Safety and Protective Efficacy of Genetically Modified Plasmodium Berghei (Pb(PfCS@UIS4)) Malaria Parasites in Healthy Volunteers
Phase: PHASE1
Conditions: Malaria,Falciparum, Plasmodium Falciparum, Plasmodium Berghei, Controlled Human Malaria Infection (CHMI)
Sponsor: Radboud University Medical Center

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CONFIDENTIAL 
 
CLINICAL TRIAL PROTOCOL 
 
 
 
 
 
 
 
 
 
 
Safety and protective efficacy of genetically modified 
Plasmodium berghei (Pb(PfCS@UIS4)) malaria parasites in 
healthy volunteers 
 
Version 4.0, 28-November-2017 
 
(“PbVac”) 

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PROTOCOL TITLE: ‘Safety and protective efficacy of genetically modified Plasmodium 
berghei (Pb(PfCS@UIS4)) malaria parasites in healthy volunteers’ 
 
Protocol ID 
PbVac 
Short title 
Safety and protective efficacy of Pb(PfCS@UIS4) 
Toetsingonline number  
NL60019.091.16 
EudraCT number  
2017-000315-17  
WIRB protocol number 
1172775 
Version 
Version 3.0 
Date 
28-02-2017 
Project leader/Clinical 
investigator 
 
Isaie J. Reuling, M.D. 
Radboudumc, Department of Medical Microbiology 
Tel: +31 (0)24 3619515 
Fax: +31 (0)24 3635153 
E-mail: isaie.reuling@radboudumc.nl 
Sponsor 
 
 
 
 
 
Principal Investigator 
 
 
 
 
Co-Principal Investigator 
 
Stichting Katholieke Universiteit  
Radboud University Medical Centre (Radboudumc) 
Department of Medical Microbiology 
Geert Grooteplein-Zuid 10 
6525 GA, Nijmegen, The Netherlands 
 
Prof. Robert W. Sauerwein, M.D. 
Radboudumc, Department of Medical Microbiology 
Tel: +31 (0)24 3610577  
Fax: +31 (0)24 3635153 
E-mail: robert.sauerwein@radboudumc.nl 
 
Perry van Genderen, MD PhD 
Department of Internal Medicine 
Havenziekenhuis 
Tel: +31 (0)10 23443616 
Fax: +31 (0)10 4121645 
E-mail: p.van.genderen@havenziekenhuis.nl 

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Clinical investigator 
 
 
 
 
Diagnostic laboratory 
investigator 
 
Gerdie M. de Jong, M.D. 
Department of Internal Medicine 
Havenziekenhuis 
Tel: +31 (0)615193584 
Fax: +31 (0)10 4121645 
Email: g.m.dejong@erasmusmc.nl 
 
Dr. Jaap van Hellemond 
Dept. Medical Microbiology & Infectious Diseases 
Erasmus MC & Havenziekenhuis 
Tel: +31 (0)6 22273138 
j.vanhellemond@erasmusmc.nl 
Funding partner 
PATH Malaria Vaccine Initiative (MVI) 
2201 Westlake Avenue, Suite 200 
Seattle, Washington 
98121, USA 
Biological evaluator 
Dr. Rob Hermsen, PhD 
Radboudumc, Department of Medical Microbiology 
Tel: +31 (0)24 3613663 
E-mail: Rob.Hermsen@radboudumc.nl 
Independent expert (s) 
Dr. Els van Nood, MD PhD 
Department 
of 
Internal 
medicine 
& 
Infectious 
Diseases 
Erasmus MC, Rotterdam 
Tel: +31 (0)6 47104092 
Email: e.van.nood@havenziekenhuis.nl 
Laboratory sites  
Safety parameters: 
Havenziekenhuis/Erasmus MC 
Dept. Clinical Chemistry, Hematology and Medical 
Microbiology & Infectious Diseases 
 
 
Immunology laboratories: 
IAVI-Human Immunology Laboratory 
Imperial College London, Faculty of Medicine 
2nd Floor Lift Bank D 

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Chelsea and Westminster Hospital 
369 Fulham Road, London, UK 
SW10 9NH 
 
Malaria Serology Lab  
Malaria Vaccine Branch 
Walter Reed Army Institute of Research 
Room 3W80 
503 Robert Grant Avenue 
Silver Spring, Maryland 20910-7500 
 
Instituto de Medicina Molecular – IMM 
Faculdade de Medicina da Universidade de Lisboa 
Miguel Prudêncio 
Av. Professor Egas Moniz 
1649-028 Lisboa 
Portugal 
 
Radboud University Medical Centre (Radboudumc) 
Department of Medical Microbiology 
Geert Grooteplein-Zuid 10 
6525 GA, Nijmegen, The Netherlands 
Pharmacy  
Pharmacy Lagaay 
Van Helmontstraat 99, 3029 AA 
Rotterdam - The Netherlands 
Pharmaceutical company 
External monitor 
CROMSOURCE 
Lange Dreef 11H 
4131 NJ Vianen, The Netherlands 
Tel: +31 (0)347 344977 ext. 2614 
Fax: +31 (0)347 34 42 89 
Mobile: +31 6 13516468 

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PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Sponsor and principal investigator: 
Robert Sauerwein, MD PhD 
Department of Medical Microbiology 
Radboud university medical center  
 
 
Co-Principal investigator: 
Perry van Genderen, MD PhD 
Department of Internal Medicine 
Havenziekenhuis 
 
 
 
Project leader: 
Isaie Reuling, MD 
Department of Medical Microbiology 
Radboud university medical center 
 
 
 
Version History 
Version 
Date 
Author(s) 
Summary of changes 
2.0 
24 January 
2017 
Isaie Reuling 
Robert Sauerwein 
Perry van Genderen 
- Information on LFT derangements in 
CHMI (section 1.5) 
- Additional inclusion criterium (section 4.2) 
- Additional biochemistry test 
measurements (flowchart) 
- Adjustment to Investigational and non-
investigatonal product (Section 6) 
- Added information on SUSARs (section 
- Minor revision in section power size 
calculation (section 4.4) 
3.0 
28 
February 
2017 
Isaie Reuling 
Robert Sauerwein 
Perry van Genderen 
- minor adjustment to section 1.5 LFT 
derangements. 
- additional safety/exclusion criteria renal 
function (section 1.5) 
4.0 
28 
November 
2017 
Gerdie de Jong  
Isaie Reuling 
Robert Sauerwein 
Perry van Genderen 
- Additional exploratory objectives and 
endpoints (Summary, section 2, section 
7.1.2.) 
-Rationale of sampling on day 4 and 5 

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post-CHMI for group 4 and 5 and 
additional details on the exploratory 
analysis (section 7.3.13). 

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TABLE OF CONTENTS 
 
1.1 
Introduction ............................................................................................................18 
1.2 
Rationale ................................................................................................................19 
1.2.1 
Pb(PfCS@UIS4) .............................................................................................20 
1.3 
Clinical experience with genetically attenuated malaria parasites ...........................21 
1.4 
Clinical experience with non-attenuated malaria parasites .....................................21 
1.5 
Safety .....................................................................................................................22 
2. 
OBJECTIVES ................................................................................................................25 
3. 
STUDY DESIGN ...........................................................................................................26 
4. 
STUDY POPULATION ..................................................................................................28 
4.1 
Population (base) ...................................................................................................28 
4.2 
Inclusion criteria .....................................................................................................29 
4.3 
Exclusion criteria ....................................................................................................30 
4.4 
Sample size calculation ..........................................................................................32 
5. 
TREATMENT OF SUBJECTS .......................................................................................33 
5.1 
Investigational product/treatment ............................................................................33 
5.2 
Use of co-intervention (if applicable) ......................................................................33 
5.3 
Escape medication (if applicable) ...........................................................................33 
6. 
INVESTIGATIONAL AND NON-INVESTIGATIONAL PRODUCT (CHALLENGE 
PRODUCT) ..........................................................................................................................35 
6.1 
Name and description of non-investigational products ............................................35 
6.1.1 
Name and description of investigational product (Pb(PfCS@UIS4)) ................35 
6.1.2 
Name and description of non-investigational product (P. falciparum NF54) .....35 
6.2 
Summary of findings from non-clinical studies ........................................................35 
6.2.1 
Summary of findings from non-clinical studies (Pb(PfCS@UIS4)) ...................35 
6.2.2 
Summary of findings from non-clinical studies (P. falciparum NF54) ...............37 
6.3 
Summary of findings from clinical studies ...............................................................37 
6.3.1 
Summary of findings from clinical studies (Pb(PfCS@UIS4)) ..........................37 
6.3.2 
Summary of findings from clinical studies (P. falciparum NF54) ......................37 
6.4 
Summary of known and potential risks and benefits ...............................................38 
6.4.1 
Summary of known and potential risks and benefits of   Pb(PfCS@UIS4) 
immunization .................................................................................................................38 
6.4.2 
Summary of known and potential risks and benefits of Pf challenge ................39 
6.5 
Description and justification of route of administration and dosage .........................40 
6.5.1 
Description and justification of route of administration and dosage 
(Pb(PfCS@UIS4)) .........................................................................................................40 
6.5.2 
Description and justification of route of administration and dosage (P. 
falciparum NF54) ...........................................................................................................41 
6.6 
Dosages, dosage modifications and method of administration ...............................41 
6.6.1 
Dosages, dosage modifications and method of administration 
(Pb(PfCS@UIS4)) .........................................................................................................41 

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6.6.2 
Dosages, dosage modifications and method of administration (P. falciparum 
NF54) 
41 
6.7 
Preparation and labelling of Investigational, Non Investigational Medicinal Product
 
42 
6.7.1 
Preparation and labelling of Investigational Medicinal Product 
(Pb(PfCS@UIS4)) .........................................................................................................42 
6.7.2 
Preparation and labelling of Non Investigational Medicinal Product (P. 
falciparum NF54) ...........................................................................................................43 
6.8 
Drug accountability .................................................................................................44 
7. 
METHODS ....................................................................................................................45 
7.1 
Study parameters/endpoints ...................................................................................45 
7.1.1 
Main study parameters/endpoints....................................................................45 
7.1.2 
Exploratory parameters/endpoints ...................................................................45 
7.2 
Randomisation, blinding and treatment allocation ..................................................46 
7.3 
Study procedures ...................................................................................................46 
7.3.1 
Screening period: Screening, Inclusion and Baseline visits .............................46 
7.3.2 
Administration of Pb(PfCS@UIS4) ..................................................................46 
7.3.3 
Controlled Human Malaria Infection ................................................................50 
7.3.4 
Treatment with Malarone® ...............................................................................51 
7.3.5 
Physical examination .......................................................................................52 
7.3.6 
Vital signs ........................................................................................................52 
7.3.7 
Patient-reported outcomes (study diary) ..........................................................53 
7.3.8 
Electrocardiogram ...........................................................................................53 
7.3.9 
Blood sampling and safety laboratory evaluations ...........................................53 
7.3.10 
Analysis of asexual parasite densities after challenge infection .......................54 
7.3.11 
Immunological assays .....................................................................................54 
7.3.12 
Cytokines ........................................................................................................55 
7.3.13 
Other ...............................................................................................................55 
7.3.14 
Case report forms and data collection .............................................................56 
7.3.15 
Flowchart trial procedures: Phase 1 ................................................................57 
Flow chart trial procedures: Phase 2 .................................................................................59 
Flow chart trial procedures: Phase 2 – adaptive design ....................................................61 
7.4 
Withdrawal of individual subjects ............................................................................63 
7.5 
Replacement of individual subjects after withdrawal ...............................................64 
7.6 
Follow-up of subjects withdrawn from treatment .....................................................64 
7.7 
Procedure in case of pregnancy .............................................................................64 
7.8 
Premature termination of the study .........................................................................64 
8. 
SAFETY REPORTING ..................................................................................................66 
8.1 
Temporary halt for reasons of subject safety ..........................................................66 
8.2 
AEs, SAEs and SUSARs ........................................................................................66 
8.2.1 
Adverse events (AEs) ......................................................................................66 
8.2.2 
Serious adverse events (SAEs) .......................................................................67 
8.2.3 
Suspected unexpected serious adverse reactions (SUSARs) .........................68 

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8.3 
Follow-up of (serious) adverse events ....................................................................69 
8.3.1 
Adverse event data collection ..........................................................................69 
8.3.2 
Assessment of causality ..................................................................................70 
8.3.3 
Follow-up of adverse events ............................................................................70 
8.4 
Local Safety Monitor (LSM) and Safety Monitoring Committee (SMC)....................71 
8.4.1 
Local safety monitor ........................................................................................71 
8.4.2 
Safety Monitoring Committee (SMC) ...............................................................71 
8.4.3 
Review of safety data by the safety monitor and SMC .....................................71 
8.4.4 
Safety holding rules .........................................................................................72 
8.4.4.1 
Individual holding rules ................................................................................72 
8.4.4.2 
Group holding rules .....................................................................................73 
8.4.5 
Deferral of study procedures for individuals .....................................................74 
9 
STATISTICAL ANALYSIS ..........................................................................................76 
9.1 
Primary study parameter(s) ....................................................................................76 
9.2 
Exploratory study parameters .................................................................................76 
10 
ETHICAL CONSIDERATIONS ...................................................................................77 
10.1 
Regulation statement .............................................................................................77 
10.2 
Recruitment and consent........................................................................................77 
10.3 
Benefits and risks assessment, group relatedness .................................................78 
10.3.1 
Ethical aspects concerning the use of human volunteers ................................80 
10.4 
Compensation for injury .........................................................................................80 
10.5 
Incentives ...............................................................................................................81 
11 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .........................82 
11.1 
Handling and storage of data and documents ........................................................82 
11.1.1 
Confidentiality..................................................................................................82 
11.1.2 
Data collection .................................................................................................82 
11.1.3 
Storage of biological samples ..........................................................................82 
11.1.4 
Database management and quality control .....................................................83 
11.2 
Monitoring and Quality Assurance ..........................................................................83 
11.3 
Amendments ..........................................................................................................84 
11.4 
Annual progress report ...........................................................................................84 
11.5 
Temporary halt and (prematurely) end of study report ............................................84 
11.6 
Public disclosure and publication policy ..................................................................84 
12 
STRUCTURED RISK ANALYSIS...............................................................................86 
12.1 
Potential issues of concern .....................................................................................86 
12.2 
Synthesis ...............................................................................................................88 
13 
Appendices ................................................................................................................90 
13.1 
Appendix 1: Systematic Coronary Risk Evaluation (SCORE) Risk Chart ................90 
13.2 
Appendix 2: Classification of Adverse Events .........................................................91 
A. 
Tables for Clinical Abnormalities ............................................................................91 
B. 
Tables for Laboratory Abnormalities .......................................................................93 
14 
REFERENCES ..........................................................................................................95 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
 
A.  
Anopheles  
ACT 
Artemisinin-based Combination Treatment 
AE 
Adverse Event 
AL 
artemether–lumefantrine  
ALT 
Alanine Aminotransferase 
ANOVA  
analysis of variance 
BMI 
Body Mass Index  
BP 
Blood Pressure 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in 
Dutch: Centrale Commissie Mensgebonden Onderzoek 
CHMI 
Controlled Human Malaria Infection 
CRF 
Case Report Form 
CPS 
chemoprophylaxis and sporozoites 
CSP 
Circumsporozoite protein 
CRO 
Contract Research Organization 
CV 
Curriculum Vitae 
ECG  
ElektroCardioGram 
EDTA 
Ethylenediaminetetraacetic acid 
eCRF 
electronic Case Report Form 
ELISA 
Enzyme-Linked Immuno Sorbent Assay 
G6PD 
deficiency 
Glucose-6-phophatedehydrogenase deficiency 
GAP 
Genetically Attenuated Parasites 
GCP 
Good Clinical Practice 
GP 
General Practitioner  
HBV 
Hepatitis B Virus 
HCV 
Hepatitis C Virus 
HIV 
Human Immunodeficiency Virus 
HTLV 
HumanT-lymphotropic Virus 
IC 
Informed Consent 
IFN-γ 
Interferon-gamma 

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IRB 
Institutional Review Board 
IV 
Intravenous 
LDH 
Lactate dehydrogenase 
LSM 
Local Safety Monitor 
mAbs 
Monoclonal antibodies 
METC  
Medical research ethics committee (MREC); in Dutch: medisch 
ethische toetsing commissie (METC) 
MFS 
Membrane Feed for Sporozoite production 
NF54 
Nijmegen falciparum strain 54 
P. 
Plasmodium 
par/ml or p/ml 
parasites per milliliter 
PATH-MVI 
PATH Malaria Vaccine Initiative 
PATH REC 
PATH Research Ethics Committee; the funding partner’s ethical 
committee. 
Pb(PfCS@UIS4) 
Transgenic Plasmodium berghei parasite expressing P. falciparum 
CS under the control of the P. berghei UIS4 promoter 
PBMC 
Peripheral Blood Mononuclear Cell 
PCR 
Polymerase Chain Reaction 
Pf 
Plasmodium falciparum 
qPCR 
Real-time Quantitative Polymerase Chain Reaction 
Radboudumc 
Radboud university medical center 
RAS 
Radiation Attenuated Parasite 
SAE 
Sanquin 
Serious Adverse Event  
Sanquin Blood Supply Foundation, who on the basis of the Blood 
Supply Act is responsible for all blood supply (blood and blood 
products) for transfusion in The Netherlands. 
SCORE 
Systematic Coronary Risk Evaluation 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
Sponsor 
 
 
 
 
 
SUSAR 
The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. 
A party that provides funding for a study but does not commission it 
is not regarded as the sponsor, and is referred to here as a funding 
partner.  
Suspected Unexpected Serious Adverse Reaction 

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SWAB 
Stichting Werkgroep Antibioticabeleid 
WIRB 
Western Institutional Review Board; PATH’s designated IRB to who 
PATH REC delegates ethical review to WIRB for vaccine studies. 
WHO 
World Health Organization 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet 
Medisch-wetenschappelijk Onderzoek met Mensen 
ZAVIN 
Ziekenhuis Afval Verwerkings Installatie Nederland (Hospital Waste 
Disposal company) 
γGT 
Gamma Glutamyl Transferase 
 
 

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SUMMARY 
 
Rationale:  
Malaria is caused by Plasmodium (P.) parasites and is one of the major infectious diseases 
in the world, with a tremendous impact on the quality of life, significantly contributing to 
ongoing poverty in endemic countries. Whole organism malaria vaccine approaches 
generate high-level (>90%) protection against malaria in humans through i) immunization 
with sporozoite forms of the parasite attenuated by irradiation or ii) when sporozoites are 
administered together with a chemoprophylactic dose of chloroquine[1, 2] In the underlying 
study, a genetically modified P. berghei parasite is used. P. berghei is one of the four 
Plasmodium species that causes malaria in rodents. The hypothesis is that immunization of 
humans with P. berghei will induce a cross-species immune response without the risk of a 
breakthrough infection. To further increase the potential for protective efficacy, the P. 
falciparum circumsporozoite (CS)- protein gene has been integrated in the P. berghei 
parasite, generating a genetically modified P. berghei parasite, abbreviated as 
Pb(PfCS@UIS4). 
 
Objectives:  
The study is divided in phase 1 and phase 2. 
 
Phase 1 
Primary objective: 
To determine the safety and tolerability of administration of Pb(PfCS@UIS4) to healthy 
volunteers delivered by infectious mosquito bites. 
 
Secondary objective: 
Immunogenicity of Pb(PfCS@UIS4) as assessed by ELISA and IFA. 
 
Exploratory objective:  
To analyse immune responses in volunteers immunized by Pb(PfCS@UIS4). 
 
Phase 2 
Primary objective: 
To determine the safety, tolerability and protective efficacy of immunization with 
Pb(PfCS@UIS4) against Controlled Human Malaria Infection (CHMI) by mosquito bite.  
 
Secondary objective: 

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Immunogenicity of Pb(PfCS@UIS4) as assessed by ELISA and IFA. 
 
Exploratory objective:  
To analyse immune responses in volunteers exposed to Pb(PfCS@UIS4) and to determine 
cytokine responses, coagulation activation and inflammation parameters  in non-immune 
individuals infected with P. falciparum during malaria liver phase 
 
Study design:  
Multicenter, open label, adaptive design, study  
Phase 1  
 
(Group 1 and 2): dose escalation, safety trial (n=6) 
 
(Group 3): dose escalation, safety trial (n=12) 
Phase 2  
 
(Group 3): protective efficacy trial  
 
(Group 4): infectivity control group, challenge 1 (n=6) 
 
(Group 5): infectivity control group, challenge 2 (n=6) 
 
Study population:  
Phase 1: Eighteen healthy male and female volunteers, aged 18 to 35 years, will participate 
in the study. 
Phase 2: The same twelve volunteers of group 3, will subsequently participate in Phase 2 of 
the study. In addition, six volunteers will be recruited as infectivity control subjects per 
challenge and will receive 5 infective mosquito bites with NF54-P. falciparum parasites. 
 
