Conditions: Malaria,Falciparum Controlled Human Malaria Infection Immunization; Infection
Keywords: Mosquito bites Sporozoite Immunization Malaria Falciparum
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 43
Healthy volunteers: yes

Brief summary:
This is an open label, randomized, controlled clinical trial. The primary aim of this project is to determine the safety and tolerability of NF135.C10 sporozoite immunization under chemoprophylaxis against homologous and heterologous challenge infection.

Detailed description:
A total of 49 healthy volunteers will be allocated to receive either three immunizations with 15 NF135.C10 infected Anopheles mosquitoes (n=30), 3 immunizations with 5 NF135.C10 infected mosquitoes (n=10) or no immunizations (n=6). Immunizations in cohort A (n=20) will be performed under mefloquine prophylaxis, spaced 4 weeks apart. In cohort B, volunteers will not take mefloquine prophylaxis, instead all volunteers will be treated presumptively on day 7 after each immunization with a curative regimen of artemether/lumefantrine, regardless of parasitaemia or symptoms.

Nineteen weeks after the last immunization, all volunteers plus naïve controls will be challenged either by the bites of 5 NF135.C10 (n=36) or 5 NF54 (n=13) infected mosquitoes. After challenge infection, volunteers will be followed up on an out-patient basis once daily for qPCR and safety lab measurements from day 6 until day 21 post challenge. All volunteers will be treated with a curative regimen of atovaquone/proguanil, either at the time of detection of blood stage parasitemia, or 28 days after challenge infection.

Arms:
arm 1: 10 volunteers will receive three immunizations with 15 NF135.C10 infected Anopheles mosquitoes under mefloquine prophylaxis. Volunteers will be challenged by the bites of 5 NF135.C10 infected Anopheles mosquitoes 19 weeks after the last immunization. arm 2: 10 volunteers will receive three immunizations with 5 NF135.C10 infected Anopheles mosquitoes under mefloquine prophylaxis. Volunteers will be challenged by the bites of 5 NF135.C10 infected Anopheles mosquitoes 19 weeks after the last immunization. arm 3: 10 volunteers will receive three immunizations with 15 NF135.C10 infected Anopheles mosquitoes and are presumptively treated with artemether/lumefantrine starting on day 7 after each immunization. Volunteers will be challenged by the bites of 5 NF135.C10 infected Anopheles mosquitoes. arm 4: 10 volunteers will receive three immunizations with 15 NF135.C10 infected Anopheles mosquitoes and are presumptively treated with artemether/lumefantrine starting on day 7 after each immunization. Volunteers will be challenged by the bites of 5 NF54 infected Anopheles mosquitoes 19 weeks after the last immunization. arm 5: Challenge infection control group: 3 volunteers will receive no immunization and will be challenged by the bites of 5 NF135.C10 infected Anopheles mosquitoes 19 weeks after the last immunization. arm 6: Challenge infection control group: 3 volunteers will receive no immunization and will be challenged by the bites of 5 NF54 infected Anopheles mosquitoes 19 weeks after the last immunization. arm 7: Challenge infection control group: 3 volunteers will receive no immunization and will be challenged by the bites of 5 NF135.C10 infected Anopheles mosquitoes 19 weeks after the last immunization.

Interventions: intervention 1: CPS-immunization intervention 2: malaria challenge infection, P. falciparum NF135.C10 intervention 3: malaria challenge infection, P. falciparum NF54 intervention 4: CPS-immunization (A/L) intervention 5: Atovaquone / Proguanil Oral Tablet [Malarone]
Intervention descriptions: intervention 1: Subjects will be immunized 3 times by exposure to the bites of P. falciparum NF135.C10 infected mosquitoes while taking mefloquine prophylaxis. intervention 2: Subjects will receive bites from 5 Anopheles mosquitoes infected with Plasmodium falciparum NF135.C10 sporozoites. intervention 3: Subjects will receive bites from 5 Anopheles mosquitoes infected with Plasmodium falciparum NF54 sporozoites. intervention 4: Subjects will be immunized 3 times by exposure to the bites of P. falciparum NF135.C10 infected mosquitoes and receive presumptive treatment with artemether/lumefantrine initiated on day 7 after each immunization. intervention 5: All participants will be treated with atovaquone/proguanil (1000/400 mg (= 4 tablets) 1×/day during 3 consecutive days) when they develop a malaria infection or on day 28 after malaria challenge infection.

Protocol document:
Prot_SAP_000.pdf: 
NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CLINICAL TRIAL PROTOCOL 
 
 
 
Safety and protective efficacy of chemoprophylaxis and sporozoite 
immunization with Plasmodium falciparum NF135 against homologous and 
heterologous challenge infection in healthy volunteers in the Netherlands 
 
CPS135 
 
 
Version 7.0 
Date: 3 November 2020 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CONFIDENTIAL


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
 
PROTOCOL TITLE 
‘Safety and protective efficacy of chemoprophylaxis and sporozoite immunization with 
Plasmodium falciparum NF135 against homologous and heterologous challenge infection in 
healthy volunteers in the Netherlands’ (CPS135) 
 
 
Protocol ID 
CPS135
Short title 
Safety and efficacy of NF135 CPS immunization 
ToetsingOnline nummer 
NL63594.091.17
Version 
7.0
Date 
3 November 2020
Protocol author 
Radboudumc
Department of Medical Microbiology 
Jona Walk, MD 
Manon Alkema, MD 
Saskia van der Boor, MD 
Sponsor 
Stichting Katholieke Universiteit Nijmegen 
Principal investigator
Radboudumc
Department of Medical Microbiology 
Matthew McCall, MD, PhD 
E-mail: Matthew.mccall@Radboudumc.nl  
Tel : +31 (0)24 3615363 
Senior Co-Investigator
Radboudumc 
Department of Medical Microbiology  
Robert Sauerwein MD PhD  
Tel : +31 (0)24 3610577 
Fax :+31 (0)24 3614666 


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
Clinical investigators 
Radboudumc 
Department of Medical Microbiology 
Tel: +31 (0)24 3619515 
Fax: +31 (0)24 3614666 
 
Saskia van der Boor, MD 
E-mail: Saskia.vanderBoor@radboudumc.nl 
 
Manon Alkema, MD 
E-mail: Manon.Alkema@radboudumc.nl 
Subsidising party 
PATH MVI
2201 Westlake Avenue, Suite 200 
Seattle, Washington 
98121, USA 
Independent expert 
Radboudumc 
Department of Internal Medicine 
Dr. Arjan van Laarhoven 
Tel : +31 24 361 69 80 
Arjan.vanLaarhoven@radboudumc.nl 
Laboratory sites 
Radboudumc Clinical Chemical Laboratory 
Eric Aaldring 
Tel: +31 (0)24 3616997 
Fax: +31 (0)24 3568408 
e-mail: Eric.Aaldring@radboudumc.nl 
 
Clinical Microbiology Laboratory 
Dr. Heiman Wertheim 
Tel: +31 (0)24 36 14281 
e-mail: Heiman.Wertheim@radboudumc.nl 
Pharmacy 
Radboudumc Clinical Pharmacy
Clinical Trial Unit 
Dr. Rob ter Heine 
Tel: +31 (0)24 3616405 
Fax: +31 (0)24 3668755 
e-mail: R.terHeine@radboudumc.nl 


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
CRO 
CROMSOURCE 
Lange Dreef 11H 
4131 NF Vianen 
The Netherlands 


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
 
 
 
 
PROTOCOL SIGNATURE SHEET 
 
 
Name 
Signature
Date 
Principal Investigator 
Matthew McCall, MD, PhD 
Department of Medical Microbiology 
Radboud university medical center 
 
 
Investigator 
Saskia van der Boor, MD  
Department of Medical Microbiology 
Radboud university medical center 
 
 
Investigator 
Manon Alkema, MD 
Department of Medical Microbiology 
Radboud university medical center 
 
 


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
1. 
TABLE OF CONTENTS 
Version 7.0 ........................................................................................................................................ 1 
PROTOCOL SIGNATURE SHEET ................................................................................................... 5 
LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS ............................................................ 9 
Objectives ....................................................................................................................................... 11 
Main study endpoints ...................................................................................................................... 12 
1. 
INTRODUCTION AND RATIONALE ........................................................................................ 13 
1.1  Introduction .............................................................................................................................. 13 
1.2  Rationale .................................................................................................................................. 13 
1.3  Controlled Human Malaria Infection ......................................................................................... 16 
1.4  Safety Aspects ......................................................................................................................... 17 
Rationale for modifications to the second immunization of cohort B ................................................ 19 
2. 
OBJECTIVES .......................................................................................................................... 20 
3. 
STUDY DESIGN ...................................................................................................................... 21 
4. 
STUDY POPULATION ............................................................................................................. 23 
4.1  Population (base) ..................................................................................................................... 23 
4.2  Inclusion criteria ....................................................................................................................... 23 
4.3  Exclusion criteria ...................................................................................................................... 24 
4.4  Sample size calculation ............................................................................................................ 25 
5. 
TREATMENT OF SUBJECTS .................................................................................................. 27 
5.1  Investigational product/treatment ............................................................................................. 27 
5.2  Mefloquine prophylaxis ............................................................................................................ 27 
5.2.2 
Contraindications .............................................................................................................. 27 
5.2.3 
Possible side effects ......................................................................................................... 27 
5.3  Atovaquone/proguanil treatment .............................................................................................. 28 
5.3.2 
Contraindications .............................................................................................................. 28 
5.3.3 
Possible side effects ......................................................................................................... 28 
5.4  Artemether/lumefantrine treatment ........................................................................................... 28 
5.4.2 
Contraindications .............................................................................................................. 29 
5.4.3 
Possible side effects ......................................................................................................... 29 
5.5  Symptomatic treatment ............................................................................................................ 29 
6. 
INVESTIGATIONAL PRODUCT ............................................................................................... 30 
7. 
NON-INVESTIGATIONAL PRODUCT ...................................................................................... 30 
7.1  Name and description of non-investigational product(s) ........................................................... 30 
7.2  Summary of findings from non-clinical studies .......................................................................... 30 
7.3  Summary of findings from clinical studies ................................................................................. 30 
NF135.C10 
NF54 ......................................................................................................................... 32 
7.4  Summary of known and potential risks and benefits ................................................................. 34 
7.5  Description and justification of route of administration and dosage .......................................... 36 
7.6  Dosages, dosage modifications and method of administration ................................................. 37 
7.7  Preparation and labelling of Non Investigational Medicinal Product .......................................... 37 
7.8  Drug accountability ................................................................................................................... 38 


