 
Effectiveness and 
Safety of a COVID-19 
Vaccine in South 
African Adults 
COVID-19 
• Biological: SARS-CoV-2 
rS/ Matrix-M1 Adjuvant 
• Other: Placebo 
Phase 2 
18 Years to 84 
Years (Adult) 
NCT03580824 
A Study to Determine if 
a New Malaria Vaccine 
is Safe and Induces 
Immunity Among 
Kenyan Adults, Young 
Children and Infants 
Malaria 
• Biological: R21 in 
Matrix- M adjuvant 
vaccine 
Phase 1 
Phase 2 
5 Months to 45 
Years (Child, 
Adult) 
NCT04583995 
A Study Looking at the 
Effectiveness, Immune 
Response, and Safety 
of a COVID-19 Vaccine 
in Adults in the United 
Kingdom 
COVID-19 
• Biological: SARS-CoV-2 
rS/ Matrix M1-Adjuvant 
• Other: Placebo 
• Biological: Licensed 
seasonal influenza 
vaccine 
Phase 3 
18 Years to 84 
Years (Adult, 
Older Adult) 
NCT02078674 
A(H7N9) VLP Antigen 
Dose-Ranging Study 
With Matrix-M1™ 
Adjuvant 
Influenza 
(Pandemic) 
• Biological: Monovalent 
Avian Influenza VLP 
(H7N9) 
• Biological: Matrix-M1™ 
adjuvant 
Phase 1 
Phase 2 
18 Years to 64 
Years (Adult) 
NCT02300142 
Rollover Trial for 
Placebo Subjects 
Previously Enrolled Into 
GEN-003-002 Study 
Genital 
Herpes 
• Biological: GEN-003 
Vaccine (30-60µg of each 
antigen) 
• Biological: Matrix-M2 
Adjuvant (25-75µg) 
Phase 2 
18 Years to 50 
Years (Adult) 
NCT01667341 
Safety and 
Immunogenicity Study 
of Therapeutic HSV-2 
Vaccine 
Genital 
Herpes 
• Biological: GEN-003 
with Matrix M-2 
• Biological: GEN-003 
Phase 1 
Phase 2 
18 Years to 50 
Years (Adult) 
NCT03026348 
Safety and 
Immunogenicity Study 
to Evaluate Single- or 
Two-Dose Regimens Of 
RSV F Vaccine With 
and Without Aluminum 
Phosphate or Matrix-
M1™ Adjuvants In 
Clinically-Stable Older 
Adults 
Respiratory 
Syncytial 
Viruses 
• Biological: RSV F 
Vaccine with Aluminum 
Phosphate Adjuvant 
• Biological: RSV F 
Vaccine 
• Biological: Matrix-M1 
Adjuvant 
Phase 2 
60 Years to 80 
Years (Adult, 
Older Adult) 
NCT02114060 
Dose Ranging Safety 
and Efficacy of 
Therapeutic HSV-2 
Vaccine 
Genital 
Herpes  
• Biological: GEN-003 
Vaccine (30µg of each 
antigen) 
• Biological: Matrix-M2 
Adjuvant (75µg) 
• And more 
Phase 2 
18 Years to 50 
Years (Adult) 

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   NCT 
Number 
Title 
Conditions 
Interventions 
Characteristics 
Population 
NCT03947190 
A Study to Determine if 
New Types of Malaria 
Vaccines Are Safe, 
Effective and Lead to 
Immunity in Kenyan 
Adults 
Malaria 
• Biological: R21/Matrix-M 
• Biological: ChAd63/MVA 
ME-TRAP 
• Biological: intradermal 
injection (ID) or direct 
venous injection (DVI) of 
PfSPZ Challenge 
Phase 2 
18 Years to 45 
Years (Adult) 
NCT03293498 
Evaluation of the Safety 
and Immunogenicity of 
a Recombinant 
Trivalent Nanoparticle 
Influenza Vaccine With 
Matrix M-1 Adjuvant 
(NanoFlu) 
Influenza 
• Biological: NanoFlu 
• Biological: Fluzone HD - 
Day 0 
• Biological: Fluzone HD - 
Day 21 
• Other: Saline - Day 21 
Phase 1 
Phase 2 
60 Years and 
older (Adult, 
Older Adult) 
NCT03658629 
Phase 2 Dose and 
Formulation 
Confirmation of Quad-
NIV in Older Adults 
Influenza, 
Human 
• Biological: NanoFlu 
(Quad- NIV) 
• Other: Matrix-M 
Adjuvant 
• Biological: Fluzone HD 
• Biological:Flublok 
Phase 2 
65 Years and 
older (Older 
Adult) 
NCT03970993 
VAC 072-An Efficacy 
Study of R21/MM in 
Different Dose 
Schedules 
Malaria 
• Biological: R21 Matrix-M 
vaccination 
• Biological: R21 Matrix-M 
vaccination and CHMI 
Phase 1 
Phase 2 
18 Years to 45 
Years (Adult) 
NCT02370589 
Study to Evaluate the 
Immunogenicity and 
Safety of an Ebola Virus 
(EBOV) Glycoprotein 
(GP) Vaccine in Healthy 
Subjects 
Ebola 
• Biological: Base Dose 
EBOV GP Vaccine 
• Biological: 2-8x Base 
Dose EBOV GP Vaccine 
• Biological: Matrix-M 
Adjuvant 
Phase 1 
18 Years to 50 
Years (Adult) 
NCT02515175 
Evaluating New 
Formulation of 
Therapeutic HSV-2 
Vaccine 
Genital 
Herpes  
• Biological: Matrix-M2 
• Biological: GEN-003 
Phase 2 
18 Years to 50 
Years (Adult) 
NCT04120194 
Phase 3 Pivotal Trial of 
NanoFlu™ in Older 
Adults 
Influenza, 
Human 
• Biological: NanoFlu 
• Biological: Fluzone 
Quadrivalent 
Phase 3 
65 Years and 
older (Older 
Adult) 
NCT04645147 
Safety and 
Immunogenicity of an 
Epstein-Barr Virus 
(EBV) gp350-Ferritin 
Nanoparticle Vaccine in 
Healthy Adults With or 
Without EBV Infection 
EBV 
• Biological: EBV gp350- 
Ferritin Vaccine 
• Other: Matrix-M1  
Phase 1 
18 Years to 29 
Years (Adult) 
NCT03146403 
Maintenance Dose 
Study of GEN-003 in 
Subjects With Genital 
Herpes Infection 
Genital 
Herpes 
• Biological: GEN-003 
• Biological: Matrix-M 
Phase 2 
Child, Adult, 
Older Adult 
NCT02905019 
A Safety and Efficacy 
Study of R21 +/- 
ChAd63/MVA ME-
TRAP 
Malaria 
• Biological: R21 with 
Matrix- M1 
• Biological: ChAd63 ME-
TRAP 
• Biological: MVA ME-
TRAP 
Phase 1 
Phase 2 
18 Years to 45 
Years (Adult) 
 
 
 

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7.4 Summary of known and potential risks and benefits 
7.4.1 
Risk Assessment 
The following section (Table 5) outlines the initial risk assessment and mitigation strategy 
for this study protocol. 
 