Intervention:  
Phase 1: A total of eighteen healthy adult volunteers will be recruited across 3 groups. Three 
volunteers (Group 1) will be exposed to bites of five Pb(PfCS@UIS4)-infected mosquitoes. 
Volunteers will be closely monitored for adverse events for a period of 28 days. Upon 
satisfactory safety assessment of group 1, an additional three volunteers (group 2) will be 
exposed to 25 Pb(PfCS@UIS4)-infected mosquito bites. Upon satisfactory safety 
assessment of group 2, an additional 12 volunteers (group 3) will be exposed to 75 
Pb(PfCS@UIS4)-infected mosquito bites. If one of the volunteers is not fit to participate in the 
study on day 0, an alternate included volunteer will replace him/her. For this purpose one 
additional volunteer will be screened for possible back up in groups 1 and 2, and two 
additional volunteers will be screened for possible back up for group 3. 
 

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All exposed volunteers are subjected to close follow-up after exposure at the 
Havenziekenhuis, with regular visits to the clinical trial centre, periodic physical 
examinations, frequent blood sampling, and recording of adverse events in a diary (see 
section 7.3. All exposed volunteers will be treated with a curative regimen of Malarone 
(atovaquone/proguanil), either at the time of detection of blood stage parasitemia by thick 
smear, or 28 days after exposure to Pb(PfCS@UIS4)-infected mosquitoes for groups 1 and 
2. Volunteers of group 3 will subsequently enter phase 2, if phase 1 is considered safe, and if 
no blood stage parasitemia is detected during follow up. End of follow-up will be 100 days 
after exposure to Pb(PfCS@UIS4) for groups 1 and 2, and group 3 in case this group is not 
proceeding to phase 2.  
 
Phase 2: The trial objective is to evaluate the safety and protective efficacy of repeated 
exposure to Pb(PfCS@UIS4)-infected mosquito bites by subsequent CHMI of test subjects. 
The same volunteers of group 3 will be exposed three additional times to the bites of 75 
Pb(PfCS@UIS4)-infected mosquitoes (n=12),at four to eight week intervals (at weeks 4, 4, 4-
8). Three to four weeks after the last exposure, all volunteers will undergo a CHMI with five 
P. falciparum (NF54)-infected mosquitoes. Six infectivity control subjects will be recruited to 
receive 5 P. falciparum (NF54) infective mosquito bites per challenge infection. 
 
Based on the first results of phase 2 an ad hoc decision will be made to include a potential 
booster immunization and/or subsequent second challenge infection with five P. falciparum 
(NF54)-infected mosquitoes. The booster immunization and/or subsequent second challenge 
infection will be initiated using the following criteria: 
1. In the event that 2 to 4 volunteers are sterilely protected, all volunteers (protected and 
unprotected) will receive a booster immunization followed by a second challenge 
infection 3 months after the booster immunization.  
2. In case ≥5 volunteers are sterilely protected, all unprotected volunteers will receive a 
booster immunization followed by a second challenge infection 3 months later, all 
protected volunteers will not be boosted and will only receive a second challenge 
infection to assess durability of protection. 
 
All exposed volunteers are subjected to close follow-up after exposure, with regular visits to 
the clinical trial centre, periodic physical examinations, frequent blood sampling and adverse 
events will be recorded in a diary. For all subjects, during this period, all relevant 
investigations will be carried out on an outpatient basis, including frequent safety analyses.  
 

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Exposed volunteers will be treated with a curative regimen of Malarone (atovaquone and 
proguanil), at the time of detection of blood stage parasitemia by thick smear during 
immunizations. During the challenge infection, all volunteers will receive a curative treatment, 
either at the time of blood stage parasitemia by qPCR or day 28 post CHMI. End of follow up 
will be 100 days after the last exposure to Pb(PfCS@UIS4) and 35 days post CHMI for the 
infectivity control subjects. 
 
Study parameters/endpoints:  
Phase 1 (Safety) 
Primary endpoints: 
 
Frequency and magnitude of adverse events in study groups. 
 
Presence of parasitemia after exposure to Pb(PfCS@UIS4), as assessed by thick 
smear. 
Secondary endpoints: 
 
Immunogenicity of Pb(PfCS@UIS4) as assessed by ELISA and IFA. 
Exploratory endpoints: 
 
Cellular and humoral immune responses after exposure to Pb(PfCS@UIS4). 
 
Phase 2 (Efficacy)  
Primary endpoints: 
 
Frequency and magnitude of adverse events after multiple exposures to 
Pb(PfCS@UIS4). 
 
Time to parasitemia after CHMI with the wild-type NF54 P. falciparum strain, as 
detected by qPCR. 
Secondary endpoints: 
 
Immunogenicity of Pb(PfCS@UIS4) as assessed by ELISA and IFA. 
Exploratory endpoints: 
 
Cellular and humoral immune responses after exposure to Pb(PfCS@UIS4) and 
challenge with P. falciparum. 
 
Cytokines response, coagulation activation and inflammation parameters after 
challenge with P. falciparum.  
 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness:  

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Benefits: There are no direct personal benefits for volunteers. Volunteers will be advised to 
take regular malaria chemoprophylaxis when travelling to malaria endemic areas in the 
future.  
Risks: Risks for volunteers are related to i) potential breakthrough blood stage infection after 
exposure to Pb(PfCS@UIS4) sporozoites, ii) potential side effects of atovaquone/proguanil 
treatment and iii) side effects related to exposure to multiple mosquito bites. Control 
volunteers from group 4 (and group 5), and immunized non-protected volunteers in Phase 2 
are expected to experience blood stage malaria infection. 
Burden:  
In Phase 1 volunteers are exposed to 5, 25 or 75 mosquito bites. After each exposure, there 
will be a short period (28 days) of intense clinical monitoring, with frequent clinical site visits 
and blood examinations. In Phase 2, volunteers are exposed to 75 mosquito bites on 3 
occasions followed by an additional 5 mosquito bites for the NF54 P. falciparum challenge 
infection. After the challenge infection there is a short period (28 days) of intense clinical 
monitoring with frequent site visits and blood examinations. Depending on the results of 
phase 2 a booster immunization and/or second challenge infection is initiated. It is expected 
that volunteers will have to visit the trial centre on 20-25 occasions for Phase 1 and 36 for 
Phase 2. If the extra booster and/or second challenge is initiated, volunteers will have 23-27 
more visits. In Phase 1, the maximum cumulative amount of blood collected will be 244 or 
321 ml for each volunteer, depending on the proceeding to phase 2. In Phase 2, the 
maximum amount of blood collected per volunteer will be 186 ml during the immunization 
and 521 ml during the challenge phase. In case a booster and/or subsequent challenge 
infection is initiated, a maximum of 508 ml of blood will be collected. In total, not more than 
1.5 L of blood will be collected per volunteer in the entire study. In addition, physical 
examinations will be performed when clinically indicated and subjects will be asked to 
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INTRODUCTION AND RATIONALE 
1.1 
Introduction 
Malaria is a common and serious tropical disease. It is a protozoan infection transmitted to 
human beings by Anopheles mosquitoes. Human malaria is caused by five species of 
Plasmodium (P.) protozoa: P. falciparum (Pf), P. vivax, P. ovale, P. malariae, and P. 
knowlesi. Malaria is a public health problem in over 90 countries worldwide, inhabited by 
some 40% of the world’s population, i.e. over 3 billion people are at risk of being infected with 
malaria. It has been estimated that the incidence of malaria in the world is around 214 million 
clinical cases each year[3]. People living in Sub-Saharan Africa account for 88% of these 
cases. Malaria mortality is estimated at 438,000 deaths worldwide per year[4]. Most malaria 
deaths occur among young children in Africa, especially in remote rural areas with poor 
access to health services. Other high-risk groups include pregnant women, non-immune 
travellers, refugees, displaced persons, and labour forces entering into endemic areas. The 
epidemiology of malaria has been changing over recent years due to a combination of 
factors including increasing resistance of malarial parasites to chemotherapy, increasing 
insecticide resistance of the Anopheles mosquito, ecological and climate changes and 
increased international travel to malaria-endemic areas.  
Parasites (sporozoite stage) are injected into the skin capillaries by a female Anopheles 
mosquito. From there, they travel via the bloodstream to the liver, where they develop and 
multiply in liver cells before re-entering the blood stream (merozoite stage) and invading 
erythrocytes for further reproduction. The liver stage of infection is obligatory, but completely 
asymptomatic. Clinical malaria is caused by the cyclical proliferation of asexual stages in 
erythrocytes, during the subsequent blood stage of infection. Malaria mortality is primarily 
due to organ dysfunction, amongst which the brain, following sequestration of infected 
erythrocytes in the micro-vasculature. 
There are several reasons why malaria continues to be one of the greatest health problems 
in worldwide. One of the main reasons is the difficulty in achieving adequate coverage with, 
and reducing pricing of, existing tools such as drugs, insecticide-treated bed nets, and, 
nowadays, counterfeit drugs. The decreasing effectiveness of existing tools (e.g. emergence 
of anti-malarial resistance by the parasite, resistance to insecticides, including pyrethroids, 
by the mosquitoes) is a major challenge. The availability of an efficacious malaria vaccine 
would certainly be a major achievement to overcome the shortcomings of current malaria 
control strategies.  
 

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1.2 
Rationale 
Both natural and controlled exposure to malaria parasites can lead to the development of 
protective immunity, providing a foothold for the development of a vaccine[2, 5-8]. The 
clinical development of a malaria vaccine has been a continuous effort over the past half 
century[9], following the traditional vaccine development approach. Different formulations of 
a number of antigens and/or adjuvants have been tested in Phase I trials but only about a 
dozen candidates have been evaluated in Phase II clinical field trials[10]. No vaccine has 
worked well and most candidates have failed completely. The most advanced vaccine to 
date, RTS,S, delays patency and reduces clinical severity, but does not provide long term 
protection against infection[11-13]. Although a milestone in itself, and potentially an 
additional tool in the combat against malaria, it is clear that vaccines with improved efficacy 
are required[14].  
 
An alternative approach to the development of a vaccine against malaria is based on the fact 
that natural exposure to Plasmodium parasites can lead to the development of protective 
immunity. Attenuated parasites may be used to immunize humans and prevent the 
development of malaria. The first initiatives to attenuate parasites have been achieved by 
parasite irradiation (RAS)[15]. More recently, there have been successful attempts to 
attenuate parasites by genetically engineering techniques resulting in genetically attenuated 
parasites (GAPs). Studies with GAPs have been shown to produce protective immune 
responses equal to, or even greater than, those produced by irradiated sporozoites in rodent 
models[16, 17]. 
 
Only one human study with a GAP has been performed, employing P. falciparum p52¯/p36¯ 
[18]. In this first-in-human study, exposure was well tolerated. All volunteers remained blood 
stage negative after low dose exposure (5 bites/volunteers). Five volunteers remained blood 
stage negative after high dose exposure, whereas one volunteer developed peripheral 
parasitemia twelve days after high exposure (200 bites/volunteer). For this reason, the study 
was discontinued prematurely. All volunteers developed antibodies to CSP. Furthermore, 
IFN-y response to whole sporozoites and multiple antigens were elicited in 5 of the 6 
volunteers, with both CD4 and CD8 cell cytokine production detected. It was concluded that 
this first generation GAP had favourable immune responses but further development of live, 
attenuated strains should be pursued.  
 
The disadvantage of using genetically attenuated P. falciparum in humans, is the risk of a 
potential breakthrough of the hepatic stage of the infection. Such a breakthrough may be 

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avoided by using a Plasmodium species that reaches the liver, induces an immune 
response, but does not result in a breakthrough that causes malaria. The rodent-infective P. 
berghei parasite, is thought to be a suitable candidate for this approach. P. berghei is well-
known since it has been widely used as a model in malaria research for over 30 years. 
Cross-species protection between different Plasmodium species has been reported in many 
studies (see Investigator’s Brochure). However, immunization of humans by P. berghei has 
never been investigated. The first study investigating the effect of a genetically modified 
Plasmodium berghei in humans is hereby proposed.  
 
To further increase immunogenicity of P. berghei, the P. falciparum CS protein gene has 
been added to the P. berghei parasite, to significantly enhance the likelihood of protection 
against P. falciparum infection. 
 
 
1.2.1 Pb(PfCS@UIS4) 
 
The Radboudumc (Nijmegen) malaria group is one of the research groups that are at the 
forefront of international research on whole organism malaria vaccines worldwide. 
Radboudumc showed that immunisation with sporozoites administered under chloroquine 
prophylaxis can induce sustained sterile protective immunity in humans[2, 19]. Radboudumc 
is an internationally recognized and world leading group in protocol design and execution of 
a malaria infection (P. falciparum - Pf) administered by mosquito bites (Controlled Human 
Malaria Infections, CHMI; [20, 21]).  
An alternative approach in the search of a malaria vaccine could be the use of a genetically 
modified P. berghei parasite. Specifically, the Pb(PfCS@UIS4) parasite, in which the Pf 
circumsporozoite protein (CS) gene has been inserted in the 230p neutral locus of P. 
berghei, in order to express PfCS in addition to the endogenous PbCS. 
Plasmodium CS is the immunodominant protective antigen in both RAS and GAS[22]. 
Previous studies have shown that protection could be achieved by immunization with CS 
alone [23], a finding that has been at the core of the development of the RTS,S vaccine, 
presently the most advanced malaria vaccine candidate (reviewed in Casares et al. 2010). 
However, it is known that CS is not the sole immunogen at play in the immunity triggered by 
a whole-organism approach. Sterile immunity has been observed in RAS hyper-immunized 
CSP-transgenic BALB/c mice that are T-cell tolerant to the CSP and cannot produce 
antibodies [CSP-Tg/JhT(−/−)][24, 25]. This suggests that the observed protection reflects the 
sum of activities of many minor protective antigens within the RAS. 

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This will be the first in-human trial of Pb(PfCS@UIS4) to evaluate the safety and protective 
efficacy of the genetically modified parasite.  
 
1.3 
Clinical experience with genetically attenuated malaria parasites 
One genetically attenuated P. falciparum parasite has been evaluated in humans before. The 
P. falciparum GAP Pf∆p52∆p36 is the only GAP so far that has been assessed in humans. 
Unfortunately, this trial had to be terminated, because of a GAP breakthrough blood infection 
in one (of six) volunteers in the high dose group, receiving 200 bites, at day 12 post-
exposure[18]. It was confirmed that the presence of parasites in the blood was the result of 
breakthrough blood infection of the genetically-attenuated parasite, not a wild-type parasite. 
There was no evidence of breakthrough blood infections in the low dose (n=6), five-bite 
group, or in the other five volunteers from the high dose group.  
Local adverse events in the five-bite group of this trial were comparable to those seen in 
standard CHMI. There were no systemic adverse events through 7 days post-dosing. In the 
high dose group, all volunteers experienced erythema and pruritus, five experienced oedema 
(of which one >90mm), one experienced induration, one experienced pain. Systemic adverse 
events were also reported within 7 days after exposure: fever, malaise (both one volunteer, 
grade 1), headache (grade 2, two volunteers), nausea and vomiting (2 volunteers, grade 2 in 
one). Because we aim for a decreased dose (38%), we expect adverse events to be less 
common and less severe. 
  
1.4 
Clinical experience with non-attenuated malaria parasites 
There is a large clinical experience with infecting humans by the bites of P. falciparum 
sporozoite-infected mosquitoes. These challenge trials have become highly standardized 
[21]. The first human malaria challenge study was performed in 1917 and, since 1986, when 
the modern protocol using laboratory-adapted P. falciparum strains was first performed by 
the US army, >3,500 subjects have been challenged by the bites of mosquitoes fed on 
cultures of P. falciparum gametocytes to produce sporozoites [26]. Infecting humans by the 
bites of P. falciparum sporozoite-infected mosquitoes is an established clinical trial 
methodology and is considered a reproducible, predictable and safe method of inducing P. 
falciparum malaria. The results of such studies were summarized in 1997 [27], in 2007 [28] 
and in 2012 [29]. Controlled human malaria infections (CHMIs) are accepted as a powerful 
tool for the evaluation of vaccine and drug efficacy, and the immunology and 
pathophysiology of early malaria infections [20]. The Radboud university medical center 
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infrastructure to conduct CHMI trials, and conducted two CHMI studies in the past within the 
same consortium. Over 350 subjects have undergone CHMI in studies conducted by 
Radboudumc since 2001. Standard operating procedures according to international 
standards are in place for both clinical and laboratory activities, and the Radboudumc 
mosquito and parasite culture was positively audited in 2014 by an international independent 
auditor. Radboudumc has developed a sensitive method of parasite detection by real-time 
quantitative PCR (18S qPCR) in whole blood that allows us to detect malaria infection in an 
early stage and is able to detect small differences in parasite density [30]. 
 
1.5 Safety 
Infection with Pb(PfCS@UIS4) is not expected to result in adverse events, other than mild 
adverse events associated with immunizations or adverse events by reactions to mosquito 
bites, as this murine Plasmodium species cannot multiply in human erythrocytes, which 
would be required for the onset of pathology. Please refer to D1. Investigator’s Brochure for 
extended safety analyses. 
 
After CHMI, most malaria-naive volunteers experience symptoms such as headache, chills or 
fatigue during 1-3 days. During the extensive experience with CHMI, severe or life-
threatening malaria has never been reported.  
In February 2008 a cardiac serious adverse event (SAE) in a twenty-year-old female 
participating in an LSA3/Alhydrogel (LSA-3 CMO-07/37; NL14715.000.06) malaria vaccine 
trial was reported to the CCMO (CCMO08.1096/MA/14715). The findings have been 
published as a case report titled “Cardiac complication after experimental human malaria 
infection: case report”, [31]. The differential diagnosis was acute coronary syndrome or 
myocarditis but the true nature and pathophysiology of the event remained unclear.  
In February 2013, in the TIP5 study (NL39541.091.12), a 23 year old healthy male volunteer 
experienced a cardiac SAE. The findings have been published as a case report titled 
“Ideopathic acute myocarditis during treatment for controlled human malaria infection: a case 
report”, [32]. This SAE was diagnosed as myocarditis, while the relation with the malaria 
infection was not resolved.  
In November 2014 a 23 year old healthy male taking part in the BMGF1 study 
(NL48301.91.14) experienced a cardiac SAE 10 days after a malaria infection under 
chloroquine prophylaxis (ChemoProphylaxis and Sporozoite immunization - CPS). This SAE 
concerned an asymptomatic hs-troponin T elevation (maximally 168ng/l) diagnosed as a mild 
myocarditis as reported to the CCMO (AE001.14.48301). Though certain cardiovascular risk 
factors were present (smoking, recent cannabis use), the temporal relationship with the 

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malaria infection and the previous cases of suspected or confirmed myocarditis after malaria 
infection, make an association with CHMI likely. 
As a result of these cardiac SAEs our safety procedures for CHMI have been strongly 
intensified. In the current trial, we will adhere to those stringent procedures that are relevant, 
including: 
1. 
Individuals are excluded from participation if they have first or second degree relatives 
who had cardiac events under the age of 50 
2. 
A positive urine toxicology test for amphetamines, cocaine and cannabis is an 
exclusion criterion 
3. 
Volunteers who took standard vaccinations within 3 months before the start of the trial 
or are planning to take standard vaccinations during the trial period up to 8 weeks 
after CHMI are excluded from participation 
4. 
Volunteers are required to stay close to the Havenziekenhuis to ensure 
maximal safety from day 5 after CHMI until treatment is finished 
5. 
Increased control of hs troponin T as a marker of cardiac damage; treatment with 
Malarone® is initiated in consultation with the cardiologist. 
6. 
Daily measurements of platelets; volunteers will be treated when platelet levels are 
<120x 10^9/L. 
7. 
Curative Malarone treatment after a single positive qPCR (>100 Pf/ml) after CHMI to 
minimize period of parasitaemia 
 
Transient, asymptomatic liver function derangements have been reported in volunteers in 
previous CHMI studies, and are likely to be related to the challenge infection (Reuling et al. 
manuscript in preparation). A retrospective analysis of 13 CHMI studies conducted in the 
Radboudumc showed that 72/120 (60%) of the volunteers that were treated at thick smear 
parasitaemia levels have mild (38%), moderate (10%) or severe (12%) increases of liver 
transaminases (ALT/AST).  The liver function test (LFT) elevations remained asymptomatic 
in all the affected volunteers, and are typically not associated with bilirubin changes. Detailed 
analysis on the LFT elevations pattern showed that these elevations exceeded the upper 
limit of normal (ULN) around the day of treatment with antimalarials to 2 days after treatment. 
The peak LFTs elevations are found between 2-14 days after treatment with values 
normalized at study end (35–42 days after challenge infection). In some volunteers, the 
elevations were also found associated with lactate dehydrogenase, suggesting that 
associated sub-clinical haemolysis might have been present during challenge. 
 