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
8. 
METHODS ............................................................................................................................... 38 
8.1  Study parameters/endpoints .................................................................................................... 38 
8.1.1 
Main study endpoint .......................................................................................................... 38 
8.1.2 
Secondary study endpoints ............................................................................................... 38 
8.1.3 
Exploratory study endpoints .............................................................................................. 38 
8.2  Randomisation and treatment allocation .................................................................................. 38 
8.2.1 
Baseline screening ........................................................................................................... 39 
8.2.2 
CPS immunization with mosquito bite ............................................................................... 40 
8.2.3 
Controlled Human Malaria Infection after drug washout (challenge).................................. 42 
8.2.4 
Treatment with atovaquone/proguanil after challenge ....................................................... 43 
8.2.5 
Re-challenge infection ...................................................................................................... 43 
8.2.6 
Physical examination ........................................................................................................ 44 
8.2.7 
Vital signs ......................................................................................................................... 44 
8.2.8 
Electrocardiogram ............................................................................................................. 44 
8.2.9 
Blood sampling and safety laboratory evaluations ............................................................. 44 
8.2.10 
Analysis of parasite densities after challenge infection ...................................................... 45 
8.2.11 
Case report forms and data collection ............................................................................... 45 
8.2.12 
Patient-reported outcomes (study diary) ........................................................................... 46 
8.2.13 
Exploratory immunological assessments .......................................................................... 46 
8.2.14 
Flow chart study design .................................................................................................... 47 
8.3  Withdrawal of individual subjects .............................................................................................. 46 
8.4  Replacement of individual subjects after withdrawal ................................................................. 47 
8.5  Follow-up of subjects withdrawn from treatment ....................................................................... 47 
8.6  Premature termination of the study .......................................................................................... 47 
9. 
SAFETY REPORTING ............................................................................................................. 48 
9.2  AEs, SAEs and SUSARs ......................................................................................................... 48 
9.2.2 
Serious adverse events (SAEs) ........................................................................................ 48 
9.3  Follow-up of adverse events .................................................................................................... 49 
9.3.2 
Assessment of causality ................................................................................................... 50 
9.3.3 
Follow-up of adverse events ............................................................................................. 51 
9.4  Local Safety Monitor (LSM) and Safety Monitoring Committee (SMC) ..................................... 51 
9.4.2 
Safety Monitoring Committee (SMC) ................................................................................. 51 
9.4.3 
Review of safety data by the safety monitor and SMC ...................................................... 51 
9.4.4 
Safety stopping rules ........................................................................................................ 52 
10.  STATISTICAL ANALYSIS ........................................................................................................ 53 
10.2 Secondary study parameter ..................................................................................................... 53 
10.3 Exploratory study parameters .................................................................................................. 53 
11.  ETHICAL CONSIDERATIONS ................................................................................................. 54 
11.2 Recruitment and consent ......................................................................................................... 54 
11.3 Benefits and risks assessment, group relatedness ................................................................... 55 
11.3.1 
Ethical aspects concerning the production of P. falciparum infected mosquitoes ............. 55 
11.3.2 
Ethical aspects concerning the use of human volunteers .................................................. 55 
11.4 Compensation for injury ........................................................................................................... 56 
11.5 Incentives ................................................................................................................................. 56 


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
12.  ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ........................................ 58 
12.1.2 
Data collection .................................................................................................................. 58 
12.1.3 
Database management and quality control ....................................................................... 58 
12.2 Monitoring and Quality Assurance ............................................................................................ 58 
12.3 Amendments ............................................................................................................................ 59 
12.4 Annual progress report ............................................................................................................. 59 
12.5 Temporary halt and (prematurely) end of study report .............................................................. 59 
12.6 Public disclosure and publication policy ................................................................................... 60 
13.  STRUCTURED RISK ANALYSIS ............................................................................................. 61 
13.2 Cardiac events following Controlled Human Malaria Infections ................................................. 63 
Case 1 ............................................................................................................................................ 64 
Case 2 ............................................................................................................................................ 64 
Case 3 ............................................................................................................................................ 65 
13.3 Transient liver function test elevations ...................................................................................... 67 
13.4 Transient serum creatinine elevations ...................................................................................... 68 
13.5 Synthesis ................................................................................................................................. 68 
14.  REFERENCES ........................................................................................................................ 69 
 


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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS 
 
 
ABR 
ABR form, General Assessment and Registration form, is the application 
form that is required for submission to the accredited Ethics Committee (In 
Dutch, ABR = Algemene Beoordeling en Registratie) 
AE 
Adverse Event 
A/L 
Artemether/lumefantrine (Riamet®) 
A/P 
Atovaquone/proguanil (Malarone®) 
AR 
Adverse Reaction 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
CV 
Curriculum Vitae 
DASS 
Depression Anxiety Stress Scales 
DSMB 
Data Safety Monitoring Board 
eCRF 
electronic Case Report Forms 
EU 
European Union 
EudraCT 
European drug regulatory affairs Clinical Trials 
GCP 
Good Clinical Practice 
IB 
Investigator’s Brochure 
IC 
Informed Consent 
IMP 
Investigational Medicinal Product 
IMPD 
Investigational Medicinal Product Dossier 
METC 
Medical research ethics committee (MREC); in Dutch: medisch ethische 
toetsing commissie (METC) 
MINI 
Mini-International Neuropsychiatric Interview 
MVI 
Malaria Vaccine Initiative 
PATH-REC 
PATH Research Ethics Committee; the funding partner’s ethical 
committee. 
PI 
Priniciple Investigator 
(S)AE 
(Serious) Adverse Event 
SPC 
Summary of Product Characteristics (in Dutch: officiële productinfomatie 
IB1-tekst) 


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Sponsor 
The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A 
party that provides funding for a study but does not commission it is not 
regarded as the sponsor, but referred to as a subsidising party. 
 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
Wbp 
Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgevens) 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch- 
wetenschappelijk Onderzoek met Mensen 
WIRB 
Western Institutional Review Board 


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SUMMARY 
 
Rationale: Malaria, a disease caused by the parasite Plasmodium, is one of the world’s major 
infectious diseases. Ultimately, the key to malaria control and hopefully eradication would be an 
effective vaccine. Chemo-Prophylaxis and Sporozoite immunization (CPS) has repeatedly shown 
to be an extremely efficient regimen for induction of long lasting sterile homologous protection. 
However, it provided only 20% and 10% sterile protection against heterologous NF135.C10 and 
NF166.C8 clones, respectively. We propose to make use of the increased liver infectivity of 
NF135.C10, to increase the late liver stage load without the need for increasing the number of 
sporozoites administered. The presumably generated higher titers and broader repertoire of 
specific antibodies can increase heterologous protection. 
Objectives 
Primary Objective: To determine the safety and tolerability of NF135.C10 sporozoite immunization 
under chemoprophylaxis 
Secondary Objectives: 
 
To 
determine 
the dose-dependent 
protective 
efficacy 
of 
NF135.C10 CPS- 
immunization against homologous controlled human malaria infection 
 
To  determine  the  protective  efficacy  of  NF135.C10  CPS-immunization  against 
heterologous NF54 controlled human malaria infection 
 
To assess the longevity of protective immunity after NF135.C10 CPS-immunization 
against homologous challenge in cohort A 
Study design and intervention: In an open label, randomized, controlled clinical trial, a maximum 
of 52 volunteers in two cohorts will be allocated to receive immunizations with  NF135.C10 infected 
Anopheles mosquitoes. In cohort A,  volunteers will receive 3 immunizations with 15 (n=10) or 5 
(n=10) NF135.C10 infected mosquitoes or no immunizations (controls, n=3). Immunizations will 
be performed under mefloquine prophylaxis, spaced 4 weeks apart. A thick smear will be performed 
on days 7-9 after each immunization and symptomatic volunteers with a positive thick smear will be 
treated with a curative regimen of atovaquone/proguanil (A/P).  
In cohort B, 20 volunteers will receive a first immunization with 15 NF135.C10 infected mosquitoes and 
a second immunization with 5 NF135.C10 infected mosquitoes. Volunteers will not take mefloquine 
prophylaxis. Instead all volunteers will be treated presumptively on day 7 after the first  immunization 
with a curative regimen of A/L, and on day 5  after the second immunization with a curative regimen of 
A/P, regardless of parasitaemia or symptoms. Seventeen - nineteen weeks after the last immunization, 
all volunteers plus 6 naïve controls will be challenged either by the bites of 5 NF135.C10 (n=36) or 5 
NF54 (n=13) infected mosquitoes. After challenge infection, volunteers will be followed up on an out-
patient basis once daily for qPCR and safety lab measurements from day 6 until day 21 post challenge. 


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All volunteers will be treated with a curative regimen of A/P, either at the time of detection of blood 
stage parasitemia, or 28 days after challenge infection. If ≥50% of subjects in the immunization groups 
in cohort A are protected against primary challenge, protected subjects, along with 3 newly recruited 
controls will undergo a (second) CHMI with NF135.C10 approximately one year after the last 
immunization.  
 