Table 5. Risk Assessment and mitigation measures 
Important 
potential/identified risk 
Data/rationale for risk 
Mitigation strategy 
Investigational study product (R0.6C vaccine) 
Important potential risk: 
First-in-human unknown 
risk 
There is no prior experience of R0.6C 
vaccine in human subjects. 
Because of lack of in human experience, 
the vaccine will be administered with a 
staggered dose escalation step of two 
dosages (30 µg and 100 µg). Subjects 
will be observed closely by qualified 
clinicians, and emergency care will be 
immediately available to subjects. A 
minimum of 14 days is required 
between product administration to 
each group. Dose-escalation will be 
stopped if any of the safety holding 
criteria are met (section 9.5.5).  
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 included. All vaccinated 
volunteers will be observed closely for at 
least 1 hour following administration of 
R0.6C with appropriate medical 
treatment readily available in case of 
severe adverse events. 
Local and systemic 
reactogenicity to 
intramuscular 
administration of R0.6C 
Generally, risks associated with 
vaccination include local 
inflammatory reactions to the injected 
product, such as pain and swelling at 
the injection site. Up to marked focal 
histopathological inflammation was 
reported in the animal toxicity 
studies, with partial recovery in 
recovery animals. Systemic effects 
generally associated with vaccination 
may include flu-like symptoms, fever, 
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 will be advised 
to inform or call the study doctor 
immediately if they experience any 
adverse events. Appropriate medication 
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chills, nausea, gastrointestinal 
symptoms, headache, malaise, 
myalgia and arthralgia. No significant 
systemic adverse effects were 
observed in repeat dose animal 
toxicity studies. 
symptomatic relief of any untoward side 
effects may be advised as necessary. 
Study procedures 
Pain when taking blood 
samples 
Because of the necessity to obtain 
frequent blood sampling for 
laboratory safety analysis, 
determining the concentration of 
R0.6C-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 taken over the whole 
study period will not exceed 500 mL. 
Pregnancy risks 
Pregnancy and lactating 
females 
Risks of the R0.6C 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 and the pregnancy test at 
the inclusion visit is negative. In case 
effective contraceptives are not 
available the clinical team may provide 
them. Volunteer who becomes pregnant 
during the trial will be withdrawn from 
any subsequent investigational product 
administration. Pregnant volunteers will 
not be withdrawn from the trial. 
Conditional on their agreement, they will 
be followed 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 

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Needlestick 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. 
 
7.4.2 
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 is not protective against malaria. Subjects may 
indirectly benefit from general medical evaluation and health screening procedures 
including testing for HIV, hepatitis B, and hepatitis C. Subject 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. Subjects will receive a financial compensation which is 
reasonable and in line with Dutch common practice (see below). 
 
7.5 Description and justification of route of administration and dosage 
Enrolled participants will receive four intra-muscular injections of the vaccine in the deltoid 
muscle, on alternating sides. The administration site minimizes the risk that the product is 
injected 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.  
Each volunteer will receive four equal doses of the vaccine on days 0, 28, 56, and 168.The 
trial is designed to evaluate the safety and tolerability in with a staggered dose escalation 
step of two dosages (30 µg and 100 µg). The interval of 4 weeks between immunizations 1, 2 
and 3 is the most commonly used interval between malaria vaccine administrations, but in 
this trial also chosen to align with the RTS,S vaccination scheme. An additional fourth 
vaccination is given on day 168 as a booster, as promising effects of this fourth, delayed 
dose were seen in a trial with the Pfs25 transmission blocking vaccine candidate[19]. The 
low dose in this protocol is based on pre-clinical data (see above), and similar to starting 
concentrations in other malaria protein vaccine trials[20, 21]. 
 
7.6 Dose preparation and administration 
Dose preparation, as described in the Pharmacy Manual, will be carried out by qualified 
personnel. The investigational product will be administered by intramuscular injection in the 
deltoid muscle in 2 different doses, 30µg R0.6C for groups 1 and 2, and 100µg R0.6C for 
groups 3 and 4. R0.6C is formulated pre-adsorbed to Alhydrogel (100ug R0.6C per  500uL of 

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[200µg R0.6C/mL, 1.6 mg Al(OH)3/mL). Each vial contains 0.8 mL vaccine. Matrix-M is 
formulated as 750 µL per vial at a concentration of 375 µg/mL. 
In groups A, a volume of 150µL of R0.6C/Alhydrogel will be aspired from the vial for the dose 
of 30µg R0.6C (groups 1A+2A) and a volume of 500µL will aspired for the dose of 100µg 
R0.6C (groups 3A+4A). 
For groups B a volume of 210 µL Matrix-M will be added to each vial containing 800 µL of 
R0.6C/Alhydrogel and mixed repeated by inversion by hand 10 times. Then 190 µL of 
R0.6C/Alhydrogel + Matrix-M formulation will be aspired for administration of the 30µg dose 
(groups 1B+2B). For the 100µg dose groups (groups 3B+4B) 630µL of R0.6C/Alhydrogel + 
Matrix-M formulation will be aspired for administration. Receipt and disposal of the 
investigational products will be done according to RUMC SOPs. All vials containing the study 
product R0.6C/Alhydrogel and Matrix-M, will be stored at 2-8°C, and storage temperatures 
will be monitored with validated temperature monitoring devices.  
 
7.7 Preparation and labelling of Investigational Medicinal Product 
Preparation and labelling of the investigational medicinal products will be done according to 
the relevant GMP guidelines. After the products are QP-released there will be no 
manufacturing steps. Dose preparation is described in the Pharmacy Manual. 
 
7.8 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 and an accurate record of the inventory, and an 
accountability record of R0.6C supplies for this study. The site pharmacist will also ensure 
the security of these documents. Partially used vials will not be used for human 
administration or for in vitro experimental studies. Unused or open products will be either 
returned to the sponsor or designee at the end of the vaccination period or destroyed 
according to the sponsor’s instructions. 
 
8. METHODS 
8.1 Study endpoints 
Primary safety Endpoints: 
1) The number of serious adverse events and solicited and unsolicited grade 3 adverse 
events possibly, probably or definitely related to the vaccine in the period from first 
R0.6C administration up to 84 days after the last immunization.  
Primary efficacy endpoints: 
1) The functional transmission reducing activity in the standard membrane feeding 
assay of volunteer sera collected two weeks after the fourth R0.6C immunization 
(I4+14), compared to baseline (I1-1)  within each of the four dose-adjuvant groups.  
 