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Volunteers treated based on qPCR threshold of 100 Pf/mL showed a lower percentage, and 
severity of LFT abnormalities. 13/58 volunteers (22%) showed LFT derangements, with 
11/58 (19%) mild, 1/58 (2%) moderate, and 1/58 (2%) severe abnormalities. Importantly, all 
volunteers showed normalized values at study end. 
 
While severe/complicated malaria is clearly associated with liver damage with sequelae after 
natural infection (WHO Severe Malaria , Tropical Medicine and International health 2014), 
such severe complications do not seem to occur in naturally acquired uncomplicated clinical 
malaria (Abro et al. J Coll Physicians Surg Pak. 2009 and Ghoda et al. Trop Gastroenterol. 
2002). In this context it is important to note that parasite densities under these conditions are 
several logs higher than observed in the CHMI model.  
 
A clear explanation for the transient elevated transaminasesin our CHMI studies is not 
obvious. Although higher parasitaemia associates with higher chances of LFT elevations on 
group level, there is no clear relationship on individual levels. Given the diversity of drug 
regimens used, it also seems unlikely to be directly related to anti-malarials. Similarly, the 
timing, differential, and limited use of paracetamol does not support a clear relationship. 
Rather a combination of the above mentioned factors, and individual susceptibility may have 
triggered the observed abnormalities. 
 
Following an independent review of the safety committee, and a hepatology expert, it was 
concluded that these transient asymptomatic and severe LFT elevations are most likely a 
direct consequence of the malaria infection rather than reflecting a direct drug-induced liver 
injury caused by the antimalarial drug or other liver noxe. Given the rapid and spontaneous 
resolution of the observations and the absence of significantly elevated bilirubin these events 
were judged to not cause permanent subclinical liver damage, and to not preclude further 
studies utilising Plasmodium falciparum challenge.  
 
As a result of the LFT elevations our safety procedures for CHMI have been intensified. In 
the current trial, we will adhere to those stringent procedures that are relevant, including: 
1. Volunteers will undergo regular safety monitoring to assess asymptomatic liver 
function test abnormalities. 
2. Avoid additional triggers that may cause elevations in liver enzymes including alcohol 
from baseline up to 1 week post treatment. 
3. Maximum dose of 3000 mg per day of paracetamol/acetaminophen 
4. Initiation of a curative antimalarial treatment at a ALT/AST levels >5x ULN 
 

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In this study, volunteers will be treated on a qPCR threshold of 100 Pf/mL after CHMI. 
 
Furthermore, transient, slight decreases in renal function was observed during CHMI. 
Therefore, extra safety measurements are included. In the current trial, we will adhere to 
those stringent procedures that are relevant, including: 
1. Exclusion of subjects with abnormal renal function at baseline. 
2. Upon inclusion: Avoid additional triggers that may cause renal dysfunction 
a. Alcohol intake from baseline up to 1 week post-treatment 
b. Heavy physical exercise 
c. Party drugs 
3. Standard monitoring of renal function at day C-1; C+6; TD, TD+2, C+35. 
 
2. OBJECTIVES 
Phase 1 
Primary objective: 
To determine the safety and tolerability of administration of Pb(PfCS@UIS4) to healthy 
volunteers delivered by infectious mosquito bite. 
 
Exploratory objective:  
To analyse immune responses in volunteers immunized by Pb(PfCS@UIS4) and challenged 
by P.falciparum. 
 
Phase 2 
Primary objective: 
To determine the safety, tolerability and protective efficacy of immunization with 
Pb(PfCS@UIS4) against Controlled Human Malaria Infection by mosquito bite.  
 
Exploratory objective:  
To analyse immune responses in volunteers exposed to Pb(PfCS@UIS4) and challenged by 
P.falciparum, and to determine cytokine responses, coagulation activation and inflammation 
parameters in non-immune individuals(controls) infected with P. falciparum during malaria 
liver phase. 

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3. STUDY DESIGN 
This is a multicenter, open label, adaptive design study, and will be divided in two phases. 
Volunteers will be recruited and screened in the Havenziekenhuis in Rotterdam. 
Administration of Pb(PfCS@UIS4) to human volunteers will take place in the Radboudumc in 
Nijmegen. After inoculation, volunteers will be monitored in the Havenziekenhuis. Clinical 
chemical and microbiological analysis and storage of the samples of the infected volunteers 
will be performed in laboratories of the Erasmus MC in Rotterdam. Some biochemical and 
microbiological analyses will be performed in the Radboudumc. Please see figure 1 for study 
flow and activities of the centers. 
 
Phase 1, will be a multicenter, dose escalation trial. There will be no control group in this 
phase.  
A total of eighteen healthy adult volunteers will be recruited and divided over 3 groups. Three 
volunteers (Group 1) will be exposed to bites of five Pb(PfCS@UIS4)-infected mosquitoes. 
Volunteers will be closely monitored for adverse events for a period of 28 days. If safe on day 
14, another group of three volunteers (group 2) will be exposed to 25 Pb(PfCS@UIS4)-
infected mosquito bites. Finally, on day 14 of group 2, if considered safe, a group of 12 
volunteers (group 3) will be exposed to 75 Pb(PfCS@UIS4)-infected mosquito bites (n=12). If 
a safety issue or a stop criterion is met in the previous group after day 14, the following group 
will immediately be treated. These volunteers will visit the study centre once daily from day 1 
until day 10 after exposure and every other day from day 12 until day 20 after exposure. If 
one of the volunteers is not fit to participate in the study on day 0, an alternate volunteer will 
replace him/her. For this purpose one additional volunteer will be screened for possible back 
up in group 1 and 2 and two additional volunteers will be screened for possible back up for 
group 3. 
 
All exposed volunteers will be treated with a curative regimen of Malarone 
(atovaquone/proguanil), either at the time of detection of blood stage parasitemia by thick 
smear or 28 days after exposure to Pb(PfCS@UIS4) infected mosquitoes for group 1 and 2. 
Volunteers of group 3 will subsequently enter phase 2, if phase 1 (until day 28) is considered 
safe and if no blood stage parasitemia is detected during follow up. End of follow up will be 
100 days after exposure to Pb(PfCS@UIS4) for groups 1 and 2.  
Depending on the results of Phase 1, Phase 2 will commence. Criteria for proceeding to the 
second phase are described in section 8.4.4. 
 
 

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Phase 2, will be a multicenter, safety and efficacy trial with an adaptive design method. A 
control group will be included for the challenge phase. The trial objective is to evaluate the 
safety and protective efficacy of repeated exposure to Pb(PfCS@UIS4)-infected mosquito 
bites by subsequent controlled human malaria infection (CHMI) of test subjects. The same 
volunteers of group 3 will be exposed three additional times to the bites of 75 
Pb(PfCS@UIS4)-infected mosquitoes (n=12),at four, four, and four to eight week interval. 
Four weeks after the last exposure all volunteers will undergo a CHMI with 5 P. falciparum 
(NF54)-infected mosquitoes. Six infectivity control subjects will be recruited to receive 5 P. 
falciparum (NF54) infective mosquito bites per challenge infection. 
 
 
 
Based on the first results of phase 2 an ad hoc decision will be made to include a potential 
booster immunization and/or subsequent second challenge infection with five P. falciparum 
(NF54)-infected mosquitoes. The booster immunization and/or subsequent second challenge 
infection will be initiated using the following criteria: 
1. In case 2 to 4 volunteers are sterilely protected, all volunteers will receive a booster 
immunization followed by a second challenge infection, 3 months after the booster 
immunization.  
2. In case ≥5 volunteers are sterilely protected, all non protected volunteers will receive a 
booster immunization followed by a second challenge infection 3 months later; all 
protected volunteers will only receive a second challenge infection to assess durability of 
protection. 
 
All exposed volunteers are subjected to close follow-up after exposure, with regular visits to 
the clinical trial centre at the Havenziekenhuis in Rotterdam, periodic physical examinations, 
frequent blood sampling and adverse events will be recorded in a diary. For all subjects, 
during this period, all relevant investigations will be carried out on an outpatient basis, 
including frequent safety analyses.  
 
Exposed volunteers will be treated with a curative regimen of Malarone (atovaquone and 
proguanil), at the time of detection of blood stage parasitemia by thick smear during 
immunizations. During the challenge infection, all volunteers will receive a curative treatment, 
either at the time of blood stage parasitemia by qPCR or 28 days post CHMI. Follow up will 
end 100 days after the last exposure to Pb(PfCS@UIS4) and 35 days post CHMI for the 
infectivity control subjects. Depending on the results of the immunizations of phase 2, the 
volunteers will receive a CHMI. Criteria for proceeding to CHMI are described in section 
8.4.4. 
 

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Figure 1: Study schedule of Phase 1 and Phase 2 
 
 
4. STUDY POPULATION 
4.1 Population (base)  
The study population will be comprised of healthy adult male and female, malaria-naïve 
subjects. A maximum of 30 subjects will be enrolled to participate in the study. The 

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investigator will ensure that all subjects being considered for the study meet the following 
eligibility criteria. Subject eligibility is to be established and confirmed by checking through all 
inclusion/exclusion criteria at both screening and baseline. A relevant record (e.g. checklist) 
of the eligibility criteria will be stored with the source documentation at the study site. 
 
4.2 Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria:  
1. 
Subject is aged ≥ 18 and ≤ 35 years and in good health.  
2. 
Subject has adequate understanding of the procedures of the study and is able and 
willing (in the investigator’s opinion) to comply with all study requirements. 
3. 
Subject is willing to complete an informed consent questionnaire and is able to answer 
all questions correctly. 
4. 
Subject is able to communicate well with the investigator and is available to attend all 
study visits, lives in Rotterdam or in proximity to the trial centre (can be on site within 1 
hour) or is willing to stay in a hotel close to the trial centre during part of the study (phase 
1: from day of immunization to day 12 post-immunization; phase 2: from day of 
immunization to day 8 post-immunization.  
5. 
The subject will remain within the Netherlands from day -1 until day +28 after 
immunization during phase 1; from day -1 until day 12 after each immunization during 
phase 2, and during the challenge period, will not travel to a malaria-endemic area 
during the study period, and is reachable (24/7) by mobile telephone throughout the 
entire study period.  
6. 
Subject agrees to their general practitioner being informed and contacted about their 
participation in the study and agrees to sign a form to request the release by their 
General Practitioner (GP), and medical specialist when necessary, to the investigator(s), 
of any relevant medical information concerning possible contra-indications for 
participation in the study.  
7. 
The subject agrees to refrain from blood donation to Sanquin or for other purposes 
throughout the study period and for a defined period thereafter according to current 
Sanquin guidelines (3 years minimum, depending on serology). 
8. 
For female subjects: subject agrees to use continuous adequate contraception** and not 
to breastfeed for the duration of study.  
9. 
Subject agrees to refrain from intensive physical exercise (disproportionate to the 
subject’s usual daily activity or exercise routine) during the malaria challenge period.  

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10. 
Subject agrees to avoid additional triggers that may cause elevations in liver enzymes 
including alcohol from baseline up to 1 week post treatment. 
11. Subject has signed written informed consent to participate in the trial. 
 
(*Acceptable forms of 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 sterilisation (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.) 
 
4.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from participation 
in this study:  
1. Any history, or evidence at screening, of clinically significant symptoms, physical signs or 
abnormal laboratory values suggestive of systemic conditions, such as cardiovascular, 
pulmonary, 
renal, 
hepatic, 
neurological, 
dermatological, 
endocrine, 
malignant, 
haematological, infectious, immunodeficient, psychiatric and other disorders, which could 
compromise the health of the volunteer during the study or interfere with the 
interpretation of the study results. These include, but are not limited to, any of the 
following.  
1.1. 
Body weight <50 kg or Body Mass Index (BMI) <18 or >30 kg/m2 at screening.  
1.2. 
A heightened risk of cardiovascular disease, as determined by: an estimated ten 
year risk of fatal cardiovascular disease of ≥5% at screening, as determined by the 
Systematic Coronary Risk Evaluation (SCORE); history, or evidence at screening, 
of clinically significant arrhythmia’s, prolonged QT-interval or other clinically 
relevant ECG abnormalities; or a positive family history of cardiac events in 1st or 
2nd degree relatives <50 years old.  
1.3. 
A medical history of functional asplenia, sickle cell trait/disease, thalassaemia 
trait/disease or G6PD-deficiency.  
1.4. 
History of epilepsy in the period of five years prior to study onset, even if no longer 
on medication.  
1.5. 
Screening tests positive for Human Immunodeficiency Virus (HIV), active Hepatitis 
B Virus (HBV), Hepatitis C Virus (HCV)  

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1.6. 
Chronic use of i) immunosuppressive drugs, ii) antibiotics, 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.  
1.7. 
Any recent or current systemic therapy with an antibiotic or drug with potential anti-
malarial activity (chloroquine, atovaquone-proguanil, arthemether-lumefantrine, 
sulfadoxine-pyrimethamine, 
doxycycline, 
tetracycline, 
piperaquine, 
benzodiazepine, flunarizine, fluoxetine, tetracycline, azithromycin, clindamycin, 
erythromycin, hydroxychloroquine, etc.) (allowable timeframe for use at the 
Investigator’s discretion). 
1.8. 
History of malignancy of any organ system (other than localized basal cell 
carcinoma of the skin), treated or untreated, within the past 5 years.  
1.9. 
Any history of treatment for severe psychiatric disease by a psychiatrist in the past 
year.  
1.10. 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, or positive urine toxicology test for 
cannabis at inclusion.  
 
2. For female subjects: positive urine pregnancy test at screening and/or at the baseline 
visits, including baseline of immunizations (I-1) and or baseline before CHMI (C-1). 
3. Any history of malaria, positive serology for P. falciparum, or previous participation in any 
malaria (vaccine) study.  
4. Known hypersensitivity to or contra-indications (including co-medication) for use of 
sulfadoxine-pyrimethamine, 
piperaquine, 
chloroquine, 
Malarone®, 
artemether-
lumefantrine, primaquine or history of severe (allergic) reactions to mosquito bites. 
5. Receipt of any vaccinations in the 3 months prior to the start of the study or plans to 
receive any other vaccinations during the study period or up to 8 weeks thereafter. 
6. Participation in any other clinical study in the 30 days prior to the start of the study or 
during the study period.  
7. Being an employee or student of the department of Medical Microbiology and Infectious 
Diseases of the Erasmus MC or Radboudumc, or the department of Internal Medicine of 
the Radboudumc or Havenziekenhuis.  
8. 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.  
 

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4.4 Sample size calculation 
Phase 1 (safety trial) is a first in-human observational trial, for which no formal power 
calculation has been performed. Based on previous experience and convention, we will start 
with a small group of three volunteers and subsequently expand to a group of twelve 
volunteers. The occurrence of (rare) AEs will follow a Poison distribution and the proposed 
sample size will only allow a crude estimation of confidence intervals. If, for example, 1 
individual (out of 12) would experience an AE, the 95% confidence interval would have an 
upper limit of 46.4%, indicating that an actual risk exceeding this percentage is ruled out with 
95% confidence. Previously performed phase 1 dose-escalation trials with malaria candidate 
vaccines used similar population sizes and examined the risk of AEs with more precision in 
follow-on trials. We envisage a similar route for the current intervention where the current 
trials form an essential first assessment of safety in a small, intensively monitored population. 
 
Phase 2 (efficacy trial) is a pilot study designed to provide proof-of-concept for the induction 
of sterile protection by transgenic P. berghei (Pb(PfCS@UIS4)) parasites against malaria 
challenge infection with the P. falciparum NF54 strain. From 13 previous CHMIs with NF54 
we know that the time to positive qPCR with >100 parasites/ml after a single challenge 
infection is on average 7.7 days, with a standard deviation of 1.46 days. In order to detect a 
difference in pre-patent period of 2.5 days between intervention and control groups (α=0.05, 
1-ß=0.80) a sample size of 10 volunteers in the treatment group and 5 volunteers in the 
control group is required. Correcting for a possible withdrawal rate of 2 volunteers in group 3, 
and 1 volunteer in the control group, we therefore chose a sample size of 12 volunteers in 
the intervention group and 6 volunteers in the control group. 
 
 
 

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5. TREATMENT OF SUBJECTS 
5.1 Investigational product/treatment 
No investigational products are applied during this study. 
 
5.2 Use of co-intervention (if applicable) 
In phase 1, all exposed volunteers of group 1 and 2 will be treated with a curative regimen of 
Malarone® (atovaquone/proguanil), either at the time of detection of blood stage parasitemia 
by thick smear or 28 days after exposure to Pb(PfCS@UIS4) infected mosquitoes. Group 3 
will only be treated in phase 1 if a blood stage parasitemia occurs as detected by thick 
smear.  
In phase 2, all volunteers will be treated with a curative regimen of Malarone® 
(atovaquone/proguanil) as soon as blood stage parasitemia is detected by thick smear 
(during the immunizations period) or qPCR (during CHMI) at any point in the trial or on day 
28 following CHMI. 
 
Pre-emptive rescue treatment with Malarone® can commence whenever deemed necessary 
by the investigator.  
 
The following list of concomitant medications or conditions are contraindications to 
atovaquone/proguanil, based on the product monograph.  
Concomitant medications: 
 
Use of anticoagulants (warfarin and other coumarin based anticoagulants). 
 
Use of rifampicin, rifabutin, tetracycline or metoclopramide. 
 
Use of indinavir. 
 
 
5.3 Escape medication (if applicable) 
Volunteers are advised to take paracetamol for complaints secondary to the CHMI (fever, 
muscle aches, headache, etc.). Paracetamol or any other symptomatic treatment will be 
supplied to the volunteers without charge. The maximum dose of paracetamol is 4 grams a 
day.  
 
Although volunteers with a previous history of severe reactions to mosquito bites will be 
excluded from the study, some volunteers may experience local reactions to the mosquito 
bites and require topical and/or oral medications. Topical tripelennamine and topical steroids 
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will be available in the clinical trials center for distribution to the volunteers. Due to the 
possible, yet unknown, effects on sporozoite migration from the skin in an area treated with 
steroids, volunteers will be asked to wait 2 hours after biting prior to applying steroid cream 
to affected areas. Additional medications available to volunteers by the clinical investigators 
will be oral cetirizine or other anti-histamines. Volunteers with exuberant reactions to 
mosquito bites may receive cetirizine prophylactically before immunization to abrogate skin 
reactions such as redness, swelling and itching. 
 
 
 
 

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6. INVESTIGATIONAL AND NON-INVESTIGATIONAL PRODUCT (CHALLENGE 
PRODUCT) 
 
Volunteers will be exposed to the bites of five, 25 or 75 Pb(PfCS@UIS4)-infected mosquitoes 
on day 0 of the study in phase 1. In phase 2, volunteers will receive three additional 
immunizations, and a possible 5th booster immunization with Pb(PfCS@UIS4), and/or a 
second challenge infection depending on the outcome of the first controlled challenge 
infection (see also section 3). Five P. falciparum NF54-infected Anopheles stephensi 
mosquitoes will be used for the purpose of controlled human malaria infections (CHMI).  
 
Pb(PfCS@UIS4 is a genetically modified P.berghei parasite line in which the Plasmodium 
falciparum CS protein gene has been inserted.  
 
6.1 Name and description of non-investigational products 
6.1.1 
Name and description of investigational product (Pb(PfCS@UIS4)) 
A genetically modified P. berghei parasite is used in this study, the Pb(PfCS@UIS4) parasite, 
where the PfCS gene has been inserted in the 230p neutral locus of P. berghei and that, 
therefore, expresses PfCS in addition to the endogenous PbCS. 
6.1.2 
Name and description of non-investigational product (P. falciparum 
NF54)  
P. falciparum isolate NF54 infected Anopheles stephensi mosquitoes for the purpose 
controlled human malaria infection (CHMI). 
 