Study population: Healthy, malaria-naive male and female volunteers aged 18-35. 
Main study endpoints 
Primary study endpoint: Frequency and magnitude of adverse events after NF135.C10 CPS 
immunization 
Secondary study endpoints: 
 
Time to blood stage parasitemia detectable by qPCR after malaria challenge infection 
 
Sterile protection after controlled human malaria infection 
Exploratory study endpoints: 
 
The phenotype and cytokine profile of P. falciparum specific T cell responses induced by 
NF135.C10 CPS immunization 
 
The antigen specificity of T cell responses induced by NF135.C10 CPS immunization 
 
The antigen specificity and/or functionality of P. falciparum specific antibodies induced by 
NF135.C10 CPS immunization 
 
The phenotype and/or function of innate and semi-innate immune responses to NF135.C10 
CPS immunization and/or CHMI, including γδT cells, invariant T cells, antigen presenting cells, 
NK cells and granulocytes 
 
Epigenetic profiles of innate immune cell subsets, with emphasis on both activation 
(H3K4me3, H3K4me1, H3K27Ac) and repression (H3K9me3, H3K27me) markers 
 
RNA transcriptome profiling through whole mRNA-sequencing, PCR and/or microarray 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: The study is associated with several short periods of intense clinical 
monitoring with frequent clinic visits and blood examinations. As it is unpredictable when 
subjects will develop a positive qPCR after challenge, it is impossible to state the exact number 
of clinical visits and blood examinations. However, in groups 1 and 2 (cohort A) the number of 
scheduled visits will be minimally 33 and maximally 71. In groups 3 and 4 (cohort B), the 
number of scheduled visits will be minimally 34 and maximally 50. For naïve controls 
(groups 5-8) the number of visits will be minimally 9 and maximally 24. The maximum amount 
of blood collected will be 500mL for the immunization period and 500mL per malaria 
challenge infection period. Additionally, periodical physical examinations will be performed 
and subjects are asked to complete a diary during the periods listed in the protocol. 


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1. INTRODUCTION AND RATIONALE 
 
 
1.1 Introduction 
Malaria, a disease caused by the parasite Plasmodium, is one of the world’s major infectious 
diseases. With approximately 429,000 deaths in 2015, it is both a chief cause of morbidity and 
mortality as well as a significant contribution to ongoing poverty in endemic countries. Most 
malaria deaths occur in Africa and the victims are predominately young children who have yet 
to develop protective immunity to the disease [1]. 
Human malaria is caused by five species of Plasmodium protozoa: P. falciparum, P. vivax, P. ovale, 
P. malariae, and P. knowlesi. Parasites (sporozoite stage) are injected into the skin by an infected 
female Anopheles mosquito. From the skin capillaries they travel via the bloodstream to the liver, 
where they develop and multiply in liver cells before entering the bloodstream again (merozoite 
stage) and invading red blood cells for further multiplication. Clinical malaria is caused only by 
the cyclical proliferation of asexual stages in red blood cells, the sporozoite and liver stages are 
asymptomatic. 
The international community spends approximately $2.9 billion a year to combat malaria using 
insecticide-impregnated bednets (ITN), insecticides and antimalarial drugs [1]. Unfortunately 
widespread implementation of these interventions are hampered by  poor health care infrastructure 
in many endemic countries. Additionally the Plasmodium parasites are rapidly developing 
resistance to antimalarial medications, including the recent development of resistance to 
artemisinin-based derivatives [2, 3], the current frontline treatment. 
Ultimately, the key to malaria control and hopefully eradication would be an effective vaccine. 
Though a number of vaccine-candidates have entered the pipeline of pre-clinical and clinical 
development, they have yet to achieve the level of efficacy necessary for effective malaria 
prevention [4]. RTS,S (Mosquirix) is the first and only malaria vaccine that recently received a 
positive opinion from the European Medicines Agency. This sub-unit vaccine, however, confers 
only modest and rather short-lasting protection [5]. 
 
1.2 Rationale 
Chemo-Prophylaxis and Sporozoite immunization (CPS), exposure to non-attenuated P. 
falciparum sporozoite- infected mosquitoes under blood stage prophylaxis, has repeatedly shown 
to induce long lasting sterile homologous protection in malaria- naive Dutch volunteers in the 
CHMI model. In three studies CPS immunization by the bites of a total of only 30-45 infected 
mosquitoes under 
chloroquine  prophylaxis (3 
immunizations with 
15 mosquitoes per 
immunization, spaced 4 weeks apart) protects 19/20 volunteers (95%) [6-8]. 


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A re-challenge of 6 CPS immunized volunteers 2,5 years after the last immunization showed that 
4 out of 6 were still protected, demonstrating the lasting protective efficacy of this immunization 
regimen [ 9 ] . In a dose-finding study, immunization with a total of 30 and 15 infected mosquitoes 
protected 9 of 10 and 5 of 10 volunteers, respectively [ 7] . In contrast, bites from 1000 
mosquitoes infected with radiation-attenuated sporozoites are required for protection [10, 11]. 
Sanaria, Inc, a United States biotech company, has incorporated the CPS approach in their product 
development portfolio as PfSPZ-CVac. GMP-compliant, cryopreserved NF54 sporozoites have 
been manufactured, and the first trial has been conducted in Germany, showing 100% protective 
efficacy against homologous challenge confirming our previous data using infected mosquito 
bites [ 1 2 ] . PfSPZ-CVac, administered by direct intravenous inoculation, is currently tested in 
malaria endemic areas. 
Induction of protection against genetically distinct (heterologous) P. falciparum strains is a major 
stumble block and critical for understanding protective efficacy as well as for clinical vaccine 
development. Recently only 20% and 10% sterile protection was obtained after CPS- immunization 
with NF54 against heterologous NF135.C10 and NF166.C8 clones [ 1 3 ] . This reduced protection 
was reflected in the in vitro finding that plasma from CPS-NF54 volunteers less efficiently 
inhibited heterologous sporozoite invasion/maturation in hepatocyte cultures. 
NF135.C10 and NF166.C8 sporozoites are more infectious for liver cells than NF54, indicated 
by a 10-100 fold higher ‘first peak’, parasitemia post-infection, as proxy for liver parasite load. 
In vitro comparison shows a 2-3 times higher invasion rate of these clones compared to NF54 
and about twice the number of merozoites per schizont [ 1 4 ] . Therefore the observed reduced 
heterologous protection could at least partly be due to increased sporozoite infectivity and 
thus  greater stringency of the challenge. However, this  seems unlikely as liver cell invasion of 
these three clones in vitro is equipotently inhibited using serial dilutions of anti-CSP monoclonal 
antibodies [13]. 
For the future clinical development, it will be essential to improve the CPS-immunization 
regimen to obtain improved heterologous protective efficacy. Though the low number of 
heterologous protected volunteers did not allow for statistical analysis to find a correlate of 
protection, volunteers that were protected against a heterologous parasite challenge had 
relatively high antibody and/or cellular responses to P. falciparum, figure 1. This suggests that 
the decreased efficacy of CPS-induced immunity against heterologous parasites can be overcome 
by increasing the intensity of these immune responses[13]. 


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Figure 1: Analysis of in vitro intra-hepatic sporozoite development inhibition by CPS-induced antibodies, 
cellular responses and protection status in vivo. 
CPS-induced antibody-mediated inhibition of challenge strain sporozoites (NF135.C10 or NF166.C8) in vitro and 
cytotoxic and cytokine-producing T cell responses to NF54 infected RBCs are shown. The 10th and 90th percentile of 
each response in all (n=19) CPS-immunized volunteers are shown as grey box-and-whisker plots. The green 
triangle represents the one CPS-immunized volunteer sterilely protected against NF166.C8 challenge infection, 
while  the  orange  square  and  upside  down  triangle  represent  the  two  CPS-immunized  volunteers  sterilely 
protected against NF135.C10 challenge infection (Source: [13]). 
 
 
This could be achieved by increasing NF54 sporozoite dose, as shown by studies using 
PfSPZ immunization of radiation-attenuated sporozoites delivered by direct venous inoculation 
[15]. However, instead we propose to make use of the increased liver infectivity of NF135.C10. 
We postulate that using these more potent parasites will generate a far greater late liver stage 
load without the need for increasing the number of sporozoites administered. Presumably this 
will generate higher antibody titers, a broader repertoire of specific antibodies and stronger T 
cell responses that may be sufficient for the induction of heterologous protection. 
In this study we will use the same numbers of infectious mosquito bites 5 and 15, that were used 
in previously published NF54 CPS immunization studies [ 7 ,  8 ] . In support of possible clinical 
development of CPS-NF135, it will be important to generate clinical data on CPS- NF135 
potency as compared to CPS-NF54. Based on previous trial results with CPS-NF54, CPS-NF135 
will be compared to both 3 x 15 mosquitoes (>95% homologous protection [8]), and a dose of 3 
x 5 mosquitoes inducing about 50% homologous protection in the CPS-NF54 model [ 7 ] . In 
addition we will investigate whether CPS-NF135 can provide superior heterologous protection 
compared to CPS-NF54. As higher sporozoite dosages are required 


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for heterologous protection [ 1 6 ] , we initially intend to use the high dose immunization regimen, 
3 x 15 NF135 infected mosquitoes, in these groups. 
As the NF135 strain has reduced sensitivity to chloroquine, but similar sensitivity to mefloquine 
as NF54 in vitro [ 1 7 ] , immunization in cohort A will be performed under mefloquine prophylaxis. 
Mefloquine has similar activity to chloroquine and we have previously demonstrated that safety 
and efficacy of NF54 CPS immunization under mefloquine is similar to immunization under 
chloroquine [18]. 
 
 
1.3 Controlled Human Malaria Infection 
Controlled human malaria infection (CHMI) has proven to be a valuable tool to study the 
pathogenesis of clinical malaria and to evaluate the efficacy of drugs and vaccines. These 
challenge trials have become highly standardized  [19] and are an important step in the 
clinical development of malaria vaccines [20]. 
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 [21]. Infecting humans by the bite 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 [22], in 2007 [23] and in 2012 [24]. 
The Radboud university medical center (Radboudumc) has the experience and infrastructure to 
conduct CHMI trials. Over 350 subjects have been enrolled in CHMIs by the Radboudumc since 
2001, and the Central Committee for Research on Human Subjects  (CCMO) has reviewed 
these protocols since 2007. 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. We have also developed a very sensitive  method of 
parasite detection by real-time  quantitative PCR (qPCR) in whole blood that allows us to detect 
malaria infection in an early stage providing early treatment and which is able to detect small 
differences in parasite density [25]. 
In two previous CHMI trials (NL48301.091.14; NL48732.091.14) qPCR was performed daily during 
follow-up and volunteers were treated with anti-malarial drugs after a single positive qPCR. 
During these two studies, 39 volunteers developed patent parasitemia. Of these volunteers 26% 
developed no malaria symptoms, 38% had only grade 1 symptoms (no influence on daily 
activities), 28% had grade 2 symptoms (some influence on daily activities) and only 13% had 
grade 3 symptoms (requiring bedrest). All grade 3 symptoms lasted for less than one day. 