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Secondary safety endpoints: 
1) The number of solicited and unsolicited grade 1 and 2  adverse events possibly, 
probably or definitely related to the vaccine in the period from first R0.6C 
administration up to 84 days after the last immunization.  
Secondary efficacy endpoints: 
1) The TRA at other timepoints (I1+14, I2+14, I3+14, I3+111 [I4-1], and I4+84) 
compared to baseline (I1-1) in each of the four dose-adjuvant groups. 
2) The anti-6C antibody quantity in volunteer sera collected two weeks after fourth 
R0.6C immunization (I4+14) and at other time points (I1+14, I2+14, I3+14, I3+111 [I4-
1], and I4+84) compared to baseline (I1-1) in each of the four dose-adjuvant 
combinations, as determined by ELISA. 
 
Exploratory Endpoints: 
1) The functional TBA in the SMFA of volunteer sera collected at different time points 
compared to baseline (I1-1) in each of the four dose-adjuvant combinations.  
2) The anti-6C antibody decay rate following R0.6C immunization for each of the four 
dose-adjuvant combinations. 
3) The anti R0.6C and anti-R0 antibody quantity in volunteer sera collected at different 
time points in each of the four dose-adjuvant combinations. 
4) The cellular immune responses in volunteers samples collected at different time 
points in each of the four dose-adjuvant combinations. 
5) The difference in TRA between the two dose groups (30 or 100µg R0.6C) and/or 
between adjuvant groups (alhydrogel and alhydrogel+Matrix-M). 
6) The difference in peak anti-6C antibody quantity between the two dose groups (30 or 
100µg R0.6C) and/or between adjuvant groups (alhydrogel and alhydrogel+Matrix-M). 
 
 
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. Subjects may choose in which of the dose groups (group 1-4) they wish 
to be enrolled. The time of notification and treatment group will be documented in the 
electronic case report form (eCRF). 
Randomization will be used only for the allocation to one of the two different adjuvant 
combinations (arms A or B). The volunteers will be allocated to one of the two adjuvant arms 
per group at random using a Mersenne-Twister random number generator implemented in R. 
Randomization tables and randomization cards will be generated using the package 
“blockrand”. Stratification will not be performed. An independent investigator at the 
Radboudumc, not involved in the clinical trial, is responsible for performing the randomization 
and for assigning volunteers according to the randomization cards. A second employee, not 

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involved in the assignment of volunteers, will check to see if randomization is done correctly. 
The randomization cards are kept in a fireproof clinical trial cabinet at Radboudumc. 
 
8.3 Study procedures 
8.3.1 
Screening visit  
The screening appointment for individual subjects is planned at least 72 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 to answer 
any questions the subjects may have, prior to obtaining informed consent and determining 
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 measure vital parameters. 
Upon arrival for the screening visit, the volunteer is asked to fill out a medical questionnaire. 
Subsequently, and prior to any 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 subject will 
be required to pass a (short) written quiz about the study in order to ensure the study 
subjects has sufficient understanding of what participation entails. 
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 an 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 screening. If certain elements of the screening procedures already indicate that 
a subject is ineligible for inclusion, screening procedures do not necessarily all have to be 
completed. The below activities will occur during the screening: 
- 
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; 
- 
Blood specimens will be collected for routine clinical laboratory testing of biochemical 
and hematological parameters, 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; 
- 
Consent to inform the general practitioner (and if necessary a medical specialist) of 
study participation and request relevant information on medical history, will be signed 

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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 in 
person or 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. 
8.3.2 
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 in 
before the screening. 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; 
- 
Recent receipt of vaccinations 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 sterility.  
 
8.3.3 
Physical examination 
A complete physical examination will include the examination of general appearance, skin, 
neck, eyes, throat, lungs, heart, abdomen, back and extremities, and a routine vascular and 
neurological examination. Height (cm) and body weight (to the nearest 0.1 kilogram [kg] in 
indoor clothing, but without shoes) will also be measured, at screening only. Body mass 
index (BMI) will be calculated using the formula: BMI = Body weight (kg) / [Height (m)]2 and 
converted to an integer. During follow-up visits a focused physical examination will be 
performed if deemed necessary by the trial physician. 

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8.3.4 
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 and 
inclusion 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 back supported 
and both feet placed on the floor, using an automated validated device, with an appropriately 
sized cuff. In case the cuff sizes available are not large enough for the subject's arm 
circumference, or in case of doubt of accuracy of the automated device a 
sphygmomanometer with an appropriately sized cuff may be used. 
If vital signs are out-of-range at screening or inclusion, 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. 
Temperature, blood pressure and pulse are measured as part of the physical examination at 
screening and inclusion. Additionally, subjects will be given a thermometer to measure their 
oral temperature on a daily basis for two weeks after each R0.6C administration, which they 
will collect in their study diaries. 
 
8.3.5 
Inclusion visit 
Subjects meeting the eligibility criteria during screening (section 5.2 and 5.3) will be invited 
back for enrolment into the study at the inclusion visit, which will occur prior to the planned 
first administration. Baseline assessments will be taken on the inclusion day. For each 
subject, study start (day 0) will be defined as the day of first R0.6C administration. For 
subjects that do not show up for the inclusion visit, or subjects that do not continue to meet 
the eligibility criteria, alternate subjects may take their place. At study inclusion, the following 
activities will occur: 
- 
Patient history will be repeated. Only subjects who still meet the inclusion criteria 
will be included to receive R0.6C vaccination; 
- 
Blood specimens will be collected for routine clinical laboratory testing of 
biochemical and hematological parameters; 
- 
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 symptom diaries and a thermometer to record any local 
and systemic symptoms and medication use; 
- 
All subjects will be issued an emergency notification card that details their 
participation in the study and provides contact phone numbers of the investigators. 
 

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8.3.6 
Administration of R0.6C TBV candidate 
Each enrolled subject will receive a total of four R0.6C vaccinations. The first vaccination will 
take place on day 0, the second on day 28 (+/- 3 days), the third on day 56 (+/- 3 days) and 
the last vaccination on day 168 (+/- 7 days). Subjects will be briefly assessed regarding any 
new medical events since the inclusion visit. The R0.6C vaccination will be prepared at bed-
side by study staff (nurse,  study physician) or by the pharmacy according to the study arm; 
arms A will receive R0.6C with Alhydrogel alone, arms B will receive R0.6C with Alhydrogel 
and Matrix-M. R0.6C will be administered intramuscular in the deltoid muscle on alternating 
sides. The site of injection will be recorded on the case report form. Subjects will be 
observed for at least 1 hour after R0.6C administration. A list of solicited local and systemic 
symptoms will be reviewed after this observation period. If the occurrence of an adverse 
event or use of medication is confirmed by the study physician, it is recorded in the eCRF. 
After leaving the hospital, subjects will be asked to examine the site of infusion and record 
local signs and symptoms for seven days including bruising, erythema, swelling or induration. 
Subjects will be asked to record any AEs in the memory aid booklet (also see section 8.3.9).  
 