6.2 Summary of findings from non-clinical studies 
6.2.1 
Summary of findings from non-clinical studies (Pb(PfCS@UIS4)) 
Pb(PfCS@UIS4) has been extensively investigated in non-clinical studies with (chimeric) 
mice, rabbits and Rhesus monkeys. The first milestones of the proof-of-principle of the 
proposed strategy have recently been completed through validation of the three core 
premises. 1) Pb(PfCS@UIS4) can infect and develop in human hepatocytes. 2) The parasite 
is unable to develop inside human erythrocytes. 3) Immunization with Pb(PfCS@UIS4) can 
elicit immune responses against P. falciparum.  
 
The non-clinical studies have demonstrated that the hepatic and mosquito infectivities of 
Pb(PfCS@UIS4) are similar to those of the wild-type parental P. berghei strain, showing that 

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the genetic modification introduced did not in any way impair the resulting parasite’s 
sporogonic development or ability to infect rodent hepatic cells in vitro, ex vivo and in vivo. 
The non-clinical data have further shown that Pb(PfCS@UIS4) parasites are able to infect 
and develop in vitro inside human hepatoma cells and human immortalized hepatocytes, as 
well as in vivo in human hepatocytes in chimeric (liver-humanized) mouse livers, warranting 
the basis for the proposed immunization approach in humans. The non-clinical studies have 
also shown that Pb(PfCS@UIS4) is unable to develop inside human erythrocytes in a blood-
humanized mouse model, which supports its safety for use in humans, in full alignment with 
the widely accepted notion that this is a non-pathogenic organism for humans. Nevertheless, 
the drug sensitivity studies further showed that, in the extremely unlikely event of a 
breakthrough infection by Pb(PfCS@UIS4), the blood stages of this parasite are sensitive to 
and can be completely eliminated by treatment with the anti-malarials Malarone© and 
Coartem©. 
 
Moreover, the non-clinical studies showed that the proposed strategy can also work in non-
natural hosts, such as rabbits, and Rhesus monkeys. These studies have shown that 
Pb(PfCS@UIS4) can infect and develop inside rabbit-, and Rhesus monkey- hepatocytes, 
both ex vivo and in vivo. Crucially, they have also shown that Pb(PfCS@UIS4) is unable to 
cause a blood-stage infection in rabbits or Rhesus monkeys, neither when infection is 
initiated by sporozoite injection nor when it is initiated by the injection of as many as 100 
million infected red blood cells in rabbits.  
 
Importantly, the non-clinical program also showed that immunization of mouse models with 
Pb(PfCS@UIS4) elicits P. berghei/P. falciparum cross-protective, as well as P. falciparum 
CS-specific cellular immune responses. Immunization experiments employing both mice and 
rabbits further showed that Pb(PfCS@UIS4) elicits P. falciparum CS-specific humoral 
responses capable of recognizing P. falciparum sporozoites. Furthermore it inhibits a P. 
falciparum infection of human immortalized hepatocytes and of human hepatocytes in 
chimeric mouse livers. The data also showed that infection with Pb(PfCS@UIS4) induces 
Type I IFN pathways in both mouse and human hepatocytes of chimeric mice. Finally, the 
data showed that immunization of monkeys with Pb(PfCS@UIS4) elicits strong immune 
responses against PfCS protein and P. falciparum sporozoites, clearly indicative of vaccine-
take and immunogenicity of Pb(PfCS@UIS4). 
 
Furthermore, biodistribution studies, drug sensitivity testing and a repeated-dose toxicology 
study have recently been completed.  

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Overall, the non-clinical program provides solid evidence that the parasite proposed to be 
used in this study fulfills all the safety requirements for human use, as it is highly unlikely to 
cause disease in humans. Besides, the parasite also meets the efficacy requirements to be 
used as a vaccine candidate, as it was shown to successfully infect human hepatocytes, a 
requirement for immunity generation by this type of vaccine candidate. Finally, the non-
clinical data shows that the parasite is able to induce immune responses that are able to 
recognize and inhibit a P. falciparum infection.  
 
Please refer to the Investigator’s Brochure for detailed description of all the non-clinical 
studies. 
 
6.2.2 
Summary of findings from non-clinical studies (P. falciparum NF54) 
The P. falciparum NF54 strain was isolated from a patient living in the Schiphol-area in the 
Netherlands. In in vitro studies, this parasite has been shown completely susceptible to 
multiple antimalarials, including chloroquine, mefloquine, atovaquone/proguanil and 
arthemeter/ lumefantrine (see D2c, ‘Product information Plasmodium falciparum infected 
Anopheles mosquitoes’). 
6.3 Summary of findings from clinical studies 
6.3.1 
Summary of findings from clinical studies (Pb(PfCS@UIS4)) 
This is the first trial of Pb(PfCS@UIS4) in humans. Therefore, no clinical studies are 
available yet. 
 
6.3.2 
Summary of findings from clinical studies (P. falciparum NF54) 
CHMIs are well accepted as a powerful tool for the evaluation of parasite development in 
humans. The Radboudumc, ErasmusMC and the Havenziekenhuis have the experience and 
infrastructure to conduct CHMIs. The Radboudumc has developed the very sensitive method 
of parasite detection by real-time qPCR available that will allow us to detect malaria infection 
in an early stage, providing early treatment.  
There is a large clinical experience with infecting humans by the bite of P. falciparum-
infected mosquitoes. Since 1986 more than 3,500 volunteers have had a CHMI by the bites 
of mosquitoes fed on cultures of P. falciparum gametocytes to produce P. falciparum 
sporozoites [26]. This has proved to be a reproducible, predictable and safe method of 

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inducing P. falciparum malaria. The results of such studies were summarized in 1997 [27], in 
2007 [33] and  in 2012 [29].  
The P. falciparum isolate NF54 has been tested for the purpose of challenge infection in 353 
volunteers in 22 CHMI studies in the Radboudumc and 2 studies in collaboration with the 
ErasmusMC and the Havenziekenhuis. In these studies generally all malaria-naive 
volunteers developed patent parasitemia after bites from five NF54 infected mosquitoes. The 
median time to blood stage parasitemia detectable by qPCR was 7.0 days. Mild-moderate 
solicited adverse events were generally experienced by all study subjects post-infection, 
most common were headache (95%), general malaise/fatigue (65%) and fever (90%). 
Gastro-intestinal complaints, including nausea, diarrhoea and abdominal pain were 
experienced by several subjects, mainly following intake of atovaquone-proguanil. Symptoms 
that were severe enough to prohibit daily activities occurred in 49% of participants. All 
volunteers have been successfully treated with anti-malarials. 
6.4 Summary of known and potential risks and benefits 
6.4.1 
Summary of known and potential risks and benefits of   
Pb(PfCS@UIS4) immunization 
There is no benefit expected for subjects participating in this study. The risk to subjects after 
immunization with Pb(PfCS@UIS4)-infected mosquitoes in this trial will be minimized by 
adherence to the inclusion/exclusion criteria and close clinical monitoring, which ensures that 
subjects with a ‘break-through’ blood stage infection will be treated at earliest stages of 
parasitemia. The risks of a CHMI for malaria-naïve subjects include i) discomfort induced by 
mosquito bites, ii) discomfort associated with periodic blood drawing and iii) risk of acquiring 
a blood stage parasitemia with Pb(PfCS@UIS4). 
Mosquito bites are known to cause mild discomfort associated with mosquito feeding. A 
small amount of inflammation and pruritus typically accompanies the bite of the insect. It is 
possible that some subjects will experience local skin induration. Anaphylaxis to the bite of a 
mosquito is extremely rare and has never been reported after CHMI. While significant allergic 
reactions are extremely rare, in the event of an allergic reaction, topical steroids, anti-
histamines, epinephrine, on-call physician and resuscitation equipment are available on site. 
The Radboudumc, an established site for CHMIs, is fully equipped to manage anaphylaxis 
and any other medical emergency.  
Frequent blood draws will be necessary to closely monitor the subjects and to perform thick 
smears for early detection of blood stage parasitemia. Universal precautions will be 
maintained for the protection of the volunteer and the study personnel. The total amount of 
blood collected will be maximally 1500 ml over the entire trial period (including screening 

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period and potential 5th booster immunization and/or 2nd challenge infection), in agreement 
with guidelines of the Sanquin blood bank.  
Intensive follow-up with thick smears performed on samples taken once daily will allow for 
detection of parasites at a very early stage. As therapy will be initiated at this early stage, 
dangerously high levels or prolonged duration of parasitemia that would put the subject at 
undue risk, will not occur. Researchers at the Radboudumc and the ErasmusMC and 
Havenziekenhuis have extensive experience with the care of clinical malaria.  
Malarone® (atovaquone-proguanil) is effective against Pb(PfCS@UIS4), as tested pre-
clinically, and is a marketed first-line treatment for malaria and will be used. Furthermore, 
Malarone® is generally well tolerated. Common adverse reactions (≥5%) in adults associated 
with Malarone® treatment include abdominal pain, nausea, vomiting, headache, diarrhoea, 
asthenia, anorexia, and dizziness. Those volunteers intolerant to Malarone® will be given 
alternative treatment according to the national malaria treatment guidelines, such as 
arthemether-lumefantrine.  
The exposure to Pb(PfCS@UIS4) -infected mosquito bites will occur at Radboudumc 
insectary, which has a double-door barrier system along with a double blower (negative 
pressure wind blockade) to prevent mosquito flight across entryways. 
6.4.2 
Summary of known and potential risks and benefits of Pf challenge 
There is no benefit expected for subjects participating in this study. The risk to subjects after 
challenge infection with P. falciparum NF54-infected mosquitoes will be minimized by 
adherence to the inclusion/exclusion criteria and close clinical monitoring, which ensures that 
subjects with malaria will be treated at earliest stages of parasitaemia. The risks of a CHMI 
for malaria-naïve subjects include i) discomfort induced by mosquito bites, ii) discomfort 
associated with periodic blood drawing and iii) risk of acquiring mild clinical P. falciparum 
malaria.  
Mosquito bites are known to cause mild discomfort associated with mosquito feeding. A 
small degree of inflammation and pruritus typically accompanies the bite of the insect. 
Anaphylaxis after mosquito bites is extremely rare and has never been reported after CHMI. 
While significant allergic reactions are extremely rare, in the event of an allergic reaction, 
epinephrine, anti-histamines, on-call physician and resuscitation equipment are available on 
site. The Radboudumc, an established site for CHMIs, is fully equipped to manage 
anaphylaxis and any other medical emergency.  
Frequent blood draws will be necessary to closely monitor the subjects and to perform qPCR 
for detection of P. falciparum parasitaemia. Universal precautions will be taken for protection 
of the volunteer and study personnel. The total amount of blood collected will be maximally 
1500 ml over the entire trial period (including screening period and potential 5th booster 

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immunization and/or 2nd challenge infection), in agreement with guidelines of the Sanquin 
blood bank. 
Intensive follow-up with qPCR performed on samples taken once daily, will ensure early 
treatment - preventing high levels or prolonged duration of parasitaemia that would put the 
subject at undue risk. Severe/complicated malaria has never been described in a CHMI. Mild 
malaria symptoms include headache, myalgia, fever, chills, sweats, nausea, vomiting, and 
diarrhoea. Researchers at the Radboudumc have extensive experience with the care of 
clinical malaria.  
Although subjects often become symptomatic with mild malaria after CHMI, rapid diagnosis 
by qPCR and/or thick smears and subsequent treatment quickly attenuates the illness so that 
the infection does not place the subject at undue risk. 
The exposure to infected P. falciparum NF54-infected mosquito bites will occur at 
Radboudumc insectary which has a double-door barrier system along with a double blower 
(negative pressure wind blockade) to prevent flight across entryways. 
 
6.5 Description and justification of route of administration and dosage 
6.5.1 
Description and justification of route of administration and dosage 
(Pb(PfCS@UIS4)) 
Pb(PfCS@UIS4) will be administered through the bites of infected Anopheles stephensi 
mosquitoes.  
Being the first in-human trial of Pb(PfCS@UIS4), three different doses were chosen. The first 
dose (5 mosquito bites) allows for optimal safety as it is a dose most commonly used for 
CHMI trials worldwide. This will allow for a very safe first evaluation of the phenotype. If 
uneventful, the dose is increased to 25 bites and, if safe, subsequently to 75 bites, which was 
chosen as the lowest dose that has the potential to induce immunity in subsequent 
immunisation trials based on data from comparable trials with non-attenuated sporozoites 
under chloroquine prophylaxis (CPS) and radiation-attenuated sporozoites (RAS). CPS is 
known to induce immunity in a subset of volunteers at a total dose of 24 mosquito bites, 
complete protection at a dose of 45 bites. RAS is likely to induce immunity in a subset of 
volunteers at cumulative doses >200 bites and full immunity at doses >1000 bites8. CPS is 
thought more efficient than Pb(PfCS@UIS4), because it allows for the complete liver stage 
development of P. falciparum parasites (and thus Pf-specific antigenic exposure), whereas 
RAS is though less efficient than Pb(PfCS@UIS4) because sporozoites will arrest very early 
in their development. With a dose of 75 bites, we envision that we will achieve a dose of 
>200 infectious bites in trials with 4  immunizations, which we believe should be sufficient to 

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obtain proof-of-concept. The potential 5th dose will function as a booster immunization for the 
volunteers that are not or only partially protected after the 4th immunization. 
6.5.2 
Description and justification of route of administration and dosage (P. 
falciparum NF54) 
Study subjects will be exposed to malaria for the purpose of challenge infection through 
(cutaneous) bites of P. falciparum NF54-infected female Anopheles stephensi mosquitoes. 
This is the natural route of infection and the one with which most experience has been 
accumulated in CHMI trials. Volunteers will be exposed to the bites of 5 P. falciparum NF54 
infected mosquitoes, which is the gold-standard dosage for CHMI studies at the 
Radboudumc, based on extensive experience with the P. falciparum NF54 strain. 
 
6.6 Dosages, dosage modifications and method of administration 
6.6.1 
Dosages, dosage modifications and method of administration 
(Pb(PfCS@UIS4)) 
Please also refer to paragraph 6.5.1. Because, at time of post-immunization dissection, not 
all 75 A. stephensi mosquitoes will be found to be infected with sporozoites and/or will not 
have taken a blood meal, a higher number of mosquitoes will be used at each immunization 
time point to achieve an end result of 75 infectious bites.  Based on a pre-clinical repeated-
dose toxicity study the average mosquito feeding rate was 80.5%, and the average mosquito 
mortality 4%. Therefore, 97 mosquitoes will be used at each immunization time point to 
obtain the targeted 75 infected mosquito bites. The mosquitoes will be placed in a container 
and allowed to feed for 15 minutes. All mosquitoes will be checked for blood meal 
assessment. When the number of blood-fed mosquitoes is below <65, <55, or <45 the 
volunteer will be exposed to extra bites of 10, 20 or 30 infected- mosquitoes respectively. 
This method of immunization will enable investigators to monitor that volunteers do receive 
approximately 75 infectious bites at each immunization, although the actual bite number may 
be slightly more or less. 
 
 
6.6.2 
Dosages, dosage modifications and method of administration (P. 
falciparum NF54) 
See also paragraph 6.5.2. Subjects receiving a challenge infection with the P. falciparum 
NF54 strain will receive 5 infectious bites from A. stephensi mosquitoes infected with P. 

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falciparum NF54 sporozoites. Previous studies have demonstrated that with this dosage, 
near 100% of malaria-naïve volunteers develop a blood stage malaria infection [34].  
 
 
6.7 Preparation and labelling of Investigational, Non Investigational Medicinal 
Product 
6.7.1 
Preparation and labelling of Investigational Medicinal Product 
(Pb(PfCS@UIS4)) 
The culture of parasites and infection of mosquitoes has been a routine procedure for over 
20 years in the Malaria Unit of the Central Animal Facility of the Radboudumc, Nijmegen. 
In vivo cultures of P. berghei parasites and Pb-infected mosquitoes have been extensively 
produced in the Malaria Unit of the Radboudumc, Nijmegen. Specifically, Pb(PfCS@UIS4- 
infected mosquitoes have already extensively been produced for the toxicology study by the 
Radboudumc. 
 
A quality control protocol of the sporozoites used for immunization in human trials is outlined 
in Figure 2. It includes (I) the creation of a Master Bank of parasites, with complete 
microbiological and genetic analysis of the samples stored there (see Master parasite stocks 
control below). (II) The use of SPF mice, subjected to complete microbiological analysis, 
which will be infected with Master Bank aliquot (see Animal quality control: SPF mice below). 
(III) The collection of salivary gland sporozoites injected by infected mosquitoes, followed by 
genetic analysis to characterize the parasite-strain identity by PCR and exclude possible 
contamination. The whole cycle will be performed at least once before the actual trial and the 
analysis in (II) and (III) will be repeated on the actual batch to be used in the trial.  
 
 
 

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Figure 2 Quality control plan for immunizing sporozoite material. 
 
Any culture material or cages with mosquitoes that contain genetically attenuated 
Pb(PfCS@UIS4) parasites will be labelled with a bright yellow label (Section D3).  
 
Please refer to D2a IMPD of Pb(PfCS@UIS4) and D2i Background Information 
Pb(PfCS@UIS4) infected Anopheles mosquitoes for a detailed description of the generation 
and production of Pb(PfCS@UIS4) and Pb(PfCS@UIS4)- infected Anopheles mosquitoes. 
6.7.2 
Preparation and labelling of Non Investigational Medicinal Product (P. 
falciparum NF54) 
The culture of parasites and infection of mosquitoes has been a routine procedure for over 
20 years in the Malaria Unit of the Central Animal Facility of the Radboudumc, Nijmegen. 
The P. falciparum NF54 isolate originates from the Schiphol area. The isolate was originally 
derived from patient material and was cultured in vitro in RPMI-1640 medium with 10% 
serum and 5% haematocrit red blood cells. Both the serum and the red blood cells are 
obtained from the Nijmegen department of the Sanquin Bloedbank region Zuid-Oost. Both 
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HCV, HumanT-lymphotropic Virus (HTLV) 1+2 and syphilis. The cultures are checked for 
bacterial, fungal and Mycoplasma contamination.  
To produce infectious gametocytes, the asexual parasites will be cultured in vitro. After 14 
days of culture, the sexual stage parasites will be harvested for feeding to 1-5 days old 
laboratory cultured Anopheles stephensi mosquitoes via a ‘membrane feeder’. The 
percentage P. falciparum-infected mosquitoes will be assessed 6-9 days after feeding and 
one day prior to the CHMI. 
Mosquitoes are kept in the same midi‐cage from Membrane Feed for Sporozoite production 
(MFS) until the day before CHMI. Mosquitoes infected with different strains are kept in 
different midi-cages, labeled with distinguishable coloured labels identifying the study, the 
strain, the date of MFS, and the feeder‐number. On the day before CHMI, a sample of 10 
mosquitoes from each batch is checked for the presence of sporozoites and a sample of 10 
mosquitoes is assessed for the average number of sporozoites per mosquito. Based on 
these results, the best batch is chosen for CHMI. Mosquitoes are then transferred from midi‐ 
cages to small CHMI‐cages. Each step in this process is performed by an experienced 
technician, and checked and recorded on standardized forms by another technician 
according to standard operating procedures. 
 
6.8 Drug accountability 
The date, time of collection and person collecting both the Pb(PfCS@UIS4)- and P. 
falciparum NF54- infected mosquitoes is filled in on a standard table. This section will be 
signed by the responsible employee. 
 

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7. METHODS 
7.1 Study parameters/endpoints 
7.1.1 Main study parameters/endpoints 
Phase 1 
Primary endpoints 
 
Frequency and magnitude of adverse events in study groups. 
 
Presence of parasitemia after exposure to Pb(PfCS@UIS4) as assessed by thick smear. 
 
Secondary endpoints: 
 
Immunogenicity of Pb(PfCS@UIS4) as assessed by ELISA and IFA. 
 
Phase 2 
Primary endpoints 
 
Frequency and magnitude of adverse events in study groups. 
 
Time to parasitemia after controlled human malaria infection with the wild-type Plasmodium 
falciparum NF54 strain, as detected by qPCR. 
 
Secondary endpoints: 
 
Immunogenicity of Pb(PfCS@UIS4) as assessed by ELISA and IFA. 
 