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1.4 Safety Aspects 
 
Adverse events in CHMIs: 
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. Real-Time Quantitative PCR (qPCR) allows for sensitive 
detection of low parasite densities, abrogating malaria infection in an early stage [ 2 6 ] . 
 
In the twenty-three CHMI studies conducted at Radboudumc prior to CPS135* (involving 376 
healthy volunteers), three cardiac events have occurred after infection with the NF54 strain with 
the confirmed or differential diagnosis of myocarditis [27, 28]. The occurrence of cardiac events 
in both natural uncomplicated malaria and CHMI has not been described elsewhere. However, 
the three cardiac events share no specific factor other than malaria infection. 
With regards to the malaria infection, these myocarditis cases do share a number of characteristics: 
i) they took place 1-5 days after start of antimalarial treatment but different anti-malarial drugs 
were used in all cases ii) there was no parasitemia detectable at the time of the event, and iii) 
next to the P. falciparum infection with NF54, other and known possibly triggering factors (e.g. 
preceding vaccinations, concomitant infections, cannabis use) were present during or preceding 
the event. More information on these SAEs is discussed in detail in section 13. 
 
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: 
 
Individuals are excluded from participation if they have first or second degree relatives 
who had cardiovascular events (including ischemic events and myocarditis) under the age 
of 50 
 
A positive urine toxicology test for amphetamines, cocaine and cannabis is an exclusion 
criterion 
 
Volunteers who took standard vaccinations within 12 weeks 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 
 
Increased control of hs troponin T as a marker of cardiac damage; treatment is initiated 
in consultation with the cardiologist 
 
Daily measurements of platelets; volunteers will be treated when platelet levels are 
<120x 10^9/L 


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Curative Malarone treatment after a single positive qPCR after CHMI to minimize period 
of parasitemia 
* Additionally, during the first immunization of cohort B of CPS135, a fourth cardiac event occurred 
after immunizing with the NF135 strain. Similar to the previous three SAEs, the event took place 
within 1-5 days after start of presumptive antimalarial treatment and there was no parasitemia 
detectable at time of the event. No other triggering factors were present during or preceding the 
event. This case is discussed in more detail in section 13.  
 
LFT elevations:  
Transient, asymptomatic liver function derangements have been reported in volunteers in 
previous CHMI studies, and are likely to be related to the challenge infection [29]. 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 parasitemia levels have mild (38%), moderate (10%) 
or severe (12%) asymptomatic increases of liver transaminases (ALT/AST). However, 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 LFT values at study end. 
A clear explanation for the transient elevated transaminases in CHMI studies is not obvious. A 
combination of the malaria infection, antimalarial use, paracetamol use, and individual 
susceptibility may have triggered the observed abnormalities. 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 
utilizing Plasmodium falciparum challenge. After review, the CCMO in the Netherlands has 
approved of further CHMI studies. 
 
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: 
• 
Regular safety monitoring to assess asymptomatic liver function test abnormalities: prior 
to CHMI, on day 6 post-CHMI, at antimalarial treatment, two days post antimalarial 
treatment and 35 days post CHMI 
• 
Avoiding additional triggers that may cause elevations in liver enzymes including 
alcohol from baseline up to 1 week post treatment 
• 
A maximum dose of 3000 mg per day of paracetamol/acetaminophen 
• 
Initiation of curative anti-malarial treatment if ALT/AST levels >5x ULN 
 
 
 


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Rationale for modifications to the first immunization phase of cohort B  
During the immunization phase of cohort A, it was observed that NF135.C10 was unexpectedly 
insufficiently susceptible to MQ prophylaxis (see also section 7.3 Summary of findings from clinical 
studies) and that all subjects required rescue treatment with A/P, resulting in heterogeneous 
exposure to liver- and blood-stage parasites which may impact on the evaluation of induced 
immunity. Since no suitable alternative registered chemoprophylactic drug exists in the 
Netherlands (NF135.C10 is resistant to CQ and A/P has a pre-erythrocytic effect), an alternative is 
to treat all subjects presumptively with a therapeutic drug on day 7 after each immunization. Such 
a presumptive therapeutic regime results in parasite killing at almost exactly the same point in its 
life cycle (i.e., as soon as parasites first emerge from the liver into the blood) and thus exposure of 
the immune system to the same range of parasite stages, as would be expected to occur under an 
effective blood-stage prophylactic (=classic CPS) regime. 
A/P itself nevertheless is unsuitable for presumptive treatment in this context, as residual 
atovaquone levels in cohort A subjects who received rescue treatment with A/P were so high that 
they plausibly interfered with liver-stage development during subsequent immunizations (see also 
section 7.3) and consequently with the development of protective immunity. A similar effect would 
be expected following presumptive treatment, even with a longer (6-week) interval between 
immunizations. A preferential drug for presumptive treatment is therefore A/L, which has no pre-
erythrocytic effect. Considerations relating to the (cardiac) safety profile of A/L are discussed in 
section 13.2 (Cardiac events following Controlled Human Malaria Infections). 
Since simultaneous intake of MQ and A/L is contra-indicated due to potential QT-prolongation as a 
result of drug-drug interactions between MQ and lumefantrine and since presumptive A/L 
treatment inhibits blood-stage multiplication, MQ prophylaxis should and can be omitted from the 
immunization regime altogether. 
 
 
Rationale for modifications to the second immunization of cohort B  
After the first immunization of cohort B, one volunteer developed a cardiac SAE. Although the 
underlying pathological mechanism remains unknown, it cannot be excluded that this may be 
directly or indirectly related to either the height of (the first wave of) parasitemia, or the level of 
systemic inflammation. Moreover, two subjects experienced recrudescent parasitemia after 
presumptive A/L treatment. Potential explanations could include de novo development of resistance 
in vivo, in particular against the lumefantrine component, and unfavorable A/L pharmacokinetics in 
these volunteers. The second immunized of cohort B will therefore take place with  5 instead of 15 
NF135 infected mosquitoes to reduce inflammation by lowering the parasite load, whilst still 
boosting immunity compared to the 1st immunization. Priming with a high dose (15 mosquitoes) and 
boosting with a lower dose (5 mosquitoes) is a common approach in vaccinology in order to improve 
the quality of memory responses by mobilizing the highest quality memory cells induced by priming. 


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This is supported by the NF54 CQ CPS model: after a second immunization a greater proportion of 
subjects developed sterile immunity compared to the first  immunization (59/67 vs 42/67, p<0.001). 
The relatively lower dose given during the second immunization is offset to an extent by the higher 
intrinsic infectivity of NF135.  
Secondly, volunteers are treated presumptively with atovaquone/proguanil (A/P) starting on day 5 
after immunization 2. A/P treatment is preferred over A/L because of  i) risk of treatment failure after 
A/L treatment, and ii) the higher inflammatory responses after A/L treatment (potentially due to more 
rapid-acting mechanism) as is also illustrated by the higher number and severity of adverse events 
following the first immunization of cohort B compared to the 15 mosquito-bite dose arm in cohort A, 
despite similar parasite densities. Treatment will be initiated on day 5 to potentially block or reduce 
release of merozoites from the liver, thus further reducing inflammation, whilst still allowing late liver-
stage development and the consequent development of pre-erythrocytic immunity. Even if merozoite 
release is not fully blocked, the presence of circulating drug concentrations upon merozoite release 
will help to ensure that intra-erythrocytic development is suppressed from the start.  
Furthermore, addition follow up visits including qPCR on days 13, 16, 20 and 23 (all +/- 1 day) 
will be added after immunization 2, to actively screen for recrudescence (as an additional 
precaution, since although recrudescence of NF135 has never been observed following A/P 
treatment, the effect of starting treatment at day 5, instead of day 7, has never previously been 
formally documented). 
Finally, the second immunization will be followed by a challenge at ~17-19 weeks after the second 
(last) immunization (allowing sufficient interval to clear residual circulating atovaquone), with 5 NF54 
or NF135 infected mosquitoes. A third immunization improves protection relatively little in the NF54 
CPS model and would be logistically infeasible due to the long half-life of atovaquone/proguanil 
following presumptive treatment.  
 
 
2. OBJECTIVES 
 
 
Primary  Objective:  To  determine  the  safety  and  tolerability  of  NF135.C10  sporozoite 
immunization under chemoprophylaxis 
 
Secondary Objectives: 
 
To determine 
the dose-dependent 
protective 
efficacy 
of 
NF135.C10 CPS- 
immunization against homologous controlled human malaria infection 
 
To  determine  the  protective  efficacy  of  NF135.C10  CPS-immunization  against 
heterologous NF54 controlled human malaria infection 


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 
To assess the longevity of homologous protective immunity after NF135.C10 CPS-
immunization, if primary homologous protection against challenge in cohort A is >50% 
 
Exploratory Objectives: 
 
To analyse P. falciparum specific T cell responses in NF135.C10 CPS-immunized 
volunteers 
 
To delineate the antibody repertoire directed against P. falciparum in NF135.C10 
CPS-immunized volunteers 
 
To evaluate changes in phenotype and function of innate and semi-innate immune 
cells following NF135.C10 immunization 
 
To explore the (innate) immunology of early malaria infection, with specific attention to 
γδ-T cells, monocytes, antigen presenting cells and natural killer cells 
 
To analyse changes in epigenetic and transcriptome profiles of (innate) immune cells 
after NF135.C10 CPS immunization and/or after malaria infection 
 
3. STUDY DESIGN 
In an open label, randomized, controlled clinical trial, a maximum of 52 volunteers in two cohorts 
will be allocated to receive immunizations with  NF135.C10 infected Anopheles mosquitoes. In 
cohort A,  volunteers will receive 3 immunizations with 15 (n=10) or 5 (n=10) NF135.C10 infected 
mosquitoes under mefloquine prophylaxis, spaced 4 weeks apart, or no immunizations (controls, 
n=3). A thick smear will be performed on days 7-9 after each immunization and symptomatic 
volunteers with a positive thick smear will be treated with a curative regimen of atovaquone/proguanil 
(A/P). Nineteen weeks after the last immunization, all volunteers plus 3 naïve controls will be 
challenged by the bites of 5 NF135.C10 infected mosquitoes.  
In cohort B, 20 volunteers will receive 1 immunization with 15 NF135.C10 infected mosquitoes and 
a second immunization with 5 NF135.C10 infected mosquitoes. Volunteers will not take mefloquine 
prophylaxis. Instead all volunteers will be treated presumptively on day 7 after the first  immunization 
with a curative regimen of A/L, and on day 5  after the second immunization with a curative regimen 
of A/P, regardless of parasitaemia or symptoms (see considerations in protocol section 1.4). Six 
controls will receive no immunization. Seventeen - nineteen weeks after the second immunization, 
volunteers plus naïve controls will be challenged either by the bites of 5 NF135.C10 or 5 NF54 
infected mosquitoes (table 1A and 1B). After challenge infection, volunteers will be followed up 
on an out-patient basis once daily for qPCR and safety lab measurements from day 6 until 
day 21 post challenge. All volunteers will be treated with a curative regimen of A/P, either at 
the time of detection of blood stage parasitemia (for treatment criteria see paragraph 8.3.3), or 
28 days after challenge infection. All volunteers will be checked for parasites by qPCR after 
treatment. 