8.3.7 
Follow up after R0.6C administration 
Follow-up visits will be carried out on an outpatient basis at the study center. Subject will be 
asked to record local signs and symptoms in their memory aid booklet (also see section 
8.3.9). The subject diary will be reviewed at each study visit and used as a base for 
discussion of possible local and systemic adverse events or medication use. In such cases, it 
may not be possible to obtain blood samples for non-safety endpoints. 
If clinically significant (laboratory) abnormalities are 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. The trial physician can decide to initiate any 
additional diagnostics (including safety laboratory evaluations) at all times. For unexpected 
laboratory abnormalities, the laboratory test will be repeated.  
8.3.8 
Unscheduled visits 
Subjects may need to present to the study center during operating hours 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. 
8.3.9 
Patient reported outcomes (study diary) 
At the inclusion visit, subjects will be issued symptom diaries. They will be asked to record all 
symptoms and medication use from the day of inclusion until end of study. Study staff will 
train subjects on use of memory aid booklets for self-assessment of solicited local and 
general adverse events through 7 days (day of R0.6C administration and subsequent 6 
days) post- administration of R0.6C; and for self-assessment of adverse events and 
concomitant medications until day 84 after the last R0.6C administration. 

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The subject diary will be reviewed at each study visit and used as a basis for discussion of 
possible adverse events or medication use If the occurrence of an adverse event or use of 
medication is confirmed by the study physician, it is recorded in the subject’s EPD and 
eCRF. At the end of the study, the diary will be collected and kept as source data. 
8.3.10 Blood sampling and safety laboratory evaluations 
During the study, blood samples will be drawn for screening, safety and research purposes. 
The blood sampling schedule in the flowchart (section 8.3.17) shows the maximum amounts 
of blood that will be drawn. 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 cumulative blood draw for each subject over the entire course of study participation is 
maximally 500mL. 
Biological safety parameters will be measured at the central laboratory of the RUMC. In the 
case where 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 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. 
Hemoglobin, hematocrit, red blood cell count, white blood cell count with differential (e.g. 
neutrophils, basophils, eosinophils, monocytes, lymphocytes) and platelet count will be 
measured at regular time points during the study. Alkaline phosphatase, total bilirubin, 
gamma-glutamyltransferase (γGT), lactate dehydrogenase (LDH), alanine aminotransferase 
(ALT), aspartate aminotransferase (AST), sodium, potassium, creatinine and urea will be 
measured at regular time points during the study. Glucose, triglycerides and cholesterol will 
be measured only at screening. 
 
8.3.11 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 screening and at baseline. A positive test for amphetamines and cocaine is 
a reason for exclusion. A positive test for cannabis on the inclusion day prior to R0.6C 
administration will also be a reason for exclusion. 
8.3.12 Pregnancy Test 
A midstream urine sample will be obtained and assessed by commercially available hCG 
urine tests. This test will be done at screening and inclusion. 
8.3.13 Analysis of transmission reducing activity 
Samples for SMFA will be collected from all subjects. Sera and/or purified IgG prepared 
from each serum will be used in the SMFA. Samples will be frozen until SMFA is planned. 
SMFA assays will be performed at Radboudumc (The Netherlands), using a qualified 

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standard membrane feeding assays using serum from subjects in cultures with P. 
falciparum and Anopheles stephensi mosquitoes. 
8.3.14 Immunological assays: ELISAs/ PBMCs  collection 
Blood samples will be taken for isolation of peripheral blood mononuclear cells (PBMCs) and 
serum (see chart 8.3.17). PBMCs and sera will be frozen and can then be used by the 
Radboudumc or its collaborators for exploratory immunological assays to further analyse 
functionality of the immune response during and after R0.6C vaccinations. An ELISA assay 
will be used to measure circulating anti-R0.6C antibody titres in the subjects’ sera at XX time 
points as shown in section 8.3.17, (sub)class and functionality (e.g. complement binding) 
may also be determined. In order to assess (antigen specific) T cell responses, the HLA-type 
of volunteers may be determined. 
Samples that are not used in aforementioned immunological and functional assays will be 
stored up to 15 years and may be used for future (immunological) research relevant in the 
field of malaria vaccination.  
8.3.15 Case report forms and data collection 
All data collected by the investigators is registered in electronic case report forms. The 
investigator’s notes are collected per subject in the coded trial EPD and are considered 
source data. Since all subjects will be healthy, there is no personal 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 
source data. The diaries, produced by the study volunteers are also considered source data 
and will be kept as source documents. 
 
8.3.16 COVID-19 related measures 
With regards to the COVID-19 pandemic, the current Radboudumc guidelines will be 
followed. During the study period subjects will be instructed to call the trial physicians at any 
time if they experience severe symptoms or symptoms possibly related to COVID-19. SARS-
CoV2 testing will be performed in accordance with current Radboudumc-wide guidelines. 
Follow-up visits not requiring blood collection for safety endpoints may be carried out by 
telephone, or at home, instead of at the clinical research center for subjects with suspected 
or confirmed SARS-CoV2 infection, until such time as they are considered no longer 
(potentially) contagious in accordance with current Radboudumc-wide guidelines. 
Vaccinations against the novel coronavirus SARS-CoV2 should preferably not take place 
within 30 days prior to or 14 days after each R0.6C vaccination.

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8.3.17 Schedule of trial visits and measurements 
 
Study Day1 
I-
120 
to 
I1 -1 
I1-1 
I1 
I1+
1 
I1+
2 
I1+
7 
I1+ 
14 
I2-1 
I2 
I2+
1 
I2+
2 
I2+
7 
I2+ 
14 
I3-1 
I3 
I3+
1 
I3+
2 
I3+
7 
I3+ 
14 
I3+ 
28 
I3+ 
56 
I4-1 
I4 
I4+
1 
I4+
2 
I4+
7 
I4+ 
14 
I4+ 
28 
I4+ 
56 
I4+ 
84 
Days after first R0.6C 
administration 
N/A N/A 
0 
1 
2 
7 
14 
27 
28 
29 
30 
35 
42 
55 
56 
57 
58 
63 
70 
84 
112 167 168 169 170 175 182 196 224 252 
Visit No. 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
25 
26 
27 
28 
29 
30 
Informed consent 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion 
criteria 
● 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographic data and 
medical history 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
●2 
Height and weight 
measurement 
●2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood pressure, 
temperature, pulse3 
● 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
●3 
Record concomitant 
medication 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
HIV, HBV, HCV  
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test (urine) 
● 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Drugs screening (urine) 
● 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Study Day1 
I-
120 
to 
I1 -1 
I1-1 
I1 
I1+
1 
I1+
2 
I1+
7 
I1+ 
14 
I2-1 
I2 
I2+
1 
I2+
2 
I2+
7 
I2+ 
14 
I3-1 
I3 
I3+
1 
I3+
2 
I3+
7 
I3+ 
14 
I3+ 
28 
I3+ 
56 
I4-1 
I4 
I4+
1 
I4+
2 
I4+
7 
I4+ 
14 
I4+ 
28 
I4+ 
56 
I4+ 
84 
Distribution of 
emergency card 
 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Distribution of diary 
 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
R0.6C administration 
 