7.1.2 Exploratory parameters/endpoints 
Phase 1 
 
To explore the composition and function of immune responses after exposure to 
Pb(PfCS@UIS4) including cytokine profile and Pb(PfCS@UIS4) induced antibodies and 
cellular (both adaptive and innate) immune responses 
 
Phase 2 
 
To explore the composition and function of immune responses after exposure to 
Pb(PfCS@UIS4) including cytokine profile and Pb(PfCS@UIS4) induced antibodies and 
cellular (both adaptive and innate) immune responses 
 
To explore the changes in metabolomics profile of urine and serum/whole blood after CHMI. 
 
To determine cytokine responses, coagulation activation and inflammation parameters in 
non-immune individuals (controls) infected with P. falciparum during malaria liver phase.  
 

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7.2 Randomisation, blinding and treatment allocation 
This is an open-label study. No randomisation or blinding will be performed. 
 
7.3 Study procedures 
7.3.1 
Screening period: Screening, Inclusion and Baseline visits 
Volunteers who wish to participate in the trial will be asked to complete an informed consent 
questionnaire. Their understanding of the trial will be tested after discussing the study with 
the investigator during informed consent, and prior to being asked to sign and date the 
consent form. Volunteers who fail to answer all questions correctly on their first attempt are 
allowed to re-take the questionnaire following further discussion with the investigator, and 
provided they subsequently answer all questions correctly, they may then complete the 
consent form and be screened for the trial. 
Subjects who sign informed consent will undergo complete screening including a medical 
history, physical examination, vital signs, ECG, urine tests and laboratory evaluations (see 
sections 7.3.5-7.3.9). If, upon physical examination, vital signs or laboratory values are out of 
the normal range a repeat measurement may be obtained. 
Subjects who meet the eligibility criteria will be invited back for enrollment into the study at 
the inclusion visit, which will occur 6 to 8 days prior to the planned immunization or challenge 
day (day 0). Some screening assessments, including physical examination, vital signs, urine 
tests and laboratory evaluations, will be repeated at this inclusion visit.  
Following this screening period of up to 120 days, subjects who continue to meet the 
eligibility criteria (section 4.2 and 4.3) will present to the study site the day before the 
immunization or challenge infection for baseline assessments. Patient history will be taken 
and all adverse events that have occurred since screening will be noted. Only subjects who 
still meet the inclusion criteria will receive bites of infected mosquitoes. For each subject, the 
study start will be defined as the day of the inclusion visit. 
 
7.3.2 
Administration of Pb(PfCS@UIS4) 
Phase 1 
On the immunization day, all subjects will be exposed to the bites of five (group 1), twenty-
five (group 2), or seventy-five (group 3) Pb(PfCS@UIS4) -infected mosquitoes in the 
Radboudumc in Nijmegen. Mosquito feeding will be allowed for 15 minutes. Volunteers will 
receive a local treatment (tripelennamine crème) for mosquito bites, if needed, and will be 
observed for 15 minutes after the feed. Directly after the feed, the mosquitoes will be 

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dissected (group 1 and group 2) or checked for blood-fed (group 3) by a technician of the 
mosquito unit according to standard operating procedures. Exposure will be repeated until 
five, twenty-five, or seventy-five infected mosquitoes have fed on each volunteer for group 1, 
2 and 3, respectively.  
As long as there are volunteers present in the mosquito unit, there will be supervision by one 
of the clinical investigators. Another clinical investigator will be on call, in case of emergency. 
Emergency aid kits will be present and readily available at any location, whenever there are 
volunteers present.  
After the immunization, subjects will be observed closely according to an intensive out-
patient follow-up schedule including frequent safety analyses (see section 7.3.14 for details). 
The study design is illustrated in more detail in the flowchart in section 7.3.14.  
Subjects are required to reside locally within close proximity to the study site (within 1 hour 
from the Havenziekenhuis). In the cases where subjects are not local, subjects will be 
provided accommodation at a hotel nearby from the immunization day until day 12 post-
immunization. For all subjects, during this period all relevant investigations will be carried out 
on an outpatient basis at the Havenziekenhuis, including frequent safety analyses (section 
7.3.14 for details).  
From the first day post-immunization up until the twentieth day post-immunization, 
assessments of parasitemia will be performed using thick smears on visit days. From day 21 
until day 28 subjects will only be seen if they have relevant symptoms. Subjects will measure 
their body temperature twice daily, and the clinical investigators will contact all volunteers by 
telephone once daily to ask if there are any symptoms or complaints. Subjects of group 1 
and 2, will visit the study site for a follow-up visit on day 1, 2 and 3 after treatment (TD+1, 2 & 
3). Subjects of group 3 will visit on day 28, and no treatment will commence if considered 
safe. All subjects of group 1 and 2 will be seen for a final control visit on day 100 after 
immunization. Group 3 will subsequently start phase 2 when phase is considered safe. The 
study design is illustrated in more detail in the flowchart in section 7.3.14.  
Thick smear assessment of parasitemia will be performed directly in volunteer samples.  
As soon as a thick smear result is deemed positive for malaria parasites, the technician will 
inform the trial clinician. Treatment will be initiated after a single positive thick smear. If 
treatment has to be initiated, the trial clinician will immediately contact the volunteer, who will 
return to the clinic to receive Malarone® treatment. Preferably, the volunteer should return 
immediately or at least within 1 hour. If the volunteer is not reachable by phone, his/her 
contact person will be called and a search is started.  

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During the entire study period subjects will be instructed to call the trial physicians at any 
time if they experience symptoms. The trial physician can decide to initiate additional 
diagnostics or treatment at all times.  
For unexpected laboratory abnormalities, the laboratory test can be repeated. If there is any 
ambiguity regarding the decision to include or exclude a volunteer, the clinical supervisor will 
discuss the case with the local safety monitor and make the final decision after that, if 
necessary with consultation of a specialist. If volunteers prove to be eligible, they will be 
invited to the next visit. If a volunteer is not fit to participate to undergo the immunization on 
the planned date, immunization may be delayed up to 10 days. 
All subjects who are exposed to Pb(PfCS@UIS4) will be treated with Malarone®. Treatment 
after immunization will be based on the above mentioned criteria. Additionally, treatment can 
be initiated in any of the following situations:  
1. 
By decision of study doctor or the safety monitor  
2. 
In consultation with the cardiologist 
3. 
On request of the volunteer  
4. 
On day 28 post immunization, if the volunteer has remained thick smear negative  
5. 
When LDH > 1000 U/l  
6. 
When thrombocytes < 120 x 109/L  
 
Phase 2 
When phase 1 is terminated and is considered safe, the same volunteers of group 3 will be 
exposed three more times to bites of 75 mosquitoes infected with Pb(PfCS@UIS4),at four, 
four, and four to eight week interval. Three to four weeks after the last exposure all 
volunteers will undergo a controlled human malaria infection (CHMI) with 5 P. falciparum 
(NF54)-infected mosquitoes. Six infectivity control subjects will be recruited to receive 5 P. 
falciparum (NF54) infective mosquito bites per challenge infection. 
 
 
 
Based on the first results of phase 2 an ad hoc decision will be made to include a potential 
booster immunization and/or subsequent second challenge infection with five P. falciparum 
(NF54)-infected mosquitoes. The booster immunization and/or subsequent second challenge 
infection will be initiated using the following criteria: 
1. In case 2 to 4 volunteers are sterilely protected, all volunteers will receive a booster 
immunization followed by a second challenge infection, 3 months after the booster 
immunization.  
2. In case ≥5 volunteers are sterilely protected, all non protected volunteers will receive 
a booster immunization followed by a second challenge infection 3 months later; all 

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protected volunteers will only receive a second challenge infection to assess 
durability of protection. 
 
One day before the immunization days, all subjects will visit the clinical trial site for baseline 
assessments in the Havenziekenhuis in Rotterdam. On the immunization days, all subjects of 
group 3 will receive bites of seventy-five (group 3) Pb(PfCS@UIS4) -infected mosquitoes in 
the Radboudumc in Nijmegen. The same procedures will be performed as during the 
immunizations in phase 1.  
After immunizations, subjects will be observed according to an out-patient follow-up schedule 
including safety analyses (see section 7.3.14 for details). The study design is illustrated in 
more detail in the flowchart in section 7.3.14. 
Subjects are required to reside locally within close proximity to the study site (within 1 hour 
from the Havenziekenhuis). In cases where subjects are not local, subjects will be provided 
accommodation at a hotel nearby from the immunization day until day 8 post-immunization. 
For all subjects, during this period all relevant investigations will be carried out on an 
outpatient basis at the Havenziekenhuis, including frequent safety analyses (section 7.3.14 
for details).  
Subjects will visit the clinical trial site on day 1, and day 8 post-immunization. Safety 
analyses and assessment of parasitemia will be performed using thick smears on visit days.  
Thick smear assessment of parasitemia will be performed directly in volunteer samples.  
From day 1 until day 20 subjects will only be seen outside the normal visits if they have 
relevant symptoms. Subjects will measure their temperature twice daily, and the clinical 
investigators will contact all volunteers by telephone once every other day to ask if there are 
any symptoms or complaints.  
During the entire study period subjects will be instructed to call the trial physicians at any 
time if they experience symptoms. The trial physician can decide to initiate additional 
diagnostics or treatment at all times.  
For unexpected laboratory abnormalities, the laboratory test can be repeated. If there is any 
ambiguity regarding the decision to include or exclude a volunteer, the clinical supervisor will 
discuss the case with the local safety monitor and make the final decision after that, if 
necessary with consultation of a specialist. If volunteers prove to be eligible, they will be 
invited to the next visit. If a volunteer is not fit to participate to undergo the challenge infection 
or immunization on the planned date, immunization or challenge infection may be delayed up 
to 10 days. 

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All subjects who are exposed to P. falciparum or Pb(PfCS@UIS4) will be treated with 
Malarone®. Treatment after challenge infection will be based on the above mentioned 
criteria. Additionally, treatment can be initiated in any of the following situations:  
1. By decision of study doctor or the safety monitor  
2. In consultation with the cardiologist 
3. On request of the volunteer  
5. When LDH > 1000 U/l  
6. When thrombocytes < 120 x 109/L  
 
End of follow up will be 100 days after the last exposure to Pb(PfCS@UIS4) and 35 days 
post CHMI for the infectivity control subjects. 
7.3.3 
Controlled Human Malaria Infection  
Three or four weeks after the last immunization of group 3, all subjects of group 3 and six 
infectivity controls will undergo a malaria challenge infection. Only subjects that met the 
inclusion criteria will undergo a malaria challenge infection. On the challenge day, all 
subjects will be exposed to the bites of five P. falciparum NF54 infected mosquitoes. 
Mosquito feeding will proceed for 10 minutes. Volunteers will receive a local treatment 
(tripelennamine crème) for mosquito bites and will be observed for 15 minutes after the feed. 
Directly after the feed, the mosquitoes will be dissected by a technician of the mosquito unit 
according to standard operating procedures. Exposure will be repeated until five infected 
mosquitoes have fed on each volunteer.  
As long as there are volunteers present in the mosquito unit, there will be supervision by one 
of the clinical investigators. Another clinical investigator will be on call, in case of emergency. 
Emergency aid kits will be present and readily available at any location, whenever there are 
volunteers present.  
After malaria challenge infection subjects will be observed closely according to an intensive 
out-patient follow-up schedule including frequent safety analyses (see section 7.3.14 for 
details). The study design is illustrated in more detail in the flowchart in section 7.3.14.  
For all subjects, during this period all relevant investigations will be carried out on an 
outpatient basis at the Havenziekenhuis, including frequent safety analyses (section 7.3.14 
for details).  
From the sixth day up until the twenty-first day post-CHMI, assessments of parasite densities 
using qPCR will be performed once daily. From day 22 until day 28 subjects will only be seen 
if they have relevant symptoms. Groups 4 and 5 will have two additional visits on day 4 and 5 
post-CHMI for explorative add-on studies. Subjects will measure their temperature twice 
daily, and the clinical investigators will contact all volunteers by telephone once daily to ask if 

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there are any symptoms or complaints. Subjects will visit the study site for a follow-up visit on 
Days 1, 2 and 3 after treatment (TD+1, 2 & 3). All subjects will be seen for a final control visit 
on day 35 after CHMI. The study design is illustrated in more detail in the flowchart in section 
7.3.14.  
qPCR assessment of parasite densities will be performed directly in volunteer samples.  
As soon as a qPCR or thick smear result is deemed positive for malaria parasites, the 
technician will inform the trial clinician. Treatment will be initiated after a single positive 
qPCR. If treatment has to be initiated, the trial clinician will immediately contact the volunteer, 
who will return to the clinic to receive Malarone® treatment. Preferably, the volunteer should 
return immediately or at least within 1 hour. If the volunteer is not reachable by phone, 
his/her contact person will be called and a search is started.  
During the entire study period subjects will be instructed to call the trial physicians at any 
time if they experience symptoms. The trial physician can decide to initiate additional 
diagnostics or treatment at all times.  
For unexpected laboratory abnormalities, the laboratory test will be repeated. If there is any 
ambiguity regarding the decision to include or exclude a volunteer, the clinical supervisor will 
discuss the case with the local safety monitor and make the final decision after that, if 
necessary with consultation of a specialist. If volunteers prove to be eligible, they will be 
invited to the next visit. If a volunteer is not fit to participate to undergo the challenge infection 
on the planned date, challenge infection may be delayed up to 10 days. 
All subjects who are exposed to P. falciparum will be treated with Malarone®. Treatment 
after challenge infection will be based on the above mentioned criteria. Additionally, 
treatment can be initiated in any of the following situations:  
1. By decision of study doctor or the safety monitor  
2. In consultation with the cardiologist 
3. On request of the volunteer  
4. On day 28 post CHMI, if the volunteer has remained qPCR-negative  
5. When LDH > 1000 U/l  
6. When thrombocytes < 120 x 109/L  
 
A second malaria challenge infection might be initiated as described in section 7.3.2, and all 
procedures will be similar. Six new infectivity controls will be included. 
7.3.4 
Treatment with Malarone® 
All volunteers will be treated with Malarone® (atovaquone/proguanil) based on the 
predetermined criteria mentioned above. The treatment will consist of the drug Malarone®. 
Dosing will be as follows: once daily 4 tablets of 250/100mg, during three days, according to 

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Dutch SWAB guidelines. This drug has been chosen because of its fast clinical response and 
the few side-effects. Furthermore, it has not been reported to have any cardiac side-effect. 
During treatment, complaints of malaria infection will be treated symptomatically. In addition 
to specific treatment with Malarone®, symptomatic treatment will be administered at the 
discretion of the study physician.  
During and one day after Malarone® treatment, qPCR is performed directly in collected blood 
samples. If qPCR remains positive after Malarone® treatment (usually the result of parasite 
debris remaining in the bloodstream) a thick blood smear will be performed to confirm the 
absence of intact malaria parasites. 
Volunteers will not be admitted to the hospital during this study, unless the study doctors or 
the safety monitor deems it necessary, or on request of the volunteer. 
 
7.3.5 
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 following formula: BMI = Body weight (kg) / [Height (m)]2 and converted to an 
integer. 
7.3.6 
Vital signs  
Vital signs including body temperature, blood pressure (BP) and pulse measurements will be 
determined and recorded at set time points during the study. 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, 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 will be measured according to local practice, consistently throughout the study. 
The thermometer used should have a precision of 0.1ºC. The same route should be used 
throughout the study. 

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7.3.7 
Patient-reported outcomes (study diary) 
At the inclusion visit, subjects will be issued symptom diaries and thermometers. They will be 
asked to record all symptoms and medication use daily from the day of inclusion until end of 
study. The subject study 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 
study file. 
Subjects will also be asked to measure their temperature orally every morning from the day 
of the immunization until day 28 (phase 1) or day 8 (phase 2), or after the malaria challenge 
infection until the third day of Malarone® (atovaquone/proguanil) treatment, and record this 
temperature in their study diaries.  
At the end of the study, the diary will be collected and kept as source data with the subject’s 
study file. 
7.3.8 
Electrocardiogram  
A standard 12 lead ECG will be performed at screening. Additional ECG assessments may 
be performed at any time throughout the study, at the discretion of the investigator. 
All assessments will occur after the subject has rested for approximately 10 min rest in the 
supine position. Calibration should be performed per the local site/requirements. Each ECG 
tracing should be labelled with the subject number and date, and kept in the source 
documents at the study site. Interpretation of the tracing must be made by a qualified 
physician and documented in the Case Report Form (CRF). Minimally, the CRF will contain 
date and time of ECG, heart rate, PR interval, QRS duration and QT interval (corrected). 
Clinically significant abnormalities should also be recorded in the CRF.  
7.3.9 
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 7.3.14) shows when blood will be 
drawn. 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, creatinine, γGT, LDH, potassium, AST, ALT, sodium, 
highly sensitive troponin T and urea will be measured at regular time points during the study. 
Glucose, triglycerides and cholesterol will be measured only at screening.  
In the event that an asymptomatic individual has evidence of an elevated troponin level, a 
second sample may be obtained to discern whether the result could represent a false 
positive (0.4% of tests). At the screening visit plasma samples will be tested for HIV, hepatitis 

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B and hepatitis C. Additionally, serum will be tested for antibodies to malaria parasites by 
ELISA. 
A midstream urine sample (approx. 30 ml) will be obtained at screening, inclusion and, for 
female subjects only, at baseline before every immunization and/or challenge infection. In 
this sample the presence of amphetamines and cocaine will be assessed; the sample taken 
at inclusion will also be tested for the presence of cannabis. Additionally, for female 
participants a commercially available hCG urine test will be used to test for pregnancy at 
screening and baseline before every immunization and/or challenge infection.  
In the case where a laboratory assessment is outside the reference range for the laboratory 
at screening and/or inclusion, a decision regarding whether the result is of clinical 
significance or not shall be made by the clinical investigator and shall be based, in part, upon 
the nature and degree of the observed abnormality.  
In all cases, the investigator must document in the source documents, the clinical 
considerations (i.e., result was/was not clinically significant and/or medically relevant) in 
allowing or disallowing the subject to continue in the study. 
7.3.10 Analysis of asexual parasite densities after challenge infection  
qPCR for assessment of parasite densities will be performed directly in volunteer samples, 
as discussed previously (see flowchart 7.3.14)  
qPCR is performed according to a standard procedure as previously described (Hermsen et 
al., 2001) with small adjustments. In short, qPCR will be performed for the multicopy 18S 
ribosomal RNA gene. All samples are spiked with the extraction control Phocine Herpes 
Virus (PhHV) to determine efficacy of DNA isolation.  
Thick smears will be performed directly in volunteer samples, as discussed previously and if 
deemed necessary by the clinical investigator (see section 8.3.2), according to a standard 
operating procedure which is based on an internationally harmonized protocol for thick 
smears in CHMIs (Moorthy et al., 1998 and WHO, 2010). Per slide, the number of fields 
correlating to 0.5 μl of blood will be read. Slides are considered positive if they contain 2 or 
more parasites in these fields. 
7.3.11 Immunological assays 
Blood samples will be taken for isolation of peripheral blood mononuclear cells (PBMCs) and 
plasma and serum (see flowchart 7.3.14).  
PBMCs and both plasma and serum will be frozen and can then be used by the 
Radboudumc or its collaborators for exploratory immunological assays to further analyze 
immunity induced by Pb(PfCS@UIS4), and to analyze the phenotype or functionality of the 
immune response during and after malaria infection. 

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Humoral assessment will include antibody assays by immunofluorescence, and ELISA’s for 
specific PfCS- and Pb-proteins. In addition, antibody functionality will be tested in a range of 
In vitro assays, such as inhibition of sporozoites invasion or maturation assays in primary 
human hepatocytes or hepatoma cell lines. 
 
Cellular assessment of parasite-specific (subset)T-cell response will be conducted by multi-
parameter flow cytometry and ELISpot assay following Pf or Pb stimulation In vitro. 
In order to assess (antigen specific) T cell responses, the HLA-type of volunteers may be 
determined. 
7.3.12 Cytokines 
Cytokine levels will be measured to assess in vivo inflammatory responses after 
immunization with Pb(PfCS@UIS4).  
In phase 1 cytokines such as, IL-1β, TNF-α, IL-6, IL-8, IL-10, IL-17, and IFNy will be 
measured by ELISA or Luminex multiplex assay, on day 1 to 10 after administration of 
Pb(PfCS@UIS4). 
 