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If ≥50% of subjects in the groups that received NF135.C10 CPS- immunization in cohort A (groups 1 
and 2) are protected against primary challenge, protected subjects along with 3 newly recruited 
controls (group 8) will undergo a (second) CHMI with NF135.C10 approximately one year after the 
last immunization. The criteria for proceeding to this second challenge are described in more detail 
in section 8.3.5. For cohort B (groups 3 and 4) there will not be a second CHMI. During the 
immunization and challenge phases, blood will also be drawn for exploratory immunology and 
parasitology objectives. These samples will be analyzed by Radboudumc and its collaborators. The 
total study period will last approximately fourteen months for cohort A (including the second 
challenge) and nine months for cohort B. An overview of the schedule is given in the flowchart in 
section 8.3.14. If one of the volunteers is not fit to participate in the study at inclusion or withdraws 
at any time before the first immunization, another volunteer who passed screening will be included 
as replacement. To allow for subjects intolerant of mefloquine to leave the study prior to the first 
CPS immunization without impacting the study sample size, in cohort A, three reserve volunteers 
will also begin mefloquine prophylaxis. If they are not used to replace a withdrawn volunteer, these 
reserve volunteers will stop mefloquine prophylaxis after the fourth dose. 
 
 
 
 
 
CHALLENGE 1 
 
CHALLENGE 2 
 
Cohort Group Number of 
volunteers 
Immunization 
Challenge 
Continuation 
criterium 
 
Volunteers 
Challenge 
 
 
Strain 
Mosquito
#
Strain 
Strain 
Immunization 
groups 
A 
1 
10 
NF135.C10 
3 x 15 
NF135.C10 
if ≥50% protected
protected 
NF135.C10 
A 
2 
10 
NF135.C10 
3 x 5 
NF135.C10 
if ≥50% protected
protected 
NF135.C10 
B 
3 
10 
NF135.C10 
1 x 15 
1 x 5 
NF54 
N/A 
N/A 
N/A 
B 
4 
10 
NF135.C10 
1 x 15 
1 x 5 
NF135.C10 
N/A 
N/A 
N/A 
Control Groups 
A 
5 
3 
- 
- 
NF135.C10 
 
 
- 
B 
6 
3 
- 
- 
NF54 
 
 
- 
B 
7 
3 
- 
- 
NF135.C10 
 
 
- 
A 
8 
3 
- 
- 
- 
 
 
NF135.C10 
Table 1: Study design. 
 
 


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4. STUDY POPULATION 
 
 
4.1 Population (base) 
The study population will be comprised of healthy, malaria naive male and female subjects 
between age 18 and 35 years old. A total of maximally 52 subjects will be enrolled to participate 
in the study. The investigator will ensure that all subjects being considered for the study meet 
the following eligibility criteria. Subject selection is to be established by checking through all 
inclusion/exclusion criteria at screening and baseline (inclusion visit). A relevant record of the 
eligibility criteria will be stored with the source documentation at the study site. 
 
4.2 Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria: 
1. 
Subject is aged ≥ 18 and ≤ 35 years and in good health. 
2. 
Subject has adequate understanding of the procedures of the study and agrees to abide strictly 
thereby. 
3. 
Subject is able to communicate well with the investigator and is available to attend all study 
visits. 
4. 
The subject will remain within the Netherlands during the challenge period, not travel to a 
malaria-endemic area during the study period, and is  reachable (24/7) by mobile telephone 
throughout the entire study period. 
5. 
Subject agrees to inform his/her general practitioner about participation in the study and to 
sign a request to release by the General Practitioner (GP), and medical specialist when 
necessary, any relevant medical information concerning possible contra- indications for 
participation in the study. 
6. 
The subject agrees to refrain from blood donation throughout the study period and for a 
defined period thereafter according to current guidelines. 
7. 
For female subjects: subject agrees to use adequate contraception and not to breastfeed for 
the duration of study. 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. 


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8. 
Subject agrees to refrain from intensive physical exercise (disproportionate to the subjects 
usual daily activity or exercise routine) during the malaria challenge period. 
9. 
Subject agrees to avoid additional triggers that may cause elevations in liver enzymes 
including alcohol from baseline up to 1 week post treatment. 
10. Subject has signed informed consent. 
 
 
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 cardiovascular events (including 
ischemia and myocarditis) 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), or active Hepatitis 
B Virus (HBV) or Hepatitis C Virus (HCV). 
1.6 
Chronic use of i) immunosuppressive drugs, ii) antibiotics or antimalarials, 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 
History of malignancy of any organ system (other than localized basal cell carcinoma 
of the skin), treated or untreated, within the past 5 years. 
1.8 
Any history severe psychiatric disease diagnosed by a psychiatrist. 
1.9 
History of drug or alcohol abuse interfering with normal social function in the period 


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of one year prior to study onset, positive urine toxicology test for cocaine or 
amphetamines at screening or inclusion, or positive urine toxicology test for cannabis 
at inclusion. 
2. 
For female subjects: positive urine pregnancy test at screening or at inclusion. 
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 Mefloquine, 
Malarone or artemether-lumefantrine, 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 90 days 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 of the Radboudumc 
or the department of Internal Medicine. 
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. 
 
 
4.4 Sample size calculation 
This is a pilot study, with the primary objective to assess the safety and tolerability of CPS 
immunization with NF135.C10 in healthy volunteers. Based on previous experience with CPS 
immunization and convention, the study will recruit 10 volunteers per intervention group. 
Cohort A – homologous protection 
The ( short term) homologous protective efficacy, protection after controlled human malaria 
infection with NF135.C10 will be compared between study groups 1 a n d  2 , and three 
unimmunized controls (Cohort A).  In five previous challenge studies using the NF135.C10 
parasite, unimmunized volunteers became parasite positive by qPCR (if once daily sampling 
was performed) at a mean of 7.1 days with a standard deviation of 0.21 days. Therefore, a delay 
in time to parasitemia (prepatent period) of 0.5 days can be detected (α=0.05, 1-ß=0.80) in the 
proposed study. 
 
Cohort B – homologous and heterologous protection  
To assess ( short term) homologous protective efficacy, protection after controlled human malaria 
infection with NF135.C10 will be compared between group 4 and three unimmunized controls 
(Cohort B). As described for homologous protection in Cohort A above, assuming a mean of 
7.1 days with a standard deviation of 0.21 days, a delay in time to parasitemia (prepatent period) 


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of 0.5 days can be detected (α=0.05, 1-ß=0.80) in a group of 10 volunteers. In case of withdrawal 
of subjects during the study prior to challenge, a delay of 0.5 days (rounded) can still be detected in 
a group of at least 7 volunteers.  
 
To asses ( short term) heterologous protective efficacy, protection after controlled human malaria 
infection with NF54 will be compared between study group 3 and three unimmunized controls. In 
nineteen previous challenge studies using  the NF54 parasite, unimmunized volunteers became 
parasite positive by PCR at a mean of 7.8 days with a standard deviation of 1.7 days. Therefore, 
a delay in time to parasitemia (prepatent period) of 3.5 days can be detected in a group of 10 
volunteers, and of 3.8 days in a group of at least 7 volunteers  (α=0.05, 1-ß=0.80).  
 
These group sizes will provide sufficient power to detect differences in prepatency of 3.8 days (or 
greater). A difference in prepatent period less than ~3.8 days would mean that the heterologous 
protective efficacy of CPS-NF135 is lower than that of CPS-NF54 (NL48732.091.14; [ 1 3 ] ). 
 
Cohort B – total protection 
The total protective efficacy of immunization against controlled human malaria infection with either 
NF135.C10 (homologous) and NF54 (heterologous) may be assessed and compared to 6 
unimmunized volunteers. Assuming on average 50% protection in immunized volunteers and 
theoretical 1% protection in control volunteers, a significant difference in proportion of protected 
volunteers between immunized and controls can be shown with a minimum of 14 volunteers 
assuming α=0.05, 1-ß=0.80. 
 
 
 
 


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5. TREATMENT OF SUBJECTS 
 
 
5.1 Investigational product/treatment 
There is no investigational product in this study. 
 
 
5.2 Mefloquine prophylaxis 
 
5.2.1 
Dosage 
Volunteers in cohort A will receive weekly mefloquine according to standard  prophylactic regimen 
(Source: LCR Richtlijn, March 2018). Volunteers will receive 250mg weekly starting three weeks 
prior to immunization, where the fourth tablet is taken the day before the first immunization, until 
four weeks after the last immunization. (Note: in cohort B, no mefloquine prophylaxis will be given.) 
 