 
● 
 
 
 
 
 
● 
 
 
 
 
 
● 
 
 
 
 
 
 
 
● 
 
 
 
 
 
 
 
Solicited local AEs 
(days 0–7) 
 
 
● 
● 
● 
● 
 
 
● 
● 
● 
● 
 
 
● 
● 
● 
● 
 
 
 
 
● 
● 
● 
● 
 
 
 
 
Solicited general AEs 
(days 0-14) 
 
 
● 
● 
● 
● 
● 
 
● 
● 
● 
● 
● 
 
● 
● 
● 
● 
● 
 
 
 
● 
● 
● 
● 
● 
 
 
 
Unsolicited AEs (days 
0-84) 
 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
Complete blood count4 
● 
● 
 
● 
● 
● 
● 
● 
 
● 
● 
● 
● 
● 
 
● 
● 
● 
● 
● 
 
● 
 
● 
● 
● 
● 
● 
 
● 
Biochemistry tests5  
● 
● 
 
● 
● 
● 
● 
● 
 
● 
● 
● 
● 
● 
 
● 
● 
● 
● 
● 
 
● 
 
● 
● 
● 
● 
● 
 
● 
PBMC isolations and 
citrate plasma 
 
● 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
● 
 
 
 
 
 
 
 
● 
 
 
 
Serology/ELISA 
 
● 
 
 
 
 
● 
 
 
 
 
 
● 
 
 
 
 
 
● 
 
●7 
● 
 
 
 
 
● 
 
●7 
● 
SMFA8  
 
● 
 
 
 
 
● 
 
 
 
 
 
● 
 
 
 
 
 
● 
 
 
● 
 
 
 
 
● 
 
 
● 
Safety Report9 
 
 
 
 
 
●8 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
●8 
End of study visit 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
● 
Daily blood volume (ml) 
12 
63 
0 
5 
5 
5 
31 
5 
0 
5 
5 
5 
15 
5 
0 
5 
5 
5 
31 
5 
4 
31 
0 
5 
5 
5 
47 
5 
4 
31 
Cumulative blood 
volume (ml) 
12 
75 
75 
80 
85 
90 
121 126 126 131 136 141 156 161 161 166 171 176 207 212 216 247 247 252 257 262 309 314 318 349 
1. 
Study days are represented as relative to the immunisation day. If an immunisation day takes place on a different day (+/-3 days is allowed for immunisation 2 and 3, +/-7 days is allowed for 
immunisation 4) the study visits related to that immunisation will shift accordingly. Moreover, following any given immunisation, for visits I#+7,I#+14 and I#+28 a relative window of +/-2 days is 

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allowed, for visits I#+56 and I#+84, a relative window of +/-10 days is allowed. 
2. 
A physical examination including height and weight and vital parameters will be performed at the screening visit. On other visits, measurements may take place at the discretion of the physician. 
3. 
Vital signs including body temperature, blood pressure and pulse will be recorded at the screening and inclusion visit. On other visits, measurem ents may take place at the discretion of the 
physician. 
4. 
CBC test includes: hemoglobin, hematocrit, platelets, red blood cell count, MCV, white blood cell count + differentiation 
5. 
Biochemistry test includes: creatinine, urea, sodium, potassium, bilirubin, AF, yGT, AST, ALT and LDH. Additional at screening: serology malaria (2mL), cholesterol, triglyceride and glucose (2mL). 
6. 
For PBMC isolations and citrate plasma collection, timepoints I2+14, I4-1 and I4+84 are optional. 
7. 
For serum samples collected at timepoints I3+56 and I4+56, +/-10 days is allowed. 
8. 
SMFA assays will be performed with serum collected on minimally 3 time points (I1-1, I3+14 and I4+14). SMFA assays for other timepoints may not be performed if the results of I3+14 and I4+14 do 
not show significant transmission reduction compared to baseline (I1-1).   
9. 
Safety report: A safety report will be prepared by the clinical investigator before initiation of a next group (2, 3 and 4), minimally 7 days after the first R0.6C administration in the preceding group. A 
safety report of all the safety data is made after the last study visit of group 4.

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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 
penalty or loss of medical benefits. 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 serious adverse event; 
- 
Any adverse event 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. Topical steroids are allowed; 
- 
Immunoglobulin and/or any blood products administered during the study period; 
- 
Pregnancy; 
- 
Completely 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. 
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. 
- 
Determine if the subject has had any reaction or AE 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. 
- 
Collect blood for biochemical and hematological clinical laboratory parameters. 
- 
Review the subject's memory aid if it is still in use at the time of withdrawal. 
- 
Document the reason for premature withdrawal on the CRF. 

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8.5 Replacement of individual subjects after withdrawal 
If an assigned subject does not present on the day of first R0.6C administration or elects 
to withdraw the consent on the day of administration, one of the reserve subjects will 
replace the subject. After subjects have received R0.6C administration they 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 safety monitor 
- 
On advice of the Safety Monitoring Committee (SMC) 
- 
On advice of the clinical investigator 
- 
On advice of the METC 
 
The investigators, local safety monitor, SMC, METC or Sponsor may decide to put the study 
on hold based on adverse events, 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) 
Adverse events are defined as any undesirable experience occurring to a subject during 
the study, whether or not considered related to the investigational product. All adverse 
events reported spontaneously by the subject or observed by the investigator or his staff 
will be recorded. Abnormal laboratory findings (e.g. clinical chemistry or hematology) or 
other abnormal assessments that are judged by the investigator to be clinically significant 
will be recorded as AEs (or SAEs if they meet the definition). The investigator will 
exercise his or her medical and scientific judgment in deciding whether an abnormal 
laboratory finding or any other abnormal assessment is clinically significant. If there are 
any severe complaints, the volunteer will be evaluated immediately by a qualified clinician 
using the appropriate clinical assessments according to standard hospital care. 
 
9.2.2 
Adverse event data collection and recording 
Adverse events will be collected at all follow-up visits, using the memory aid booklets as a 
basis (see section 8.3.9), and whenever a subject reports signs or symptoms to the trial 
physician between visits.  
 
After administration with R0.6C the following systemic signs and symptoms will be solicited 
until 14 days after administration: 
Fever, headache, myalgia, fatigue, chills, and rash. The time points that these symptoms are 
solicited are indicated in section 8.3.17. 
 
After the administration of R0.6C the following local signs and symptoms will be solicited until 
7 days after administration: 
Pain, itching, swelling, induration and redness at injection site. The time points that these 
symptoms are solicited are indicated in section 8.3.17. 
 