7.3.13 Other 
During the P. falciparum challenges, sampling of material such as urine, serum, citrated and 
EDTA whole blood will be collected on a daily basis, for add on studies on pathogenesis of 
early malaria. In these samples the vasopressin response, haemostasis and cholesterol 
metabolism will be studied by determination of several parameters with standard clinical 
laboratorial techniques and ELISAs. Furthermore, the deformability of erythrocyte will be 
measured by a Rheo scan.  
 
Investigation of early malaria pathogenesis and the host immune responses during liver 
phase has never comprehensively been studied in humans.  Recently, leukocyte counts and 
leukocyte differentiation were proven to change during malaria liver phase [35]. To 
investigate the changes in proteins and metabolites in blood metabolomics during the liver 
phase in more detail, volunteers of group 4 and 5 will be exposed to two extra visits with 
blood sampling (day 4 and 5 post-CHMI). The following parameters will be determined: 
cytokine levels (e.g. IL-6; IL-10, IL-12, TGF-β, IFN-γ and TNF-α as measured by ELISA or 
Luminex multiplex assay), and coagulation factors (e.g. prothrombin fragment 1.2, D-dimer, 
platelet count, von Willebrand factor, von Willebrand factor pro-peptide will be measured by 
ELISA). In addition, standard hematology- and biochemistry tests will be assessed on these 

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time-points. The samples that will be taken are: citrate vial 2.7 ml; CTAD vial 2.7 ml; EDTA 
vial 4.0; serum vial 5.0 ml and urine sample 10 ml. Results of tests will only be analyzed after 
day 35 of the challenge to avoid bias in clinical evaluation or approach.  
 
 
7.3.14 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 in subject study files and are considered source data. Since 
all subjects will be healthy, there is no medical file for the study subjects, with exception of 
the medical file in case of adverse events/reactions resulting in a medical consultation or 
hospitalization. In this case the medical file will also be considered as the source data. The 
diaries, produced by the study volunteers are also considered source data. They will be kept 
as source documents together with the investigator’s notes. 
 

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7.3.15 Flowchart trial procedures: Phase 1  
 
 
Screening  
Inclusion 
Baseline 
Administration 
Pb(PfCS@UIS4) 
 
EOS 
Visit Number 
V1 
V2 
V3 
V4 
V5-14 
V15-19 
V20-23 
V24 
V25 
Trial timeline 
-120 to -11 
-8 to -6 
-1 
0 
1-10 
12-20 
28-31 
(TD+1-3) 
35 
100 
Number of visits 
1x 
1x 
1x 
1x 
10x 
5x 
4x 
1x 
1x 
Informed consent 
X 
 
 
 
 
Eligibility criteria 
X 
 
X 
 
 
 
Demographic data, Medical history 
X 
 
 
 
 
Physical examination and vital signs 
X 
X 
X8 
X8 
X8 
X8 
X8 
ECG 
X 
 
 
 
 
Temperature 
X 
X 
X 
X 
X 
X 
X 
X 
Immunization by mosquito bites 
 
 
X 
 
 
 
Collecting (serious) adverse events 
as necessary9 
Treatment Day(TD; Malarone®) 
 
 
X10 
X10 
X10 
 
 
Haematology tests1  
X 
X 
X 
X 
X 
X 
X 
X 
Biochemistry tests2  
X 
X 
X11 
X11 
X 
X 
HsTropT + LDH  
 
 
X 
X 
X 
X 
 
 
Serology3  
X 
X 
 
 
 
Pregnancy and toxicology urine test4 
X 
X 
X 
 
 
 
Parasitology5 (thicksmear) 
 
 
X 
X 
X 
X 
X 
Parasitology5 (qPCR) 
 
 
X 
 
 
 
Exploratory immunology 
 
 
X 
X 
X 
X 
X 
X 
Safety report6 
 
 
X 
 
 
X 
Total blood volume collect (ml)7 
15 
11.5 
73 
0 
163.5 
9 
49 
1 Hemoglobin, hematocrit, platelets, red blood cell count, white blood cell count + differentiation (differentiation only on inclusion and day 100) 
2 Creatinine, urea, sodium, potassium, bilirubin, AF, γGT, AST, ALT, LDH and hs-Trop-T. Additional at screening: cholesterol, triglyceride + glucose 
3 HIV, HBV, HCV, P. falciparum(screening only) 
4 Toxicology urine tests at screening and inclusion visits only. Pregnancy test for female subjects at screening and baseline visits (before every 

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immunization/challenge) 
5 Thicksmear will be taken from same sample as qPCR.18S qPCR for blood stage P. berghei will only be assessed on samples retrospectively. 
6 A safety report will be compiled upon all subjects of group 1 and 2 having completed day 12 after immunization, day 28 for group 3, and at the end of the study, 
7 Total blood volume: 321ml 
8 On indication. 
9 Only SAE’s that are possibly or probably related to trial will be collected prior to inclusion. 
10 If positive thicksmear or on day 28 post-immunization 
11 Biochemistry tests will be performed on day 2, day 4, day 12 post immunization and TD+2. 

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Flow chart trial procedures: Phase 2 
 
Inclusion  
Immunization  2  
Immunization  3  
Immunization  4  
CHMI  
EOS 
Visit Number 
V21 
V22 
V23 
V24 
V25 
V26 
V27 
V28 
V29 
V30 
V31 
V32 
V33 
V34 
V35-V50 
V51-V54 
V55 
V56 
Trial timeline 
-29 
30 
31 
38 
57 
58 
59 
66 
113 
114 
115 
122 
134 
135 
141-156 
163-166 
170 
214 
Number of visits 
1x 
1x 
1x 
1x 
1x 
1x 
1x 
1x 
1x 
1x 
1x 
1x 
1x 
1x 
16x14 
4x 
1x 
1x 
Informed consent 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility criteria 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographic data, Medical history 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination and vital signs 
X9 
X9 
X9 
X9 
 
X9 
X9 
X9 
 
X9 
X9 
X9 
 
X9 
X9 
X9 
X9 
ECG 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Temperature 
X 
X 
X 
X 
 
X 
X 
X 
 
X 
X 
X 
 
X 
X 
 
 
Immunization with Pb(PfCS@UIS4) 
 
X 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
Challenge with P. falciparum 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Collecting (serious) adverse events 
as necessary10 
Treatment Day(TD; Malarone®) 
 
 
X11 
X11 
X11 
X11 
X11 
X11 
 
X11 
X11 
 
 
X12 
X12 
 
 
Haematology tests1  
X 
 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
 
X 
X 
X 
X 
Biochemistry tests2  
X 
 
X 
 
X 
 
 
 
X 
X 
 
 
X13 
X 
X 
HsTropT + LDH  
 
 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
Serology3  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy and toxicology urine test4 
X 
 
X 
 
 
X 
 
 
 
X 
 
 
 
 
 
Parasitology5 (thicksmear) 
 
 
X 
X 
X 
X 
 
 
X 
X 
 
 
 
 
 
 
Parasitology5 (qPCR Pb) 
X 
 
X 
X 
X 
X 
X 
X  
 
X 
X 
 
 
 
 
X 
X 
Parasitology6 (qPCR Pf) 
 
 
 
 
 
 
 
 
X 
 
X 
X 
X 
X 
Exploratory immunology 
X 
 
X 
 
 
 
X 
 
 
 
X 
 
X 
X 
 
X 
Safety report7 
 
 
X 
 
 
X 
 
 
 
X 
 
 
 
 
X 
Total blood volume collect (ml)8 
49 
0 
6.5 
6.5 
49 
0 
6.5 
6.5 
49 
0 
6.5 
6.5 
73 
0 
330.5 
58.9 
58.9 
1 Hemoglobin, hematocrit, platelets, red blood cell count, white blood cell count + differentiation (differentiation only on inclusion and day 100) 
2 Creatinine, urea, sodium, potassium, bilirubin, AF, γGT, AST, ALT, LDH and hs-Trop-T. Additional at screening: cholesterol, triglyceride + glucose 
3 HIV, HBV, HCV, P. falciparum(screening only) 
4 Pregnancy test for female subjects at baseline visits (before every immunization/challenge). Toxicology urine tests only at baseline prior challenge infection 
5 Thicksmear will be taken from same sample as qPCR.18S qPCR for blood stage P. berghei will only be assessed on samples retrospectively. 

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6 If positive qPCR for P. falciparum. Thicksmear only on indication (sample taken from qPCR sample). 
7 A safety report will be compiled upon all subjects per group having completed day 28 after immunizations (with exception of day 21 for the 4th immunization) and at the end of the study. 
8 Total blood volume: 707.3ml 
9 On indication. 
10 Only SAE’s that are possibly or probably related to trial will be collected prior to inclusion. 
11 If positive thicksmear  
12 If positive qPCR for bloodstage P. falciparum 
13 Biochemistry tests on  C+6, TD TD+2 
14 Two additional visits for group 4 are scheduled (visit 34a and 34b) on day 4 and 5 after infection. Not included during these extra visits: physical examination and vital signs, qPCR Pf, HS Trop T, LDH. 

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Flow chart trial procedures: Phase 2 – adaptive design 
  
 
 
 
Immunization 5  
CHMI 2 
EOS 
Visit Number 
V57 
V58 
V59 
V60 
V61 
V62 
V63-78 
V79-V82 
V83 
 
Trial timeline 
225 
226 
227 
234 
309 
310 
316-331 
338-341 
345 
 
Number of visits 
1x 
1x 
1x 
1x 
1x 
1x 
16x14 
4x 
1x 
 
Informed consent 
 
 
 
 
 
 
 
 
 
 
Eligibility criteria 
X 
 
 
 
 
 
 
 
 
 
Demographic data, Medical history 
 
 
 
 
 
 
 
 
 
 
Physical examination and vital signs 
X9 
 
X9 
X9 
X9 
 
X9 
X9 
X9 
 
ECG 
 
 
 
 
 
 
 
 
 
 
Temperature 
X 
 
X 
X 
X 
 
X 
X 
 
 
Immunization with Pb(PfCS@UIS4) 
 
X 
 
 
 
 
 
 
 
 
Challenge with P. falciparum 
 
 
 
 
 
X 
 
 
 
 
Collecting (serious) adverse events 
as necessary10 
Treatment Day(TD; Malarone®) 
X11 
X11 
X11 
 
 
X12 
X12 
 
 
Haematology tests1  
X 
X 
X 
X 
 
X 
X 
X 
 
Biochemistry tests2 ( 
 
X 
X 
 
 
X13 
X 
 
HsTropT + LDH  
X 
X 
 
X 
 
X 
 
 
 
Serology3  
 
 
 
 
 
 
 
 
Pregnancy and toxicology urine test4 
X 
 
 
X 
 
 
 
 
 
Parasitology5 (thicksmear) 
X 
X 
 
 
 
 
 
 
Parasitology5 (qPCR Pb) 
X 
X 
X 
 
 
 
 
X 
 
Parasitology6 (qPCR Pf) 
 
 
X 
 
X 
X 
X 
 
Exploratory immunology 
X 
 
 
X 
X 
 
X 
X 
 
 
Safety report7 
X 
 
 
X 
 
 
 
 
 
Total blood volume collect8 
49 
0 
6.5 
6.5 
57 
0 
330.5 
59 
 

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1 Hemoglobin, hematocrit, platelets, red blood cell count, white blood cell count + differentiation (differentiation only on inclusion and day 100) 
2 Creatinine, urea, sodium, potassium, bilirubin, AF, γGT, AST, ALT, LDH and hs-Trop-T. Additional at screening: cholesterol, triglyceride + glucose 
3 HIV, HBV, HCV, P. falciparum(screening only) 
4 4 Pregnancy test for female subjects at baseline visits (before every immunization/challenge). Toxicology urine tests only at baseline prior challenge infection 
5 Thicksmear will be taken from same sample as qPCR.18S qPCR for blood stage P. berghei will only be assessed on samples retrospectively. 
6 If positive qPCR for P. falciparum. Thicksmear only on indication (sample taken from qPCR sample). 
7 A safety report will be compiled upon all subjects per group having completed day 28 after immunizations and at the end of the study. 
8 Total blood volume: 508.5ml 
9 On indication. 
10 Only SAE’s that are possibly or probably related to trial will be collected prior to inclusion. 
11 If positive thicksmear  
11 If positive qPCR for bloodstage P. falciparum 
13 Biochemistry tests on C+6, TD, and TD+2 
14 Two additional visits for group 5 are scheduled (visit 62a and 62b) on day 4 and 5 after infection. Not included during these extra visits: physical examination and vital 
signs, qPCR Pf, HS Trop T, LDH.  

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7.4 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.  
The investigator can decide to withdraw a subject from the study for urgent medical reasons.  
Volunteers can be withdrawn from the study procedures at the discretion of the clinical 
investigator or the local safety monitor for the following reasons:  
 
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.  
 
The use of concomitant, chronic medication active on the immune system (e.g. steroids, 
immunosuppressive agents) or with known antimalarial activity against P. falciparum. 
 
Pregnancy.  
 
Withdrawal of informed consent by volunteer. 
 
Completely lost to follow-up. 
 
Ineligibility (either arising during the study or retrospectively, having been overlooked at 
screening). 
 
If, on balance, 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. 
 
If withdrawal of a subject occurs for any reason, the investigator must make every effort to 
determine the primary reason for a subject’s withdrawal from the study and record this 
information in the study file. However, in accordance with the principles of the current version 
of the Declaration of Helsinki, a subject does have the right to withdraw from the study at any 
time and for any reason, and is not obliged to give his or her reasons for doing so. 
If it is felt that inclusion of the study subject’s data for analysis is compromised, the subject 
will be terminated from the study and data will not be included in analysis. This does not 
preclude the ethical responsibility of the investigators to ensure the safety of the subject and 
ensure they receive curative therapy for malaria, and follow the subject for cardiac 
manifestations of disease. All data generated before withdrawal will be included in final study 
analysis. Blood samples collected before withdrawal will be used/stored unless the subject 
specifically requests otherwise. 
For subjects who are 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 

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(i.e. documented phone calls and e-mails) will be undertaken to locate or recall him or at 
least to determine his health status. The investigator should show "due diligence" by 
documenting in the source documents steps taken to contact the subject, e.g. dates of 
telephone calls, registered letters, etc. 
 
7.5 Replacement of individual subjects after withdrawal 
If a subject withdraws before or on inclusion day he/she will be replaced with an alternate 
volunteer who passed screening, if possible. 
 
7.6 Follow-up of subjects withdrawn from treatment 
In the event that a volunteer discontinues the study for any reason, he/she will be required to 
complete all safety follow-up as appropriate, as determined by the principal investigator. All 
volunteers who have been exposed to the bites of infectious mosquitoes are required to take 
a curative regimen of Malarone® (or alternative effective anti-malarial treatment should 
Malarone® be contra-indicated, at the discretion of the clinical investigator).  
 
7.7 Procedure in case of pregnancy 
Pregnancy itself is not considered an adverse event, but any pregnancy complication or 
elective termination of a pregnancy for medical reasons will be recorded as an adverse event 
or serious adverse event. 
 
The investigator will record the pregnancy information on a pregnancy notification form (as 
described in standard operating procedures) and submit it to the local safety monitor, and 
sponsor within 24 hours of learning of a subject’s pregnancy. The subject will be followed to 
determine the outcome of pregnancy. At the end of the pregnancy, information on the status 
of the mother and child will be forwarded to the sponsor. Follow-up will generally be no 
longer than six to eight weeks following delivery date. 
 
Volunteers who become pregnant during the study will not receive additional immunizations 
or challenges, but may continue other study procedures at the discretion of the investigator. 
 
All female volunteers should refrain from getting pregnant until the last study visit.  
 
 
7.8 Premature termination of the study 
The study may be discontinued by the sponsor:  

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 
On advice of the safety monitor. 
 
On advice of the Safety Monitoring Committee (SMC). 
 
On advice of the clinical investigator. 
 
On advice of the CCMO. 
 
If the predefined holding criteria are met (section 8.4.4) 
 
The safety monitor, SMC, CCMO or investigators may decide to put the study on hold based 
on adverse events, pending discussion with the Sponsor / SMC / CCMO / safety monitor / 
investigators. Following discussion, it may be decided to terminate the study. Safety 
reporting procedures are described in section 8. 
 

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8. SAFETY REPORTING 
8.1 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 jeopardize subject health or 
safety. The sponsor will notify the CCMO without undue delay of a temporary halt including 
the reason for such an action. The study will be suspended pending further review by the 
accredited METC, except insofar as suspension would jeopardize the subject’s health. The 
investigator will take care that all subjects are kept informed. 
PATH REC (PATH Research Ethics Committee) and WIRB will also be notified of any 
decisions to prematurely suspend or terminate the study. 
 
 
8.2 AEs, SAEs and SUSARs 
8.2.1 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. An AE can therefore 
be any unfavourable and unintended sign (including an abnormal laboratory finding), 
symptom, or disease (new or exacerbated) temporally associated with the use of a medicinal 
product or study intervention. AEs may include events that occur as a result of protocol-
mandated procedures (i.e. invasive procedures, modification of subject’s previous 
therapeutic regimen). 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, haematology, urinalysis) 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 judgement in deciding whether an abnormal laboratory finding or other 
abnormal assessment is clinically significant. 
 
If there are any severe complaints not typical for malaria infection, such as chest pain, the 
volunteer will be evaluated immediately by a qualified clinician using the appropriate clinical 
assessments (e.g. ECG or measurement of cardiac enzymes) according to standard hospital 
care. 
 

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Any hs-troponin value above 14ng/L will be recorded as adverse event (grade 1). For any hs-
troponin value above 60ng/L the cardiologist will be consulted, an electrocardiogram will be 
made (grade 2) and the local safety monitor will be informed within 24 hours. The limit of 
detection of the hs-troponin T assay is 3 ng/L. The 99th percentile cut-off point is 0.014 ng/mL 
(14ng/L). Further procedures, such as cardiac MRI’s will be performed on advice of the 
cardiologist. Any hs-troponin above 100 ng/L will be considered a grade 3 (severe) adverse 
event. 
 
8.2.2 Serious adverse events (SAEs) 
A serious adverse event (SAE) is any untoward medical occurrence or effect that  
 
results in death; 
 
is life threatening (at the time of the event); 
 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
 
results in persistent or significant disability or incapacity; 
 
is a congenital anomaly or birth defect; or 
 
any other important medical event that did not result in any of the outcomes listed above 
due to medical or surgical intervention but could have been, based upon appropriate 
judgement by the investigator. 
An elective hospital admission will not be considered as a serious adverse event. 
 
To ensure no confusion or misunderstanding of the difference between the terms ‘serious’ 
and ‘severe’, which are not synonymous, the following note of clarification is provided:  
 
The term ‘severe’ is often used to describe the intensity (severity) of a specific event (as in 
mild, moderate, or severe myocardial infarction); the event itself, however, may be of 
relatively minor medical significance (such as severe headache). This is not the same as 
‘serious’, which is based on patient/event outcome or action criteria usually associated with 
events that pose a threat to a patient's life or functioning. Seriousness (not severity) serves 
as a guide for defining regulatory reporting obligations. (Example: a grade 3 AE is not 
necessarily a SAE.) 
 
The principle investigator of each participating centre is responsible for reporting of the 
Serious Adverse Event to the sponsor within 24 hours of occurrence of the event. The 
investigator (on behalf of the sponsor) will report the SAEs through the web portal 
ToetsingOnline to the accredited METC that approved the protocol, within 15 days after the 

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sponsor has first knowledge of the serious adverse events. The Sponsor will notify the SMC 
of the SAE within 24 hours of being notified of the SAE.  
 
SAEs that result in death or are life threatening should be reported in an expedited fashion. 
The expedited reporting to the accredited METC will occur not later than 7 days after the 
responsible investigator has first knowledge of the adverse event. This is in the form of a 
preliminary report, a complete report will be provided by the sponsor no later than 15 days 
after first knowledge of the SAE.  
 
8.2.3 Suspected unexpected serious adverse reactions (SUSARs) 
Adverse reactions are all untoward and unintended responses to an investigational product 
related to any dose administered. 
 
Unexpected adverse reactions are SUSARs if the following three conditions are met: 
1. the event must be serious (see chapter 9.2.2); 
2. there must be a certain degree of probability that the event is a harmful and an 
undesirable reaction to the medicinal product under investigation, regardless of the 
administered dose; 
3. the adverse reaction must be unexpected, that is to say, the nature and severity of 
the adverse reaction are not in agreement with the product information as recorded 
in the Summary of Product Characteristics (SPC).  
 