5.2.2 
Contraindications 
 
Severely decreased liver function 
 
Severe haemolysis 
 
Hypersensitivity for quinine or related substances 
 
A history   of depression, suicidal 
tendencies, 
anxiety   disorders, psychoses, 
schizophrenia or other psychiatric disorders 
 
A history of convulsions 
 
5.2.3 
Possible side effects 
The most commonly reported frequencies of side-effects of mefloquine are registered for 
curative and prophylactic dosages combined, however the frequencies of adverse events are lower 
for prophylactic dosages than curative regimens. (Source: farmacotherapeutischkompas.nl; 
SmPC Mefloquine) 
Very frequent side effects (>10%): 
 
Sleeplessness 
 
Abnormal dreams 
Frequent side effects (1-10%): 
 
Anxiety 
 
Depression 
 
Dizziness and vertigo 
 
Headache 
 
Vision abnormalities 
 
Nausea, vomiting, abdominal pain and diarrhea 


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 
Pruritus 
Recently, a Cochrane Database Systematic Review analysed the frequency of side-effects 
compared 
with other 
malaria 
prophylactic 
drugs, 
including 
atovaquone-proguanil 
and 
chloroquine, drugs more commonly used in CHMI studies [30]. This meta-analysis concluded that 
there was a very low incidence of severe adverse events  with mefloquine, and no difference 
in severe adverse events when comparing mefloquine and chloroquine as prophylaxis. When 
comparing mefloquine with atovaquone-proguanil, the best estimates of absolute effect sizes for 
mefloquine versus atovaquone-proguanil were 6% versus 2% for discontinuation of the drug, 
13% versus 3% for insomnia, 14% versus 7% for abnormal dreams, 6% versus 1% for anxiety, 
and 6% versus 1% for depressed mood. 
 
 
5.3 Atovaquone/proguanil treatment 
 
5.3.1 
Dosage 
The treatment will consist of the drug A/P. Dosing will be as follows: once daily 4 tablets of 250 
atovaquone/100mg proguanil for three consecutive days according to Dutch SWAB guidelines. 
 
5.3.2 
Contraindications 
 
Severely reduced kidney function (creatinine clearance <30 ml/min) 
 
Concomittent use of metoclopramide, tetracycline, rifampicine, rifabutine, efavirenz, 
nevirapine or protease inhibitors 
 
5.3.3 
Possible side effects 
Very frequent side effects (> 10%): 
 
Headache 
 
Nausea, vomiting, diarrhoea and abdominal pain 
Frequent (1-10%): 
 
Allergic reactions, rash, pruritus 
 
Fever, anorexia 
 
Coughing 
 
Abnormal dreams, sleeplessness 
 
Depression 
 
Dizziness 
 
Hyponatremia, elevated liver enzymes 
 
 
5.4 Artemether/lumefantrine treatment 
 


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5.4.1 
Dosage 
The treatment will consist of the drug A/L and will be taken on day 7 after the first immunization in 
cohort B only. Dosing will be as follows:  4 tablets of 20mg artemether and 120mg lumefantrine 
taken on T= 0, 8, 24, 36, 48 and 60 hours, according to Dutch SWAB guidelines. 
 
5.4.2 
Contraindications 
 
(History of) Congestive heart failure with reduced left ventricular ejection fraction. 
 
Symptomatic arythmia’s, clinically relevant bradycardia 
 
Disturbances in electrolyte balance 
 
Prolonged QT-interval 
 
Concomitant use of other drugs that induce QT-prolongation (including potentially 
mefloquine), CYP2D6 metabolized drugs with small therapeutic window or CYP3A4 
inducers.  
 
5.4.3 
Possible side effects 
Very frequent side effects (> 10%): 
 
Headache, dizziness  
 
Palpitations 
 
Nausea, vomiting, abdominal pain 
 
Fatigue, asthenia, loss of appetite  
 
Myalgia, arthralgia 
Frequent (1-10%): 
 
Paresthesia, clonus, walking disorder 
 
Insomnia 
 
Cough 
 
Diarrhea 
 
Skin rash, itching 
 
Prolonged QT interval (ECG) 
 
5.5 Symptomatic treatment 
Volunteers will be provided tripelennamine crème for the local treatment of mosquito 
bites.Volunteers in cohort A and during the 1st immunization of cohort B will be provided 
paracetamol for complaints secondary to CHMI and onset of parasitemia (fever, muscle aches, 


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headache, etc.). The maximum dose of paracetamol is 3 grams a day. Given their anti-
inflammatory effect, volunteers in cohort B from immunization 2 onwards will be provided with 
NSAIDs. Should volunteers experience severe nausea, domperidon tablets or suppositories can 
be prescribed by the study physician. The maximum dose of domperidon is 10mg 3 times daily 
orally or 30mg 2 times daily rectally. Tripelennamine crème, paracetamol or any other 
symptomatic treatment will be supplied to the volunteers by the study physician. 
 
6. INVESTIGATIONAL PRODUCT 
Not applicable, there is no investigational product used in this study. 
 
7. NON-INVESTIGATIONAL PRODUCT 
 
 
7.1 Name and description of non-investigational product(s) 
P. falciparum isolate NF135.C10 infected mosquitoes for the purpose of CPS-immunization and 
controlled human malaria infection. 
P. falciparum isolate NF54 infected mosquitoes for the purpose of controlled human malaria 
infection. 
 
7.2 Summary of findings from non-clinical studies 
The P. falciparum NF135 strain (clone C10) was isolated from a patient visiting Cambodia 
[ 1 7 ] . In in vitro studies, this parasite has been shown susceptible to multiple antimalarials in 
vitro, 
including 
A/P 
(IC50 
atovaquone 
1.277nM, 
IC50 
proguanil 
2.8*106nM), 
arthemeter/lumefantrine (IC50 artemisinin 24.36nM, IC50 lumefantrine 188.8nM) and mefloquine 
(IC50 115.4nM), but it has reduced sensitivity to chloroquine. The P. falciparum NF54 strain was 
isolated from a patient living in the Schiphol-area in the Netherlands. This parasite is susceptible 
to A/P, arthemeter/lumefantrine, mefloquine and chloroquine in vitro.  
 
7.3 Summary of findings from clinical studies 
Safety findings 
CHMIs are well accepted as a powerful tool for the evaluation of parasite development in 
humans. Radboudumc has the experience and infrastructure to conduct CHMIs. Radboudumc 
also developed  a very sensitive method of parasite  detection  by real-time qPCR that will allow 
us to detect malaria infection in an early stage providing early treatment and to detect small 
differences in parasite density [26]. There is a large clinical experience with infecting humans by 
the  bite of P. falciparum- infected mosquitoes. Since 1986 more than 3500 volunteers have had 
CHMI by the bites of mosquitoes fed on cultures of P. falciparum gametocytes to produce P. 


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falciparum sporozoites [21]. This has proved to be a reproducible, predictable and safe 
method of inducing P. falciparum malaria. The results of such studies were summarized in 1997 
[22], in 2007 [23] and in 2012 [24]. 
 
When treatment after CHMI was initiated based on thick blood smear, mild-moderate solicited 
adverse events were generally experienced  by all study subjects post-infection, included 
particularly headache (95%), general malaise/fatigue (65%) and fever (90%). Gastro-intestinal 
complaints, including nausea, diarrhea 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. When treatment was initiated based a single 
positive qPCR, 75% of participants experienced any solicited adverse event, but severe adverse 
events occurred in only 10% of participants. 
Since 2007 the Radboudumc has performed 
CPS immunization with the NF54 parasite on 
healthy volunteers. A total of 62 subjects have 
received CPS immunization by bites from 3x 
12-15  NF54-infected  mosquitoes  (EHMI8,  
NL14967.091.06;  EHMI9,  NL33904.091.10; 
ZonMw1, 
NL34273.091.10; 
BMGF1,   
NL48301.091.14; BMGF2b, NL48732.091.14).  
Combined  data  of  30 subjects from three 
studies showed that all volunteers experienced mild (possibly or probably)  related  adverse  
events  (AEs) 
during   CPS  immunization,   57%  (17/30) 
experienced 
moderate, 
and 
23% 
(7/30) 
experienced severe AEs (table 2). Severe 
AEs were malaise, headache, fatigue, fever 
and myalgia. The majority of AEs occurred after the first immunization (97% of subjects), while 
33% (10/30) of subjects experienced AEs after the second and third immunization (see table 2). A 
single volunteer in the BMGF1 study (NL4830.091.14) experienced a serious adverse event 
after CPS immunization, diagnosed as an asymptomatic myocarditis. This event is discussed in 
detail in section 1.5 and section 13. 
 
This is the first study of CPS immunization with the NF135.C10 strain. However, the NF135.C10 
strain has been used extensively in CHMI, with 29 volunteers receiving a challenge with 5 infected 
mosquitoes 
(TIP1, 
NL303050.058.09; 
TIP3, 
NL41004.078.12; 
BMGF2a, NL48704.000.14;   
Immunization 
 
1st 
 
2nd  
3rd  
Any  
Grade 
No. %
No. % No. % No. % 
1 
27
90 10
33 9 
30 30
100
2 
16 
53 3 
10 2 
7 
17 
57 
3 
6 
20 0 
0 
1 
3 
7 
23 
Any
29
97 10
33 10 
33 30
100
Table  2: 
Subjects  with  AEs  during  CPS
immunization. 


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BMGF2b,   NL48732.091.14).   In   26 volunteers that developed parasitemia after challenge with  5  
NF135.C10  (n=13)  or  NF54  (n=13)  infected mosquitoes,  there  was  no  difference  in  reported  
adverse  events  or  elevation  of  liver enzymes, table 3 and figure 2.  
 
 
 
Challenge 
NF135.C10 
NF54 
Grade 
No. 
% 
No. % 
1 
2 
3 
11 
5 
1 
85 
38 
8 
11 
4 
0 
85 
31 
0 
Any 
11 
85 
11 85 
Table 3: Subjects with AEs after challenge 
 
 
Figure 2: Graphs show the number of ALT or AST elevations in volunteers undergoing a malaria challenge infection 
with NF54 (n=13) or NF135.C10 (n=13), and treated at first positive qPCR (above 100 parasites per milliliter). Data 
from the BMGF1 (NL48301.091.14) and BMGF2b (NL48732.091.14). Dotted lines show the upper limit normal for male 
(blue) and female (pink) volunteers.  
 