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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 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) 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/reactions (ICD-10 terminology will be included in the eCRF) 
2. Duration (date and time of occurrence and end date and time) 
3. Intensity (see explanation directly below) 
4. Relationship with the intervention (see section 9.2.3) 
5. Action taken, including treatment 
 
The intensity of the symptoms will be ranked as (1) mild, (2) moderate, (3) or severe, 
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 
 
If an adverse event changes in intensity during the specified reporting period, a new 
description of the adverse event 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, respectively. 
 
9.2.3 
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 adverse event with the study procedures 
will be categorized as: 

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Definitely: 
administration of the IP is the cause, another etiology causing the adverse 
event is not known. 
Probable: 
administration of the IP 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 IP, 
however, there is an alternative etiology that is more likely. 
Unlikely: 
a relationship to the administration of IP is unlikely, however, it cannot be ruled 
out. 
Not related:  a relationship to the administration of the IP cannot be reasonably established; 
another etiology is known to have caused the adverse event or is highly likely 
to have caused is. 
 
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. 
 
9.2.4 Serious adverse events (SAEs) 
A serious adverse event 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 been based upon 
appropriate judgement by the investigator. 
An elective hospital admission will not be considered as a serious adverse event. The 
investigator will report all SAEs to the sponsor without undue delay after obtaining knowledge 
of the events (within 24 hours).  The sponsor will report all 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 serious 
adverse events.  
 

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9.2.5 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 section 9.2.4); 
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: 
- 
Summary of Product Characteristics (SPC) for an authorised medicinal 
product; 
- 
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. As this is an open label study in which the sponsor, investigator and the SMC are 
not blinded, the code would not have to be broken in the case of a SUSAR. 
 
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. 
After termination of the trial, the investigator should assure that the subject is referred for 
medical follow-up, as appropriate. 
 
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 and 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 DSMB, the sponsor will send the advice to the 
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 adverse events and volunteer safety. He/she is independent of the 
sponsor and the investigator. The local safety monitor is notified of all grade 3 adverse 
events probably or definitely related to R0.6C administration and persisting at grade 3 for >48 
hours. 
9.5.2 
Safety Meetings 
The SMC will review safety data at pre-defined time points throughout the study, specifically: 
prior to commencing the first administration in the subsequent dose group ( and ≥7 days of 
after the first R0.6C-administration of all subjects in the preceding dose-group). A minimum 
of two SMC meetings must take place: a meeting prior to initiation of the first 100µg R0.6C 
dose group (group 3), and a meeting after completion of follow up in all groups. The chair of 

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the SMC will determine for the other time points whether a meeting will be held, or whether a 
recommendation from all members may be formalized through e-mail. The frequency of 
these reviews may be adapted upon SMC recommendation if deemed necessary. 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 (section 9.5.5) and SMC charter. If desirable, the SMC can have an 
additional meeting before the start of the trial, to discuss the protocol, the trial, future 
meetings and to have the opportunity to clarify any aspects with the sponsor. 
9.5.3 
Safety Reports 
Safety reports will be prepared by the clinical investigator for review by the committee prior to 
each 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 ICD-10 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 there are safety concerns and whether 
the study should continue without change, be modified, or be terminated. If at any time a 
decision is made to permanently discontinue administration of study R0.6C administrations in 
all subjects, the Sponsor will notify the CCMO expeditiously. 
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 SMC advice 
to the CCMO, including a note to substantiate why (part of) the advice of the SMC will not be 
followed. 
9.5.4 
Inter-group progression 
Progression to the next dosage group will depend in all cases upon a positive review by the 
SMC of safety data of the previous dosage group. The data to be reviewed before each dose 
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administration) and clinically significant laboratory tests collected at 1, 2 and 7 days following 
R0.6C administration for all subjects in the specified dose group. The SMC will review the 
AEs (local, general, and laboratory) and will make recommendations regarding the 
continuation of the study for each group. 
9.5.5 
Safety holding rules 
The study may be placed on safety hold at any time for the following reasons: 
• On advice of the safety monitor; 
• On advice of the Principal/Clinical investigators; 
• On advice of the SMC; 
• On advice of the CCMO; 
• If holding rules are met (see below). 
 
If the following holding rules are met, administration of R0.6C will be held for all remaining 
administrations in that dosage group and subsequent groups: 
• 
One or more participants experience a SAE that is determined to be at least 
possibly related to the administration of R0.6C 
• 
Two or more subjects experience a grade 3 adverse event (local, clinical 
systemic or laboratory systemic) possibly, probably or definitely related to R0.6C 
administration and persisting at grade 3 for >72 hours 
If considered necessary by the investigators or SMC, remaining administrations in lower 
dosage groups may also be put on hold. 
 
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 R0.6C dose 
• 
Relationship of the AE or SAE to (one of) the adjuvants 
• 
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 
R0.6C 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 

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to resume R0.6C administration. It may be decided to proceed with only R0.6C adjuvated 
with Alhydrogel (groups A) or only R0.6C adjuvated with Alhydrogel and Matrix-M (groups B). 
If the CCMO has recommended safety hold, re-initiation of the study will require CCMO 
concurrence. The CCMO 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 CCMO within 24 hours. The PI, local safety monitor, 
SMC or CCMO may stop or suspend the use of this product at any time. 

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10. STATISTICAL ANALYSIS 
All data will be collected and verified prior to analysis. All data will be analysed in accordance 
with the Statistical Analysis Plan. Final analyses of all data will occur after study completion and 
final verification of data according to GCP. All data analyses will be conducted using IBM 
SPSS, R, SAS or Graphpad using the latest version available. Detailed statistical 
procedures, listings, table shells, and figures will be provided in a SAP prior to analysis. The 
SAP will be finalized before study close-out and database lock. The following key statistical 
components will be considered and a detailed description will be documented in the SAP: 
• 
Primary and secondary endpoints and how they will be measured 
• 
Statistical methods and tests that will be used to analyze the endpoints 
• 
Strategy that will be used if the statistical test assumptions are not satisfied 
• 
Indication of whether the comparisons will be using one-tailed or two-tailed t-
test (with justification of the choice) and the level of significance to be used 
• 
Identification of whether any adjustments to the significance level or the overall 
P-value will be made to account for any planned or unplanned subgroup 
analyses or multiple testing 
• 
Specification of potential adjusted analyses and a statement with which 
covariates or factors will be included 
• 
Planned exploratory analyses and justification of their importance 
• 
Any subgroup effects with biological justification and support from within and 
outside the study 
 
10.1 Primary study parameter(s) 
The number of subjects enrolled, completed, or withdrawn will be summarized. Reasons for 
withdrawal, when known, will be provided. Demographic data will be summarized by 
descriptive statistics and will include total number of observations (n), mean, standard 
deviation (SD) and range for continuous variables, and number and percentages for 
dichotomous variables. The primary safety, and reactogenicity outcomes will include all 
subjects who meet the eligibility criteria, received the study product R0.6C, and for whom 
safety data are available. 
 