The sponsor will report expedited all SUSARs through the web portal ToetsingOnline to the 
CCMO. The expedited reporting of SUSARs through the web portal ToetsingOnline is 
sufficient as notification to the competent authority. 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 
to treatment allocation, the code would not have to be broken in the case of a SUSAR.  
 
Any SAEs, SUSARs or AEs that suggest the research places subjects or others at a greater 
risk of harm (including physical, psychological, economic, or social harm) than was 
previously known or recognized, or that require a change to the protocol or consent will be 
reported to PATH REC and/or WIRB in accordance with the reporting requirements of each. 
 

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8.3 Follow-up of (serious) adverse events 
8.3.1 
Adverse event data collection 
Safety assessments will be performed and recorded by the investigators. All adverse 
events/reactions (solicited and unsolicited), noted by the investigators will be accurately 
documented in the case report form by the investigators. For each event/reaction the 
following details will be recorded:  
1. Description of the event(s)/reaction(s)  
2. Date and time of occurrence  
3. Duration 
4. Intensity 
5. Relationship with the intervention 
6. Action taken, including treatment 
7. Outcome 
In addition, symptoms will be ranked as (1) mild, (2) moderate, or (3) severe, depending on 
their intensity according to the following scale: 
 
Mild (grade 1): awareness of symptoms that are easily tolerated and do not interfere with 
usual daily activity  
 
Moderate (grade 2): discomfort that interferes with or limits usual daily activity 
 
Severe (grade 3): disabling, with subsequent inability to perform usual daily activity, 
resulting in absence or required bed rest 
If an AE changes in intensity during the specified reporting period, a new description of the 
AE will be added. Interrupted AEs are registered as one AE if the interruption is <24 hours. 
 
When an AE/SAE occurs, it is the responsibility of the investigators to review all 
documentation (e.g. hospital progress notes, laboratory, and diagnostics reports) related to 
the event. The investigators will then record all relevant information regarding an AE/SAE on 
the CRF or SAE Report Form, respectively. The investigator will attempt to establish a 
diagnosis of the event based on signs, symptoms, and/or other clinical information. In such 
cases, the diagnosis should be documented as the AE/SAE and not the individual 
signs/symptoms. 
 
After every immunization the following signs and symptoms will be solicited at any visit after 
immunization in phase 1 or at any visit in phase 2 until CHMI: 
Fever (by examination), rash, urticaria, pruritis, edema, headache, fatigue, malaise, rigors, 
myalgia, arthralgia, dizziness, sweats, cough, nausea, vomiting, abdominal pain, diarrhoea,  

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The following signs and symptoms local to the site of mosquito bites will be solicited after 
every immunization: Tenderness, induration (assessed by palpation), bruising/extravasated 
blood, erythema, swelling (assessed by direct or lateral visualization), and pain, pruritis 
(subjective symptoms). 
 
The following signs and symptoms will be solicited after CHMI in stage B: 
Fever (by examination), rash, urticarial, pruritis, edema, headache, fatigue, malaise, rigors, 
myalgia, arthralgia, dizziness, sweats, cough, nausea, vomiting, abdominal pain, diarrhoea, 
chest pain, palpitations, shortness of breath. 
8.3.2 
Assessment of causality 
The investigators are obligated 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 of the underlying diseases, 
concomitant therapy, other risk factors and the temporal relationship of the event to the 
challenge will be considered and investigated. The relationship of the adverse event with the 
study procedures will be categorized as: 
Probable 
An adverse event that follows a reasonable temporal sequence from 
the study procedure and cannot be reasonably explained by the 
known characteristics of the subject’s clinical state. 
Possible 
An adverse event for which insufficient information exists to exclude 
that the event is related to the study procedure. 
Not related 
An event for which sufficient information exists to indicate that the 
aetiology is unrelated either because of the temporal sequence of 
events or because of the subject’s clinical state or other therapies. 
 
8.3.3 
Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been reached. 
AEs that result in a subject’s withdrawal from the study or that are present at the end of the 
study will be followed up (if the volunteer consents to this) until a satisfactory resolution or 
stabilisation occurs, or until a non-study related causality is assigned.  
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. 
AEs and SAEs will be reported until end of study within the Netherlands, defined as the last 
patient visit. 

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8.4 Local Safety Monitor (LSM) and Safety Monitoring Committee (SMC) 
8.4.1 
Local safety monitor 
For this study, a local safety monitor will appointed, who is based in the Havenziekenhuis 
and will be involved in the review of severe and serious adverse events and volunteer safety. 
He/she is an experienced clinician qualified to evaluate safety data from clinical studies with 
malaria infections. He/she is independent of the sponsor, funding partner, and the 
investigators. 
8.4.2 
Safety Monitoring Committee (SMC) 
An independent Safety Monitoring Committee (SMC) will be appointed, including at least 3 
individuals. Their main responsibility will be assessing any severe or serious adverse events 
and, if necessary, halting further study procedures. A safety report including a list of all 
reported adverse events and any safety laboratory values outside the normal range will be 
prepared on specific days as detailed in section 7.3.15.  
The advice(s) of the SMC will only be sent to the sponsor of the study and the funding 
partner, PATH. Should the sponsor decide not to fully implement the advice of the SMC, the 
sponsor will send the advice to the CCMO, including a note to substantiate why (part of) the 
advice of the SMC will not be followed. 
8.4.3 
Review of safety data by the safety monitor and SMC 
A safety report including a list of all reported adverse events and any safety laboratory values 
outside the normal range will be prepared specific days as detailed in section 7.3.15. 
These reports will be prepared by a clinical investigator and sent to the safety monitor, all 
clinical investigators involved and the funding partner, PATH. The safety monitor will review 
the safety data within 2 workdays and if warranted instruct the site to take appropriate action.  
In addition, safety data for all participants will be assessed by the SMC at the end of the 
study. Responsibilities of the SMC are described in the SMC Charter. The advice(s) of the 
SMC will only be communicated to the CCMO when the sponsor does not follow this. With 
this notification a statement will be included indicating whether the advice will be followed. 
Any highly sensitive troponin T value greater than 60 ng/l will be reported to the safety 
monitor within 24 hours. Any safety laboratory values that lead to immediate malaria 
treatment will be reported to the safety monitor within 24 hours.  
 

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8.4.4 
Safety holding rules 
The study may be placed on safety hold 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. 
 
When individual or group holding criteria are met (see section 8.4.4.1 and 8.4.4.2). 
 
In phase 1, the trial will proceed from group 1 to group 2 to group 3 as long as the holding 
criteria are not met. At any time if holding criteria are met, an ad hoc meeting of the SMC will 
be called. 
 
The safety monitor, CCMO, or investigators may decide to put the study on hold based on 
adverse events, pending discussion with the safety monitor, CCMO, and investigators. In 
addition, the PI can always decide based on characteristics, duration and severity of 
signs/symptoms to treat and stop the trial for individual cases. The PI will identify when 
stopping rule criteria are met and alert the appropriate parties. The SMC will be responsible 
for granting approval for proceeding from phase 1 to phase 2, and before the challenge 
infection. If the CCMO has recommended safety hold, re-initiation of the study will require 
CCMO concurrence. The CCMO, PATH REC and WIRB will be informed of a safety hold by 
the sponsor. Following discussion, it may be decided to terminate the study. 
 
8.4.4.1 Individual holding rules 
The following Individual Holding Rules will apply to each subject. The PI will monitor the 
individual holding rules, and will make recommendations regarding the continuation of 
immunizations in a subject. If the individual holding rules are met, no further immunizations 
will be administered to that subject until the investigators have conferred with the local safety 
monitor and a full written report has been submitted to PATH. 
 
Local Reactions: The investigator, at his or her discretion, can hold an individual’s 
immunization based on a local reaction. 
 
Clinical systemic adverse events (AEs): Immunization possibly or probably related 
unsolicited Grade 3 AEs beginning within 28 days following immunization and 
persisting at Grade 3 for greater than 48 hours.  

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 
Laboratory systemic AEs: Immunization possibly or probably related Grade 3 
abnormalities beginning within 7 days following immunization and persisting at Grade 
3 for greater than 48 hours.  
 
Systemic AEs:  Acute systemic allergic reaction or anaphylactic shock following the 
administration of Pb(PfCS@UIS4). 
 
Positive urine pregnancy test. 
 
Serious adverse event (SAE) determined to be possibly or probably related to the 
administration of Pb(PfCS@UIS4). 
 
The occurrence of a Pb(PfCS@UIS4) blood stage infection (detected by thick smear) 
in one of the volunteers in phase 1 or 2. 
 
If a subject has an acute illness (moderate or severe illness with or without fever) or a fever 
(oral temperature greater than or equal to 38.0°C) at the scheduled time of administration of 
Pb(PfCS@UIS4), the subject will not receive the immunization at that time. The immunization 
may be administered to that subject at a later date within the time window specified in the 
protocol or the subject may be withdrawn from the study at the discretion of the investigator.  
 
After an individual holding rule is activated, a thorough review by the PI, local safety monitor, 
and sponsor will occur. Immunization of the subject may resume only if all parties agree it is 
safe to resume immunization. 
 
All immunized subjects will be followed for safety until resolution or stabilization (if 
determined to be chronic sequelae) of their AEs. 
 
8.4.4.2 Group holding rules 
The following holding rules apply to each group of subjects. Solicited and unsolicited adverse 
events and safety laboratory data will be recorded and tracked by the PI after each 
immunization.  The local safety monitor will review the AEs (local, clinical systemic, and 
laboratory systemic) and will make recommendations regarding the continuation of the study 
for each group. If the group holding rules are met, no further immunizations will be 
administered in any group until the investigators have conferred with the local safety monitor 
and a full written report has been submitted to PATH. 
 
Local Reactions: Upon investigator discretion. 
 
Clinical systemic AEs: immunization possibly or probably related unsolicited Grade 3 
AEs in the same organ system beginning within 28 days following immunization and 

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persisting at Grade 3 for greater than 48 hours in 2 or more subjects within group 1 or 
within group 2, or 4 or more subjects within group 3 following each immunization. 
 
Laboratory systemic AEs: immunization possibly or probably related Grade 3 
abnormalities beginning within 7 days following immunization and persisting at Grade 
3 for greater than 48 hours in 2 or more subjects within group 1 or within group 2, or 4 
or more subjects within group 3 following each immunization. 
 
SAE determined to be possibly or probably related to the immunization. 
 
The occurrence of a Pb(PfCS@UIS4) blood stage infection (detected by thick smear) 
in one of the volunteers in phase 1 or 2. 
After a group holding rule is activated, a thorough review by the PI, local safety monitor, and 
sponsor will occur. Immunization of the subjects within the affected group and the other 
immunization groups may resume only if the local safety monitor, the PI, and the sponsor 
agree it is safe to resume immunizations and the following considerations are discussed: 
 
Relationship of the AE or SAE to the immunization. 
 
Relationship of the AE or SAE to the immunization dose (ie, only associated with high 
dose group). 
 
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. 
All immunized subjects will be followed for safety until resolution or stabilization (if 
determined to be chronic sequelae) of their AEs.   
 
 
8.4.5 
Deferral of study procedures for individuals 
Deferral of trial procedures for individual subjects could be considered when health problems 
based on history and clinical examination (physical examination and laboratory tests) are 
present. Details regarding these problems will be assessed in concurrence with appropriate 
specialists and if necessary with the local safety monitor. Subsequently, a volunteer-specific 
decision regarding deferral or continuation of study procedures for this particular individual 
will be made e.g. a subject with a positive pregnancy test before immunization will obviously 
be excluded from the study, while a subject suffering from a clinically significant common 
cold will be able to continue study procedures possibly in the next group once symptoms 
diminish. If a volunteer is not fit to participate to undergo the challenge infection of 
immunization on the planned date, challenge infection may be delayed up to 10 days. 
 

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9 STATISTICAL ANALYSIS 
 
9.1 Primary study parameter(s) 
All volunteers exposed to Pb(PfCS@UIS4) will be included in the intention-to-treat analysis.  
The primary endpoint is evaluated by tabulating all adverse events for each volunteer. 
Adverse events will be analysed by calculating the proportion of volunteers in each group 
who reported mild, moderate or severe adverse events. The frequency of signs and 
symptoms will be compared between groups with the Fisher’s exact test.  
Presence of parasitemia after exposure to Pb(PfCS@UIS4) will be assessed by thick smear. 
After challenge infection, the time to P. falciparum positivity (positive qPCR >100 Pf/ml) will 
be compared between groups with the Mann-Whitney U test. 
 
9.2 Exploratory study parameters 
In the exploratory immunological analyses we will assess differences by comparing mean 
values between the groups using either the Fisher’s exact test or Wilcoxon signed rank test, 
paired if pre-exposure values are compared with post-exposure values, unpaired if 
comparisons are made between groups. For discrete variables (e.g. the number of positive 
assays), non-parametric tests or, depending on the distribution, analyses based on count 
data such as Poisson regression are used. 

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10  ETHICAL CONSIDERATIONS  
10.1 
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 Ethics Committee (EC) / 
Institutional Review Board (IRB) approvals, including WIRB approval, of the protocol and any 
subsequent amendments in compliance with local law before the start of the study. 
 
10.2 
Recruitment and consent 
As soon as the study is approved by the CCMO and WIRB, healthy volunteers 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 Erasmus 
University. Furthermore, a Facebook page (link: http://www.facebook.com/malariavaccin) 
showing the advertisement text will be designed 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 which contains a link to a 
short online form. This short online form requests the interested volunteer to provide their 
contact details and answer some initial general health and medical questions. When 
seemingly suitable volunteers 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. 
Volunteers 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 and WIRB-approved informed consent. Informed 
consent must be obtained after the informed consent questionnaire and before conducting 
any study-specific procedures (i.e. any 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 informed consent 
questionnaire and health questionnaire answers will be discussed, and inclusion and 
exclusion criteria will be checked. Also, a letter for the general practitioner will be signed and 
sent after screening. Again, the investigator will answer all questions the volunteer has. The 

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investigator will emphasize that participation in the study is entirely voluntary and that the 
subject may withdraw at any time, without any obligation to declare their reason for 
withdrawal. However, if the volunteer has undergone exposure to Pb(PfCS@UIS4) or CHMI 
and not completed a course of appropriate antimalarial therapy then the volunteer will need 
to maintain contact with the investigators for monitoring and treatment.  
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 volunteers. Volunteers 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.  
 
10.3 
Benefits and risks assessment, group relatedness 
In order to obtain information on the capacity of potential immunization strategies to induce 
protection, immunization strategies will need to be tested in human subjects. Of course, the 
compelling need for a malaria vaccine needs to be balanced with the potential risks and 
discomforts for the volunteers. Explorative studies of potential pre-erythrocytic malaria 
vaccine candidates are a potentially powerful tool in the difficult decision making process of 
continuing investigation into vaccine candidates. This safety trial will be pivotal to the 
development of Pb(PfCS@UIS4) and will be used as a go/no-go criterion for further pursuit 
of Pb(PfCS@UIS4) as a potential vaccine candidate.  
There are no direct benefits to participation in the trial for volunteers. Despite their 
participation, volunteers will be advised to take regular malaria chemoprophylaxis when 
travelling to malaria endemic areas in the future.  
Risks for volunteers of phase 1 are related to exposure to a i) potential break-through blood 
stage infection after exposure to Pb(PfCS@UIS4) sporozoites, ii) the side effects of 
Malarone® (atovaquone/proguanil) and iii) side effects related to exposure to multiple 
mosquito bites.  
The volunteers of phase 2 will be exposed to the same risks as phase 1. However, the risk of 
blood stage infection after administration of Pb(PfCS@UIS4) is considered lower, since an 
absence of break-through infections in phase 1 is a criterion for proceeding to phase 2. 
Instead, volunteers of phase 2 have the additional risk iv) to develop blood-stage malaria 
after wild type P.falciparum NF54 infection.  
  
i) Risks related to administration of Pb(PfCS@UIS4)  
Based on preclinical assessment the occurrence of ‘break-through’ blood infections by 
Pb(PfCS@UIS4) is highly unlikely (please refer to D1 Investigator’s Brochure for a detailed 

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description of the pre-clinical studies. To ensure prompt treatment of a possible blood stage 
infection and adequate counselling and support, volunteers will be subjected to an intense 
clinical monitoring schedule with frequent site visits and blood examinations, comparable to 
protocols used in CHMI studies.  
Volunteers with breakthrough blood infections of Pb(PfCS@UIS4) will be treated with 
Malarone® (atovaquone/proguanil). In vitro and in vivo drug tests show that Pb(PfCS@UIS4) 
is sensitive to drugs such as atovaquone/proguanil and artemether/lumefantrine (please also 
refer to D1 Investigator’s Brochure). It is thus expected that a 100% cure rate will be met with 
these drugs, similar to blood infections of P. falciparum NF54 in CHMI studies.  
 
ii) Risks related to exposure to atovaquone/proguanil treatment 
Side effects associated with atovaquone/proguanil treatment are mainly gastrointestinal such 
as nausea, with vomiting, abdominal pain and diarrhea, and only present in a minority of 
cases. Headache, dizziness, anorexia and allergic reactions have been described. However, 
previous experience with Malarone® (atovaquone/proguanil) treatment in CHMI has 
demonstrated that side-effects are generally mild-to-moderate and limited to the days of 
treatment only (3 days). In case of unacceptable side effects, artemether/lumefantrine can be 
used as alternative treatments. 
 
iii) Side effects related to exposure to multiple mosquito bites 
The exposure of test subjects to mosquito bites has been shown to induce local adverse 
events such as itching, redness and swelling, the severity of which varies among test 
subjects. Topical tripelennamine and topical steroids such as 1% hydrocortisone cream and 
for more severe reactions, clobetasol 0.05% cream will be available in the clinical trials 
center for distribution to the volunteers. Additional medications available to volunteers by the 
clinical investigators will be oral cetirizine or other anti-histamines. Volunteers with exuberant 
reactions to mosquito bites may receive cetirizine prophylactically before immunization to 
abrogate skin reactions such as redness, swelling and itching. Systemic adverse events 
elicited by mosquito bites have not been found previously. It is not expected that bites of 
mosquitoes infected with Pb(PfCS@UIS4) will alter these events. In addition to local events, 
the administration of irradiated sporozoites by mosquito bite has led to the occurrence of mild 
headache and/or malaise in a limited number of volunteers. 
 
iv) Risks related to exposure to blood stage NF54 parasites (phase 2 only) 
There is a broad experience with the use of NF54 to induce blood stage malaria infection 
during CHMI the Radboudumc, ErasmusMC and Havenziekenhuis. 

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The occurrence of cardiac events in volunteers after blood stage malaria infection during 
CHMI trials in the Radboudumc has raised discussion about the safety of such controlled 
malaria infection trials. Based on recommendations of the CCMO and an External Scientific 
Advisory Committee to the European Malaria Vaccine Development Association, additional 
safety measures have been added to our protocols, including the early detection of blood 
infections by PCR followed by swift treatment of trial participants with antimalarial drugs. 
These safety measures have also been implemented in the current trial. For a more detailed 
discussion of these events, see section 1.5 and section 12. 
 
10.3.1 Ethical aspects concerning the use of human volunteers 
Infection of humans with malaria has been carried out for nearly a century, including for 
therapeutic use as treatment for neurosyphilis and later for drug and vaccine evaluation. The 
ability to carry out this type of work is largely based on the relatively low morbidity and the 
lack of mortality seen in these studies since the advent of feeding mosquitoes on P. 
falciparum gametocyte cultures in 1986 [26]. The occurrence of three cardiac events in 
volunteers participating in phase I/IIa malaria vaccine trials in Nijmegen raised intense 
discussion about the safety of malaria challenge trials with respect to cardiac events. Based 
on recommendations of the CCMO and an External Scientific Advisory Committee to the 
European Malaria Vaccine Development Association, this malaria challenge trial protocol has 
been adjusted (see also section 1.4). 
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. Of course, 
the compelling need for a malaria vaccines and treatments needs to be balanced with the 
potential risks and discomforts of the volunteers. Explorative studies looking for new or 
complementary vaccines are of paramount importance with the potential of large-scale 
application in endemic countries. These are considered essential tools to consolidate recent 
gains of malaria control in recent years and move towards malaria elimination. 
The study will be undertaken in accordance with Good Clinical Practices (GCP), according to 
the standards defined in the EEC directive 91/507/EEC, and in the Directive on Good Clinical 
Practice in Clinical Trials (ICH GCP, 75/318/EEC, January 1997) and under the principles of 
the Declaration of Helsinki; ethical permission will be sought from the CCMO the Netherlands 
and from the funding partner’s designated IRB, WIRB. 
 