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Efficacy findings 
The P. falciparum isolate NF54 has been tested in 18 studies involving 251 volunteers. In these 
studies generally all unimmunized volunteers developed patent parasitemia with NF54. The P. 
falciparum isolate NF135.C10 has been tested in 5 studies involving 58 volunteers. In a single 
CHMI study (TIP1, NL30350.058.09, [17]), only 3 out of 5 volunteers developed patent 
parasitemia with NF135.C10. However, it is unlikely that this is an inherent characteristic of this 
strain. In the same study, 1 out of 5 volunteers in the NF54 group also failed to develop 
parasitemia, in contrast to 17 other studies when NF54 generated 100% parasitemia. Instead, 
technical problems with the parasite culture because of interrupted gas supply likely affected the 
culture medium quality. These problems have since been solved and have not recurred in the 
4 subsequent studies using NF135.C10, where all volunteers challenged with 5 mosquitoes 
developed patent parasitemia. 
In 2012 CHMI with NF135.C10, NF166.C8 and NF54 was compared in malaria naive volunteers 
(TIP3, NL41004.078.12). Five out of five subjects in each group developed patent parasitemia 
(assessed by microscopy, not by qPCR) following bites of 5 NF54, NF135.C10 or NF166.C8 
infected mosquitoes, respectively; median [range] pre-patent periods for these strains were 10.5 
[9.0-10.5], 7 [6.5-8.5] and 7 days [6.5-8.5] [14]. There was no statistically significant difference 
in frequency, duration or gradation of adverse events between the three groups. 
 
Summary of findings from CPS135 – Cohort A 
During immunization in cohort A of the CPS trial, all n=20 subjects taking mefloquine prophylaxis 
required rescue treatment with A/P due to positive thick smear in combination with symptoms  
following their first immunization and n=6 and n=9 required A/P rescue treatment following their 2nd 
and 3rd immunizations, respectively. MQ levels at time point of parasite release from the liver were 
found to be in the expected range for subjects taking MQ prophylaxis. Circulating breakthrough 
parasites that were re-cultured from subjects showed similar in vitro MQ IC50s (med 56 [range 11 – 
194nM]) as compared to the parent strain used for inoculation. These results are indicative of in vivo 
resistance of NF135.C10 to MQ and of as yet unexplained discrepancy between in vitro IC50s and 
in vivo resistance. Moreover, residual atovaquone levels following rescue treatment and measured 
immediately prior to 2nd and 3rd immunization, were also considerably higher (med 54.5 ng/mL [range 
<20-225ng/mL]) than IC50s atovaquone for NF135.C10 liver- and blood-stage parasites in vitro.  
 
Summary of finding CPS135 – Cohort B immunization 1 
After the first immunization in Cohort B, two volunteers developed recrudescent parasitemia on day 
19 and day 21 after treatment on day 7 with A/L. Both subjects were successfully treated with rescue 


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treatment (standard curative 3‐day regimen of A/P per protocol), with good clinical and 
parasitological response (qPCR negative by 23 days post‐inoculation onwards). A/L treatment failure 
has been reported more frequently in large European Males [31]. Drug susceptibility of the 
recrudescent isolate in comparison to the parent strain, as well as circulating A/L levels in these two 
plus the remaining Cohort B volunteers is pending. 
 
 
7.4 Summary of known and potential risks and benefits 
Cohort A 
The risks associated with both mosquito feeding and exposure to P. falciparum are discussed 
below. In this part of the trial, the risk of developing clinical malaria is low, as volunteers will be 
taking standard mefloquine prophylaxis and the P. falciparum strain used, NF135.C10, is sensitive 
to mefloquine. In cohort A, thick smears (TS) will be made  on day 7, 8 and 9 after each 
immunization (the days with highest parasitemia after immunization)  in order to monitor that 
volunteers do not develop parasitemia. Thick smears made on day 7 and 8 will be read only of 
subjects are symptomatic, all thick smears on day 9 will be read regardless of symptoms. A thick 
smear will always be read in any volunteer with a temperature above 38.0° Celsius after any 
immunization. Symptomatic volunteers with a positive thick smear during the immunization phase 
of cohort A will be treated with A/P. In asymptomatic parasitaemic volunteers A/P treatment will be 
initiated in case of parasite increment, or if judged clinically prudent by the investigator.  
Mefloquine is a marketed medication registered for use as a malaria prophylactic agent for 
Plasmodium strains sensitive to it. Common side effects include sleeplessness and vivid 
dreaming (>10%), psychiatric symptoms such as fear and depression (1-10%), headache (1- 10%) 
and gastro-intestinal symptoms (1-10%). In line with clinical practice, all volunteers will be screened 
for contra-indications in their medical and family history to mefloquine (section 5.2.2). Volunteers 
with contra-indications will not be given mefloquine. Furthermore, ten days after start of mefloquine, 
the presence of any side-effects possible related to mefloquine (section 5.2.3) will be discussed 
in a follow-up visit. Volunteers will be asked to complete the Depression Anxiety Stress Scales 
( DASS) questionnaire and 20 items on positive symptoms from the Community Assessment of 
Psychic Experience (CAPE) questionnaire to detect changes in emotional states of depression, 
anxiety, stress and psychotic symptoms. These questionnaires will be taken during subsequent 
follow-up visits only on indication. 
 
Cohort B 
In cohort B, all volunteers will in presumptively receive a curative treatment of A/L on day 7 after 
the first immunization, and A/P on day 5 after the second immunization. Daily qPCR will be 


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performed from day 6-10 following the first immunization, and from day 6-10, day 13, 16, 20 and 
23 (+/- 1 day) after the second immunization In addition, a thick smear will be performed in any 
volunteer with a temperature above 38.0° Celsius or if judged clinically prudent by the investigator. 
Volunteers with recrudescent parasitaemia during the immunization phase of cohort B will be 
treated with A/P, or an appropriate alternative. 
 
In general:  
There is no benefit expected for subjects participating in this study. The risk to subjects after 
exposure to P. falciparum infected mosquitoes in this trial will be minimized by adherence to the 
inclusion/exclusion criteria and close clinical monitoring, which ensures that subjects with malaria 
are detected and treated early. The risks associated with CPS immunizations are those related 
to exposure to P. falciparum infected mosquitoes and those associated with mefloquine 
prophylaxis and/or treatment with A/P and A/L. 
 
The risks of a CHMI for malaria-naïve subjects include the discomfort sustained by mosquito bites, 
the discomfort associated with periodic blood draws and the risk of acquiring clinical  P.  
falciparum malaria. 
The exposure to infected P. falciparum 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. 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. 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, 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 early detection of P. falciparum  parasitemia after 
challenge infection. Universal precautions will be maintained for the protection of the volunteer and 
the study personnel during venapuncture. Throughout this study, the amount of blood collected will 
be maximally 500 mL during the immunization period and 500 mL during each challenge period. This 
amount is similar to widely accepted guidelines used by the Sanquin blood bank. 
Intensive follow-up with qPCR 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. 
Severe malaria has never been described in a CHMI. Mild malaria symptoms include headache,  


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myalgia,  fever, chills, sweats, nausea, vomiting, and diarrhea. 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 treatment quickly 
attenuates the illness so that the infection does not place the subject  at  undue  risk.  A/P, which  
is  a  marketed  first-line treatment  for  malaria  will  be  used to treat subjects after the second 
immunization in cohort B, after the challenge and in case of treatment failure of mefloquine or A/L,  
and  is  generally  well  tolerated.  Common  adverse reactions  (≥5%)  in  adults  associated  with  
A/P   treatment  include  abdominal  pain, nausea, vomiting, headache, diarrhoea, asthenia, 
anorexia, and dizziness. Those volunteers intolerant of A/P will be given alternative treatment 
according to the national malaria treatment guidelines. 
 
Additional information about the potential risks associated with CHMI and a summary of 
relevant reported Serious Adverse Events is given in section 1.5 and section 13. 
 
 
7.5 Description and justification of route of administration and dosage 
 
Study subjects will be exposed to malaria through (cutaneous) bites of P. falciparum-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. The dosages selected for this study will allow us 
to directly compare the relative homologous and heterologous protective efficacy of NF135.C10 CPS 
immunization within the context of this trial, as well as to compare this ratio to that induced by NF54 
CPS immunization in previous studies. 
 
Cohort A 
Study subjects will be immunized 3 times with the bites of 5 or 15 NF135.C10 P. falciparum 
infected mosquitoes at intervals of four weeks. Experience from CHMI studies by our group has 
shown immunization with three monthly bites from  15 NF54 P. falciparum infected mosquitoes  
to be a very effective dosage to ensure the volunteers are fully protected against homologous 
challenge [ 6 - 8 ] . While immunization with three monthly bites from 5 NF135.C10-infected 
mosquitoes leads to 50% homologous protection [7]. In contrast, immunization with three monthly 
bites from 15 NF54 infected mosquitoes leads to only 10-20% protection against NF135.C10 and 
NF166.C8 challenge [13].  
 
 
 


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Cohort B 
Subjects in cohort B will be exposed to 15 NF135.C10 infected mosquitoes after the first 
immunization and 5 NF135.C10 infected mosquitoes after the second immunization, an approach 
commonly adopted in vaccinology (protocol section 1.5).  
 
Challenge 
The gold-standard dosage for CHMI studies at the Radboudumc is five infective bites per subject, 
based on extensive experience with the NF54, NF135.C10 and NF166.C8 strains. 
 
 
7.6 Dosages, dosage modifications and method of administration 
. No dose modification will take place. 
 
7.7 Preparation and labelling of Non Investigational Medicinal Product 
The culture of P. falciparum 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 isolates used in this study were originally derived from patient material (see also 
section 6.3) and were 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 are negative for malaria and 
Hepatitis B surface Antigen (HBsAg), and seronegative for HIV, HCV, HEV, HumanT-lymphotropic  
Virus (HTLV) 1+2 and syphilis. The cultures are checked for bacterial, fungal and Mycoplasma 
contamination. 
To produce infectious gametocytes for this study, 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 A. 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.  Criteria that must be met for use of mosquitoes for CHMI are provided 
in the Product Information. Mosquitoes are then transferred from midi‐  cages to small CHMI‐


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cages. Each step in this process is performed by an experienced technician, and checked and 
recorded on standardized forms by another technician. 
 
 
 
7.8 Drug accountability 
The date, time of collection and person collecting the mosquitoes is filled in on a standard table. 
This form will be signed by the responsible employee. 
 