Safety endpoints 
For the safety analysis, data from all subjects who received at least one dose of R0.6C and 
for whom safety data are available will be included. All analyses will be descriptive. Data will 
be presented by dose, overall/dose and overall/subject. Results will be summarized by study 
group. The percentage of subjects with at least one local AE (solicited or unsolicited), with at 
least one general AE (solicited or unsolicited) and with any AE during the solicited follow-up 
period will be tabulated with exact 95% CI (two-sided). No multiplicity adjustment will be 
implemented in analysis. The same calculations will be performed for AEs rated as grade 3. 

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The percentage of subjects reporting each individual solicited local and general AE during 
the solicited follow-up period will be tabulated with exact 95% CI. The same tabulation will be 
performed for grade 3 AEs and for AEs with relationship to R0.6C administration. The reports 
of unsolicited AEs will be reviewed by a physician and will be categorized by ICD-10. The 
percentage of subjects with at least one report of unsolicited AE and reported up to 28 days 
after R0.6C administration will be tabulated with exact 95% CI. The same tabulation will be 
performed for grade 3 unsolicited AEs and for unsolicited AEs with a relationship to R0.6C 
administration. SAEs will be described in detail. Withdrawals due to AEs/SAEs will also be 
summarized. Vital signs which are outside of the normal range and clinically significant will 
also be listed in tables. The frequency of signs and symptoms will be compared between 
groups with the chi- square test or fishers exact test. Serious adverse events (SAEs) 
occurring at any point during the trial will be summarized and relatedness to vaccine will be 
assessed. 
Any clinically important deviations in routine laboratory test results and/or vital signs as 
determined by the investigator will be listed. Isolated laboratory abnormalities will be reported 
as AEs if they are considered clinically relevant by the investigator. Vital signs which are 
considered clinically relevant by the investigator will be summarized. All adverse events will 
be listed by participant and will include details of onset time, duration, severity and 
relationship to the study product. 
 
Efficacy endpoints 
SMFA will be performed to determine TRA of R0.6C. Differences will be assessed by 
comparing mean values between time points using either a two-tailed student’s t-test or a 
non-parametric equivalent. Paired tests will be used if pre-intervention values are 
compared with post-intervention values. For discrete variables (e.g. the number of positive 
assays), the chi-squared test or Fisher’s exact test will be used (two-tailed). 
 
10.2 Interim analysis  
There are no statistical criteria for study termination in this clinical trial. Safety and 
reactogenicity data will be evaluated after each R0.6C administration before proceeding to 
the next group. A summary report of AEs will be provided to the SMC after all four 
groups have completed follow-up. 

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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 ICH Good Clinical Practice, 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 CCMO 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. Furthermore, a Facebook page (link: 
http://www.facebook.com/malariavaccin) showing the advertisement text will be used to 
inform people about the trial. This brief advertisement will indicate a telephone number to call 
and an e-mail address for contact to request further information. It will furthermore indicate a 
website (www.malariavaccin.nl) which contains a form. This short questionnaire will be 
completed using the online form. When seemingly suitable subjects contact investigators via 
e-mail, telephone or the online form, they will be invited to an information meeting during 
which the study will be explained to them by the study investigator. Directly after the meeting 
they will be provided with documents to review at home (the information sheet, 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 investigator, and any 
follow-up discussion if necessary, the volunteer will be given sufficient time to consider 
participation (also see section 8.3.1). 
 
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, CCMO- 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. In addition, a letter for the general practitioner will be signed and sent 
after screening. Again, the investigator will answer any questions the volunteer has. The 
possibility of withdrawal from the study, at any time, without penalty and without any 

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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. 
11.3 Benefits and risks assessment 
Testing in human subjects remains the only reliable and convincing way to obtain 
information on the immunological responses that are important for protection against 
malaria. Explorative studies looking for new and complementary candidate malaria 
interventions are of paramount importance with the potential of large-scale application in 
endemic countries. Of course, the compelling need for new methods of malaria 
interventions needs to be balanced with the potential risks and discomforts for the subjects. 
Risks for subjects are related to administration with R0.6C TBV candidate. There are no 
direct benefits to participation in the trial for subjects. Subjects will be advised to take regular 
malaria chemoprophylaxis when travelling to malaria endemic areas in the future.  
 
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). This insurance provides cover for damage 
to research subjects through injury or death caused by the study. The insurance applies to 
the damage that becomes apparent during the study or within 4 years after the end of the 
study. 
 
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. 
 

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11.5 Incentives 
Enrolled subjects will receive up to €1305,- in compensation for their time and for the 
inconveniences of taking part in this study. These amounts are based on predefined criteria 
described in our department standard operation procedures (SOP): 
• 
Full day visit (i.e. CHMI, immunization, vaccination)   €125 / visit (4 immunizations) 
• 
Brief visit (<1 hour) 
 
 
 
         €25 / visit (25 visits) 
• 
Bonus for study length 
 
 
 
         €20 / month (9 months) 
 
Travel expenses will not be additionally reimbursed, and 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 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 medical treatment with full costs covered by the insurance of Radboudumc. 

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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 electronic CRF (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. 
 
12.2 Monitoring and Quality Assurance  
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). 
 
Monitoring 
The investigator will give the 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 variables will be assessed for minimally 50% of included subjects. 
 
To ensure that the study is conducted in accordance with ICH-GCP and regulatory 
requirements, monitoring responsibilities will be provided by the Radboud Technology 
Center Clinical Studies (RTCCS). 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; 

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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 submission). 
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. RTCCS 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. 
To ensure the quality of clinical data for all subjects, source data verification will be 
performed on subject data received by RTCCS. During this review, subject data will be 
checked for consistency, omissions, and any apparent discrepancies. In addition, the 
data will be reviewed for adherence to the protocol and ICH-GCP. To resolve any 
questions arising from the source data verification process, data queries and/or site 
notifications will be sent to the site for resolution as soon as possible and within the period 
described in the monitoring plan; 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 RTCCS. 
 
12.3 Amendments  
Amendments are changes made to the research after a favourable opinion by the CCMO has 
been given. All amendments will be notified to the CCMO that gave a favourable opinion.  
 
A ‘substantial amendment’ is defined as an amendment to the terms of the CCMO 
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 CCMO and to the competent authority. 
Non-substantial amendments will not be notified to the CCMO and the competent authority, 
but will be recorded and filed by the sponsor.  
 

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12.4 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the CCMO 
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 CCMO 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 CCMO 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 
CCMO 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 CCMO and the Competent Authority.  
 
12.6 Public disclosure and publication policy 
The study is entered by the sponsor into Clinicaltrials.gov registry and registered under 
NCT04862416. 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 make every effort to publish the results in a peer-reviewed journal. 