10.4 
Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of the 
WMO. 

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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. 
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. 
 
The insurance applies to the damage that becomes apparent during the study or within 4 
years after the end of the study. 
 
10.5 
Incentives 
Enrolled volunteers of phase 1 will receive up to 1000,- Euros in compensation for their time 
and for the inconveniences of taking part in this study. Volunteers of phase 2 (Group 3) will 
receive an additional compensation up to 2300,- Euros. Volunteers that will receive a booster 
immunization and/or a second challenge infection will receive an additional compensation up 
to 900,- Euros or 1400,- Euros. Infectivity controls will receive up to 800,- Euros. This is 
based on compensation fees for procedures as below: 
 
Inconvenience of blood tests and/or visits: 20,- Euros per blood sampling and/or visit 
 
Mosquito immunization: 400,- Euros 
 
CHMI: 300,- Euros  
 
Compensation length: 20,- Euros per month 
Travel expenses will not be additionally reimbursed, and compensation will not be provided 
to volunteers who are not enrolled i.e. screen failures. Eligible volunteers who are enrolled at 
the inclusion visit as back-ups, but who are not challenged on Day 0 will be compensated 
50,- Euros. These compensation amounts are reasonable and in line with Dutch common 
practice. In case of unexpected medical complications, there will be access to state-of-the-art 
medical treatment with full costs covered by the insurance of Radboudumc. 

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11 ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
 
11.1 
Handling and storage of data and documents 
11.1.1 Confidentiality 
All parties agree to adhere to the principles of medical confidentiality in relation to clinical 
study subjects involved in this trial, and shall not disclose the identity of subjects to third 
parties without prior written consent of the subject. 
All data will be anonymised; volunteer data will be identified by a unique study number in the 
CRF. Separate confidential files containing identifiable information will be stored in secured 
locations. All plasma samples, or other biological samples, with exception of those taken for 
safety diagnostics, will be labeled with the volunteer study identification number. Samples 
taken for safety diagnostic (processed by the central clinical laboratory of the 
Havenziekenhuis) will be labeled with part of the subject identification code, study 
identification name and a fictitious birth date (only using the subjects actual birth year). The 
samples will not be labeled with volunteer names or actual birth dates. 
The subject identification code will be kept by the principal investigator.  
The investigator will maintain and retain appropriate medical and research records and 
essential documents for this trial in compliance with ICH GCP Guidelines, any regulatory 
requirements, and institutional requirements for the length of storage and for the protection of 
confidentiality of volunteers. The investigator will permit direct access to study records and 
source documents to authorized representatives of the sponsor, ethical committee(s) / 
institutional review board(s), regulatory agencies, authorized individuals from PATH, and the 
external monitor(s), for the purposes of quality assurance reviews, audits / inspections, and 
evaluation of the study safety and progress. Direct access includes examination, analysis, 
verification, and reproduction of de-identified records and reports that are important to the 
evaluation of the trial. Data and biological samples will be stored for 15 years. 
11.1.2 Data collection 
Designated trial staff will enter the data required by the protocol into the electronic CRF 
(eCRF).  
 
11.1.3 Storage of biological samples  
Biological samples, such as immunology samples and leftovers, obtained during the study 
will be properly stored and governed in a coded manner. Sera and plasma samples will be 
stored in -80°C freezers and isolated cells in liquid nitrogen.  All samples will have a 

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studycode, subjectcode and a randomizationcode, which enables it to be traced to the 
human subject. The key to the code is only accessible by the investigators (PI, investigator 
and co-investigator) and the local safety monitor. Biological samples will be stored for a 
maximum period of 15 years, after which they will be destroyed. The subjects will be asked 
for their consent for retention and analysis of the material. Appropriate ethical approval will 
be obtained prior to future use of stored samples. 
11.1.4 Database management and quality control 
All data should be recorded, handled and stored in a way that allows its accurate reporting, 
interpretation and verification. 
An external monitor will review the data entered into the eCRFs by investigational staff for 
completeness and accuracy and will instruct the site personnel to make any required 
corrections or additions. Queries are made during each monitoring visit. Designated 
investigator site staff are required to respond to the queries and confirm or correct the data. 
Medical history/current medical conditions and adverse events will be coded using the ICD-
10 terminology. 
 
11.2 
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, the progress of 
enrolment, and to ensure that Malarone® is being dispensed and accounted for according to 
protocol.  
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, 
electrocardiograms, 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. The 
only exception is the data from the quantitative PCR, which is loaded from the PCR machine 
directly into the eCRF. As with all parts of the eCRF, there is an audit trail in place to register 
every data entry. The investigator will also keep the original informed consent form signed by 
the subject (a signed copy is given to the subject). 
The investigator will give the external monitor access to all relevant source documents to 
confirm their consistency with the eCRF entries. According to the NFU risk classification 
system, this clinical trial has been classified as ‘middle risk’. The monitor will perform full 
verification for the presence of informed consent, adherence to the inclusion/exclusion 

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criteria and documentation of SAEs. The recording of data that will be used for all primary 
and safety variables will be assessed for 25% of included subjects (i.e. 8 subjects). 
 
11.3 
Amendments  
Amendments are changes made to the research, and will only be made after favourable 
opinions / approvals by the CCMO and WIRB have been given - except where necessary to 
eliminate apparent immediate hazards to the subject(s). All amendments will be submitted to 
CCMO and WIRB for review and approval.  
 
11.4 
Annual progress report 
The 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.  
Continuing review reports will be submitted to WIRB, in accordance with its reporting 
requirements. 
 
11.5 
Temporary halt and (prematurely) end of study report 
The investigator will notify the CCMO of the end of the study within a period of 8 weeks. The 
end of the study is defined as the subject’s last visit on day 100 after the last exposure to 
Pb(PfCS@UIS4) and 35 days post-CHMI for the infectivity control subjects.  
The investigator 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 investigator will 
notify the CCMO within 15 days, including the reasons for the premature termination. 
Within one year after the end of the study, the investigator will submit a final study report with 
the results of the study, including any publications/abstracts of the study, to the CCMO. 
PATH REC and WIRB will also be notified of any decisions to prematurely suspend or 
terminate the study, and a study closure report will be submitted to WIRB when all subjects 
have finished their final visits and follow-up and when the sponsor has indicated the study is 
closed. 
 
11.6 
Public disclosure and publication policy 
The study will be publicly disclosed and registered on clinicaltrials.gov before inclusion of the 
volunteers.The final report will be prepared by the investigators at the Radboudumc, 
Havenziekenhuis and the ErasmusMC. It will be signed by the project leader or the principal 

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investigator. The investigators will make every effort to publish the results in a peer-reviewed 
journal. 

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12 STRUCTURED RISK ANALYSIS  
12.1 
Potential issues of concern 
a. Level of knowledge about mechanism of action 
In this study we will investigate the safety and tolerability of administration of a genetically 
modified P. berghei parasite, Pb(PfCS@UIS4), by mosquito bite.  
All pre-clinical data indicate that Pb(PfCS@UIS4) is a safe antigen delivery platform, i.e. not 
able to develop within human erythrocytes. It is expected that sporozoites of 
Pb(PfCS@UIS4) are able to invade the liver, but are unable to develop a parasitemia. 
However, as this is the first in-human trial of Pb(PfCS@UIS4), the exact phenotype of the 
parasites (sporozoites, liver stages) in humans is unknown. If a break-through blood infection 
would occur, medical vigilance to ensure early detection of such cases is crucial to the safety 
of volunteers. Changes in the liver stage development of the genetically modified 
Pb(PfCS@UIS4) strain may result in prolonged duration of the liver stage or the release of 
very low numbers of merozoites from the liver, ultimately delaying time to patency. A 
stringent follow-up schedule until anti-malarial treatment is thus required for all volunteers.  
In addition, exposure of humans to a great number of sporozoites/attenuated liver stages 
parasites could induce an immune response, which may present with clinical symptoms.  
 
b. Previous exposure of human beings with the test product(s) and/or products with a similar 
biological mechanism 
There is extensive clinical experience of controlled human P. falciparum malaria infections by 
the bite of infected mosquitoes. Since 1986 more than 3,500 volunteers have had CHMI by 
the bites of mosquitoes fed on cultures of P. falciparum gametocytes to produce P. 
falciparum sporozoites[26]. This has proved to be a reproducible, predictable and safe 
method of inducing P. falciparum infections. The results of such studies were summarized in 
1997 [27], in 2007 [33] and in 2012 [29]. Furthermore, there has been one preceding clinical 
trial with a genetically modified Plasmodium falciparum performed with Pf∆p52∆p36 (also 
refer to Section 1.3) which was administered by a similar route through mosquito bites. In the 
high dose group (200 bites) all volunteers experienced erythema and pruritus, five 
experienced edema (of which one >90mm), one experienced induration, one experienced 
pain. Systemic adverse events were also reported within 7 days after exposure: fever, 
malaise (both one volunteer, grade 1), headache (grade 2, two volunteers), nausea and 
vomiting (2 volunteers, grade 2 in one). In the Pf∆p52∆p36 trial one volunteer in the high-
dose group experienced a breakthrough blood stage infection (confirmed by PCR). The 
clinical signs and symptoms of this breakthrough blood infection were similar to the wild-type 
NF54 parental strain.  

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This is the first-in-human trial of Pb(PfCS@UIS4).  
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo human 
cell material? 
Several animal studies and in vitro assays have been performed demonstrating the capability 
of generating an immune response without generating a disease-triggering human blood-
stage infection (see D1. Investigator’s Brochure).  
 
d. Selectivity of the mechanism to target tissue in animals and/or human beings 
The life-cycle of malaria parasites follows a fixed and pre-determined course in the human 
host. P. falciparum sporozoites delivered by mosquito bite are only able to invade into and 
develop within hepatocytes. These developments are constrained by multiple parasite-host 
ligand interactions and the parasite’s ability to manipulate the host cell’s internal environment 
[36]. It has been shown that the sporozoites of Pb(PfCS@UIS4) invade hepatocytes similarly 
to sporozoites of P. berghei, both in in vitro cultures of primary hepatocytes and in chimeric 
mice engrafted with human liver tissue. Furthermore, biodistribution- and toxicology studies 
have been performed with Pb(PfCS@UIS4), and no irregular or unexpected target tissues 
have been described. Please see D1. Investigator’s Brochure for a detailed description. 
 
e. Analysis of potential effect 
Safety and tolerability of Pb(PfCS@UIS4) will be assessed by recording adverse events, 
reported at any time during the trial either at a visit or by writing in the diary. Vital signs, 
including temperature, will be recorded at every visit following exposure. Assessment of 
complete blood count will be performed at any time point blood is drawn (daily or every other 
day after exposure), blood chemistry analysis will be performed frequently after exposure. 
The maximum parasitemia in subjects undergoing CHMIs is equivalent to ~50 parasites/μL, 
the detection level of thick smear microscopy is ~5 parasites/μL).  
In this study we will use thick smear analysis after exposure of Pb(PfCS@UIS4) and qPCR 
as main tool to detect blood-stage malaria parasites after CHMI, which has an even greater 
sensitivity (detection limit 20 parasites/ml). 
 
 
f. Pharmacokinetic considerations 
Not applicable. 
 
g. Study population 

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Included subjects are healthy young adult volunteers, who have been extensively screened 
for any evidence of co-morbidity, in particular cardiovascular risk factors. Female subjects of 
child-bearing age are screened for pregnancy by urine test and are required to use 
contraception throughout the study period. Please refer to 4.2 Inclusion criteria for 
acceptable forms of contraception. See section 7.7 for procedure to be followed when a 
volunteer is pregnant during the study. 
 
h. Interaction with other products 
Concurrent use of drugs potentially interacting with Malarone® (atovaquone-proguanil) (e.g. 
artemether-lumefantrine, rifampicin, metoclopramide, oral anti-coagulants and certain anti-
retrovirals) are contra-indicated. 
 
i. Predictability of effect 
All pre-clinical data indicate that the Pb(PfCS@UIS4) sporozoites can invade human 
hepatocytes but not develop within human erythrocytes. Therefore, it is expected that 
sporozoites of Pb(PfCS@UIS4) are able to elicit an immune respons in the skin and liver 
needed for pre-erythrocytic protection against P. falciparum, without causing disease 
because of the inability of developing parasitemia.  
 
j. Can effects be managed? 
Subjects are frequently seen on an outpatient basis for clinical and parasitological 
assessment to ensure treatment is started if necessary at the earliest possible time point. In 
the unlikely event that a break-through blood stage infection occurs, volunteers will be 
curatively treated with a regimen of Malarone® (atovaquone/proguanil). Pb(PfCS@UIS4) has 
been tested for susceptibility to atovaquone/proguanil in vitro, and in vivo in mice 
drugsensitivity studies. Should treatment with this drug need to be discontinued prematurely 
in any subject for whatever other reason (e.g. intolerability), artemether/lumefantrine will act 
as a back-up drug, and is a good alternative. Artemether/lumefantrine has also been shown 
to be effective against Pb(PfCS@UIS4) in drug sensitivity studies (see D1 Investigator’s 
Brochure). 
 
12.2 
Synthesis 
The extensive pre-clinical evaluation, indicate that Pb(PfCS@UIS4) can be safely used in 
healthy volunteers in this trial and the risks are therefore considered low. They will, 
furthermore, be minimized by strict adherence to the inclusion/exclusion criteria and close 
clinical monitoring. 

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13 Appendices 
13.1 
Appendix 1: Systematic Coronary Risk Evaluation (SCORE) Risk Chart 
The SCORE risk chart, made by the European Society of Cardiology, comprised of gender, 
age, total cholesterol, systolic blood pressure and smoking status, with relative risk chart, 
qualifiers and instructions. 
For the Netherlands, the low risk chart (Figure 1) is used. To use this chart, we should find 
the cell nearest to a person’s age, cholesterol, and systolic blood pressure values. The risk in 
the chart is 10-year total risk.  
 
 
Figure 1. SCORE – European Low Risk Chart 
 
 

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13.2 
Appendix 2: Classification of Adverse Events 
The following tables are adapted from the Food and Drug Administration’s Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers 
Enrolled in Preventive Vaccine Trials. 
 
A. Tables for Clinical Abnormalities 
Local Reaction to 
mosquito bites 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Pain 
Does not interfere with activity 
Repeated use of nonnarcotic pain reliever > 24 
hours or interferes with activity 
Any use of narcotic pain reliever or 
prevents daily activity 
Tenderness 
Mild discomfort to touch 
Discomfort with movement 
Significant discomfort at rest 
Pruritis 
No interference with activity 
Some interference with activity 
Significant; prevents daily activity 
Induration/Swelling * 
<15 cm and does not interfere 
with activity 
≥15 cm or interferes with activity 
> 15 cm or prevents daily activity 
*Induration/Swelling should be evaluated and graded using the functional scale as well as the actual measurement 
 
 

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Vital signs 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Fever (oC) 
38.0 – 38.4 
38.5 – 38.9 
≥39.0 
Tachycardia – beats per minute 
101 – 115 
116 – 130 
>130 
Bradycardia – beats per minute 
50 – 54 
45 – 49 
< 45 
Hypertension (systolic) –mmHg 
141 – 150 
151 – 155 
>155 
Hypertension (diastolic) – mmHg 
91 – 95 
96 – 100 
>100 
Hypotension (systolic) – mmHg 
85 – 89 
80 – 84 
< 80 
Respiratory rate – breaths per minute 
17 – 20 
21 – 25 
>25 
* Subject should be at rest for all vital sign measurements.  
** Auricular temperature. 
*** When resting heart rate is between 60 – 100 beats per minute. Use clinical judgement when characterizing bradycardia among some healthy subject 
populations, for example, conditioned athletes. 
 
Systemic 
(General) 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Rash 
No interference with activity 
Some interference with activity 
Significant; prevents daily activity 
Urticaria 
No interference with activity 
Some interference with activity 
Significant; prevents daily activity 
Pruritis 
No interference with activity 
Some interference with activity 
Significant; prevents daily activity 
Edema 
No interference with activity 
Some interference with activity 
Significant; prevents daily activity 
Headache 
No interference with activity 
Repeated use of nonnarcotic pain reliever > 24 
hours or some interference with activity 
Significant; any use of narcotic pain reliever or 
prevents daily activity 
Fatigue 
No interference with activity 
Some interference with activity 
Significant; prevents daily activity 
Malaise 
No interference with activity 
Some interference with activity 
Significant; prevents daily activity 
Rigors 
No interference with activity 
Some interference with activity 
Significant; prevents daily activity 

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Myalgia 
No interference with activity 
Repeated use of nonnarcotic pain reliever > 24 
hours or some interference with activity 
Significant; any use of narcotic pain reliever or 
prevents daily activity 
Arthralgia 
No interference with activity 
Repeated use of nonnarcotic pain reliever > 24 
hours or some interference with activity 
Significant; any use of narcotic pain reliever or 
prevents daily activity 
Nausea/Vomiting 
No interference with activity or 1 – 
2 episodes/24 hours 
Some interference with activity or > 2 episodes/24 
hours 
Prevents daily activity, requires outpatient IV 
hydration 
Abdominal pain 
No interference with activity 
Repeated use of nonnarcotic pain reliever > 24 
hours or some interference with activity 
Significant; any use of narcotic pain reliever or 
prevents daily activity 
Diarrhea 
2 – 3 loose stools or < 400 
gms/24 hours 
4 – 5 stools or 400 – 800 gms/24 hours 
6 or more watery stools or > 800gms/24 hours 
or requires outpatient IV hydration 
Chest pain 
No interference with activity 
Repeated use of nonnarcotic pain reliever > 24 
hours or some interference with activity 
Significant; any use of narcotic pain reliever or 
prevents daily activity 
Palpitations 
No interference with activity 
Some interference with activity 
Significant; prevents daily activity 
Shortness of 
breath 
No interference with activity 
Some interference with activity 
Significant; prevents daily activity 
 
B. Tables for Laboratory Abnormalities 
Haematology 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Erythrocytes 
(.1012/L) 
Male 
4.0 - 4.4 
3.5 – 3.9 
< 3.5 
Female 
3.5 – 3.9 
3.0 – 3.4 
< 3.0 
Hematocrit 
(%) 
Male 
0.35 – 0.39 
0.30 – 0.34 
< 0.30 
Female 
0.30 – 0.34 
0.25 – 0.29 
< 0.25 
Hemoglobin 
(mmol/L) 
Male 
 8.0 – 8.5 
7.0 – 7.9 
< 7.0 
Female 
7.0 – 7.5 
6.0 – 6.9 
< 6.0 
 MCV (fL)  
60 – 79 
40 – 59 
< 40 

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 MCH (fmol) 
1.50 – 1.69 
1.25 – 1.49 
< 1.25 
 MCHC (mmol/L) 
15.0 – 18.9 
10.0 – 14.9 
< 10  
Leukocytes 
(.109/l) 
Leukocytosis 
11.01 – 15.00 
15.01 – 20.00 
> 20.00 
Leukopenia 
3.00 – 3.99 
2.00 – 2.99 
< 2.00 
Thrombocytopenia (.109/l) 
75 – 150 
50 – 74 
< 50 
 
Serum 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Alkaline phosphatase 
1.1 – 2.0 x ULN 
2.1 – 5.0 x ULN 
> 5.0 x ULN 
ALT 
1.1 – 2.5 x ULN 
2.6 – 15.0 x ULN 
> 15.0 x ULN 
AST 
1.1 – 2.5 x ULN 
2.6 – 15.0 x ULN 
> 15.0 x ULN 
Bilirubin (Total) 
1.1 – 1.25 x ULN 
1.26 – 3 x ULN 
> 3 x ULN 
Creatinine 
(umol/L) 
Male 
105 – 123 
124 – 142 
> 142  
Female 
91 – 109 
110 – 128 
> 128 
Gamma GT 
1.1 – 2.5 x ULN 
2.6 – 15.0 x ULN 
> 15 x ULN 
Potassium – Hyperkalemia 
(mmol/L) 
5.1 – 5.2 
5.3 – 5.4 
> 5.4  
Potassium – Hypokalemia 
(mmol/L) 
3.3 - 3.4  
3.1 – 3.2 
< 3.1 
Sodium - Hypernatremia (mmol/L) 
146 – 147 
148 – 149 
> 149 
Sodium - Hyponatremia (mmol/L) 
132 – 134 
131 – 130 
< 130 
Ureum 
22 – 25 
26 – 29 
> 29 
*ULN is the upper limit of the normal range. 
 
 

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