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13. STRUCTURED RISK ANALYSIS  
 
13.1 Potential issues of concern 
a. Level of knowledge about mechanism of action 
The R0.6C fusion protein is a chimera consisting of the 6-cysteine C-terminal fragment of 
PfS48/45 (6C) coupled to the N-terminal region of asexual stage Glutamate Rich Protein 
GLURP (R0) produced in Lactococcus lactis. Immunisation with R0.6C in rodents induces 
functional antibodies against the 6C subunit. Anti-6C antibodies (Abs) are ingested during 
the blood meal and can bind male sexual forms in the mosquito gut, preventing their 
fertilisation of female gametes and thus ookinete and oocyst development.  
 
b. Previous exposure of human beings with the test product(s) and/or products with a 
similar biological mechanism 
There is no in human experience with the R0.6C/AlOH and R0.6C/AlOH + Matrix-M TBV 
candidates. However, the GLURP-R0 region, which constitutes 76.7% of R0.6C, forms part 
of the malaria vaccine candidate, GMZ2, which has been tested in both European and 
African clinical trials as listed in section 7.3.  
To date 994 individuals, of whom the majority are African children below 5 years of age, have 
been immunized with GMZ2/AlOH. The GMZ2/AlOH formulation has shown excellent safety 
and tolerability confirming the safety of the expression system and the safety of the GLURP-
R0 portion of R0.6C. 
The Matrix-M adjuvant technology [17] is a promising technology which has been explored 
for various infectious diseases and has a good safety profile in humans [18].  To-date Matrix-
M has been utilized in more than 25+ clinical trials, including multiple Phase III trials (Table 4, 
section 7.3). In these studies, Matrix-M has been combined with multiple malaria vaccine 
candidates, influenza, covid-19, and other disease indications. 
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo 
human cell material? 
Immunisation with R0.6C in rodents induces functional antibodies against the 6C subunit, the 
sera of vaccinated animals were able to induce >99% transmission blocking activity in the 
standard membrane feeding assay (SMFA) with cultured gametocytes[7]. Anti-6C antibody 
titres were further increased by immunising with R0.6C adjuvanted with Alhydrogel or 
Alhydrogel and Matrix-M[8].   
 
d. Selectivity of the mechanism to target tissue in animals and/or human beings 
The recombinant protein, R0.6C, is based on the genome of P. falciparum. The amino acid 
sequence of both R0 (GLURP) and 6C (Pfs48/45) was searched (BLAST) against the 
genome of homo sapiens (TAXID9606) for an in silico safety assessment of cross-reactivity. 
In those searches, 6C showed no homology to any human protein, and R0 demonstrated no 
significant homology to human proteins.  
 

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Humans have been exposed to the GLURP-R0 region and the Pfs48/45-6C domain since 
Plasmodium falciparum was first transmitted to humans. There are no recognized 
autoimmunity related to antibodies elicited by natural exposure in endemic populations 
suggesting that anti-R0 and anti-6C antibodies do not cross react with human epitopes. 
Since R0.6C is strongly recognized by the reduction sensitive mAb45.1, it is highly likely that 
recombinant Pfs48/45-6C adopts its native fold and primarily elicit antibodies against native 
epitopes of Pfs48/45. Moreover, the GLURP-R0 region, which constitute 76.7% of R0.6C, 
forms part of the malaria vaccine candidate, GMZ2, which has been tested in both European 
and African clinical trials (also see section 7.3). The GMZ2/AlOH formulation has shown 
excellent safety and tolerability suggesting that the R0 portion of R0.6C elicits antibodies 
against native epitopes of GLURP and does not elicit cross-reactive antibodies against 
human protein sequences. 
 
In a rabbit toxicology study of R0.6C-AlOH NZW Rabbits received a total of 4 IM doses of 
100 µg R0.6 with or without 50 µg Matrix-M. There were no related mortalities, clinical signs, 
or effects on body weights or food consumption.  Hematology, clinical chemistries, 
ophthalmology, gross pathology and organ weights were unaffected at any dose. 
Histopathological evaluation of a panel of tissues revealed no effects associated with 
treatment with R0.6C-AlOH with or without Matrix-M other than histopathology changes with 
marked focal inflammatory response at sight of injection. Partial recovery was recorded when 
comparing the site dosed 3 days prior to necropsy with the site dosed 46 days prior to 
necropsy. All animals in the two test article immunized groups (immunized with R0.6C-AlOH 
or R0.6C-AlOH + Matrix-M) showed significant and consistent antibody responses (≥ 
409.600 fold dilution) at the terminal bleed (day 46 for main animals and day 71 for recovery 
animals). 
 
e. Analysis of potential effect 
The R0.6C dose administered in the rabbit toxicity study (100 μg/rabbit) translates to a 
human equivalent dose of 774 μg for a 60 kg human, providing, a safety margin of >7x 
higher than the highest dose to be administered in this first in human trial (100 μg). 
 
f. Pharmacokinetic considerations 
Biodistribution studies of R0.6C have not been conducted because the vaccine does not 
include whole/live parasite material and is not capable of replication or productive infection. 
The route of administration (IM) is well understood. Consistent with International Council for 
Harmonisation (ICH) guidelines, formal pharmacokinetic studies were not conducted. 
The antibodies induced by the R0.6C TBV candidate will be evaluated in human subjects by 
analysis from human sera collected at several time points after R0.6C administration. See 
sections 8.1 and the schedule 8.3.17 for details. 
 
g. Study population 
Included subjects are healthy young adult subjects, who have been extensively screened for 
any evidence of co-morbidity including but not limited to: immune deficiency, hypersensitivity 

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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 
R0.6C is not known to interact or be incompatible with any other products. 
 
i. Predictability of effect 
The SMFA will be used to evaluate the transmission reducing abilities of the R0.6C TBV 
candidate. SMFA is the most widely used assay to determine the TRA and an accepted 
surrogate of TRA for public health interventions [5]. Laboratory reared Anopheles mosquitoes 
are fed through membrane feeders with in vitro cultured gametocytes to which serum or 
purified anti-R0.6C antibodies in various concentrations are added. Subsequently, the oocyst 
density in mosquitoes are compared between the experimental and control group by means 
of microscopy of the mosquito gut, parasite DNA detection or immune-assays [5, 9]. The 
SMFA can also be used to determine the transmission blocking activity (TBA) by comparing 
the number of infected mosquitoes between the experimental and control group. 
Because the mode of action of TBVs is so explicit, there is a highly informative endpoint in 
the SMFA that is a clear indicator of later public health impact. For other vaccines, there is no 
such clear in vitro assay. The TRA measured in the SMFA assay around which there is 
confidence that blocking activity is present was set by consensus at 80% reduction in oocyst 
intensity, however even TBVs with a TRA of less than 80% could eliminate plasmodium at 
low transmission levels over consecutive transmission seasons[10]. 
 
Additionally, anti-6C, anti-R0 and anti-R0.6C antibody quantities will be measured by ELISA 
from subject sera collected at pre-specified time points (also see section 8.1 and 8.3.17). 
 
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 
Please see section 7.4 for a summary of known and potential risks and benefits that includes 
mitigation strategies. 
 
 
 
 
 
 
 
 

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