 
8. METHODS 
 
8.1 Study parameters/endpoints 
 
8.1.1 Main study endpoint 
 Frequency and magnitude of adverse events after NF135.C10 CPS immunization 
 
8.1.2 Secondary study endpoints 
 Time to blood stage parasitemia detectable by qPCR after malaria challenge 
infection 
 Sterile protection after controlled human malaria infection 
 
 
8.1.3 Exploratory study endpoints 
 The phenotype and cytokine profile of P. falciparum specific T cell responses 
induced by NF135.C10 CPS immunization 
 The antigen specificity of T cell responses induced by NF135.C10 CPS immunization 
 The immunoglobulin repertoire and functional activity of P. falciparum specific sera 
and monoclonal antibodies induced by NF135.C10 CPS immunization and derived 
from plasmablasts 
 The phenotype and/or function of innate and semi-innate immune responses to 
NF135.C10 CPS immunization and/or CHMI, including γδT cells, invariant T cells, 
antigen presenting cells, NK cells and granulocytes 
 Epigenetic profiles of innate immune cell subsets, with emphasis on both activation 
(H3K4me3, H3K4me1, H3K27Ac) and repression (H3K9me3, H3K27me) markers 
 RNA transcriptome profiling through whole mRNA-sequencing, PCR and/or 
microarray 
 
8.2 Randomisation and treatment allocation 
This is an open-label study. For logistical reasons the trial will need to be performed in two time-


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separated  cohorts  (cohort  A:  group  1,  2,   5 and 8;  cohort  B:  group  3,  4,  6 and  7). 
Volunteers will be recruited separately for groups 1 and 2, groups 3 and 4, group 5, groups 6,  
7, and group 8. Between these groups (i.e. group 1 and group 2), included volunteers will be 
randomly assigned. Volunteers will be allocated to a group according to a randomization list 
derived from the Microsoft Excel command ASELECTUSSEN (0,1000). Every volunteer number 
will be linked to a random number between 0 and 1000. For example, the ten lowest numbers 
will be assigned group 1, and the ten highest numbers will be assigned to group 2. If two 
identical numbers are produced, the whole procedure is repeated. One of the investigators at 
the Radboudumc will be responsible for performing the randomization and for assigning volunteers 
according to the randomization list. A second employee, not involved in the assignment of 
volunteers, will check to see if randomization is done correctly. The master randomization list is 
kept in a fireproof clinical trial cabinet at the Radboudumc Malaria Unit. 
 
8.2.1 
Baseline screening 
Subjects who sign informed consent will undergo complete screening including a patient 
history, physical examination, vital signs and laboratory evaluations  (see sections  8.3.6- 8.3.9). 
If physical examination, vital signs or laboratory values are out of the normal range a repeat 
measurement may be obtained. 
Following a screening period of up to 120 days, subjects who meet the eligibility criteria 
(Section 4.2 and 4.3) will present to the study site 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 be included into the study. Subjects  that wish to 
participate in cohort A will be screened for contra-indications  to mefloquine. Volunteers with 
contra-indications to mefloquine (section 5.2.2) will not be allowed to take mefloquine 
and will therefore not be included in an immunization group in cohort A. For each subject in 
cohort A, study start will be defined as the day of the first dose of mefloquine prophylaxis. In cohort 
B, mefloquine prophylaxis will not be a part of the immunization procedure, therefore study start 
(inclusion) will be defined as the day before the first immunization. 
Mefloquine prophylaxis has been associated with mild side effects including vivid dreams, 
anxiety, headache and gastro-intestinal symptoms in 1-10% of individuals (see also section 5). 
In most cases (around 80%) these side effects present during the first two to three weeks of use. 
For this reason, ten days after start of mefloquine, the presence of any side- effects 
possible related to mefloquine (section 5.2.3) will be discussed in a follow- up visit and 
volunteers will be asked to complete the DASS questionnaire and 20 items on positive 
symptoms from the CAPE questionnaire to detect changes in emotional states of 
depression, anxiety, stress and psychotic symptoms. To allow for subjects intolerant of 


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mefloquine to leave the study prior to the first CPS immunization without impacting the study 
sample size, three reserve volunteers will also begin  mefloquine prophylaxis. If they are not 
used to replace a withdrawn volunteer, these volunteers will stop mefloquine prophylaxis after 
the fourth dose. They will be compensated with 100 euros and offered a chance to participate in 
the challenge infection in study groups. 
 
8.2.2 
CPS immunization with mosquito bite 
On the first day of the study, subjects in cohort A will be seen by the investigators to initiate 
mefloquine prophylaxis. Volunteers that will undergo CPS immunization in cohort A(groups 1 
and 2) will receive 250mg mefloquine once a week according to a standard prophylactic regime. 
They will receive four doses prior to the first CPS immunization  (on  day  22). Volunteers  will 
continue mefloquine prophylaxis throughout CPS immunization and for four weeks after the last 
immunization, for a total duration of 16 weeks. In cohort B mefloquine prophylaxis will not be a part 
of immunization procedures, for subjects in this cohort the day before the first immunization will mark 
the first day of the study. 
For subjects in cohort A, sufficient prophylaxis is a requirement for receipt of the next immunization 
and will be evaluated prior to each immunization. Compliance with the prophylaxis is monitored 
at all study visits. Additionally, study subjects will receive a weekly reminder via either e-mail 
or text message. Twenty-two days after initiation of mefloquine prophylaxis, subjects of cohort A 
will receive CPS immunization with bites from 5 or 15 NF135.C10 P. falciparum infected A. 
stephensi mosquitoes, depending on allocation (see section 3). This procedure will be repeated 
three times at four week intervals. CPS immunizations of cohort B will consist of bites from 15 
(immunisation 1) and 5 (immunisation 2) NF135.C10 P. falciparum infected A. stephensi 
mosquitoes. 
On each of the  days of immunization, the volunteers will be exposed to the bites of 5 or 15 
NF135.C10 P. falciparum infected A. stephensi mosquitoes. Mosquitoes will be prepared by 
technicians of the Radboudumc malaria unit and placed in identical boxes, numbered to 
correspond with the participant’s study code. Treatment allocation will not be blinded. The 
infections will be performed by placing a box containing mosquitoes on the forearm of the 
volunteer. (For more information concerning the production of mosquitoes, please refer to 
section ‘Mosquito preparation’ under 6.3.5). Directly after the feed, the mosquitoes will be 
dissected by a technician of the mosquito unit. This will be done to assure that the mosquito has 
fed and the presence of sporozoites in the salivary glands of the mosquitoes. Exposure will be 
repeated until the exact number of infected mosquito bites has been reached. In a previous 
study, the prophylactic dose of mefloquine was sufficient to overcome bites of 15 NF54 infected 
mosquitoes [18], and the in vitro sensitivity of NF135.C10 to mefloquine is similar to NF54 (see 


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Product Information). 
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. 
All volunteers in cohort A and B will be seen by the trial clinicians on days 6 – 10 after each 
immunization for safety assessments (see flow chart, section 8.3.14). Additionally, volunteers of 
cohort B will also be seen on 13, 16, 20 and 23 (all +/- 1 day) for follow-up visits including qPCR 
after the second immunization.  
For cohort A, to ensure no break-through infections occur under mefloquine prophylaxis, on day 7 
and 8 after each immunization blood will be taken for thick smears and only immediately read in 
subjects with symptoms that could be related to parasitemia. Additionally, blood will be taken for 
thick smears and read on day 9 after each immunization. A thick smear will also be performed 
in any volunteer with a temperature  above  38.0°  Celsius  after  immunization.  If  a symptomatic 
subject  develops a positive thick blood smear at any time point after CPS immunization, the subject 
is immediately treated with A/P, during three days (see also section 8.3.3). Should an 
asymptomatic volunteer be blood smear positive on day 9, the day 7 blood smear will be read to 
detect if parasitemia is increasing. In asymptomatic parasitaemic volunteers A/P treatment will be 
initiated in case of parasite increment, which  is defined as two consecutive positive thick smears 
of which the second thick smear has a higher parasitemia than the first, or if judged clinically 
prudent by the investigator. If there is no increase, the volunteer will continue with regular follow-
up. Further blood smears may be performed in volunteers with a positive thick smear without 
treatment. If a treatment with A/P is required during the immunization phase, the subject will 
continue with the next immunization(s) and a blood sample will be taken to retrospectively 
determine atovaquone levels at the time of the subsequent immunization.  
Subjects that will receive immunizations in cohort B (groups 3 and 4) will not receive mefloquine 
prophylaxis. Instead,  subjects in cohort B will be treated presumptively with a curative regimen of 
A/L on day 7 after the first immunization, and A/P on day 5 after the second immunization. To 
ensure treatment is adequately initiated, the first dose of A/L or A/P will be taken under direct 
observation by one of the study clinicians during the study visit on day 7 or day 5, respectively. 
Treatment effectiveness will be monitored by performing daily qPCR on day 6 through day 10 after 
immunization 1 and additionally day 13, 16, 20 and 23 after the second immunization. Should 
volunteers develop a temperature above 38.0° Celsius before day 7, or when judged clinically 
prudent by the investigator, a thick smear will be performed. If the thick smear is positive, the 
volunteer will start with a curative treatment of A/P, or an appropriate alternative.  
During the entire study period all subjects of cohort A and B will be instructed to call the trial 
physicians at any time if they experience symptoms. The trial physician can at all times decide 


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to initiate  any additional diagnostics (including laboratory safety evaluations and/or diagnostics 
for malaria parasites) considered clinically indicated.  Blood will be drawn for retrospective qPCR 
on day 6 through 10 after each immunization in each cohort. Subjects in cohort A will continue 
mefloquine prophylaxis until four weeks after the last CPS- immunization. 
 
8.2.3 
Controlled Human Malaria Infection after drug washout (challenge)  
Seventeen - nineteen weeks after the last CPS immunization, all subjects from group 1, 2, 3 and 
4, plus nine newly recruited subjects in group 5, 6 and 7, will undergo malaria challenge infection. 
On the challenge day, all subjects will be exposed to the bites of five NF135.C10 or NF54 strain 
P. falciparum infected mosquitoes. Mosquito feeding will be allowed for 10 minutes. Volunteers 
may 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 as described above in section 7. 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 8.3.9 for details). From 
the sixth day until the twenty-first day post-CHMI, assessments of parasite densities using 
qPCR will be performed once daily. Subjects will also visit the clinical site for a follow-up visit on 
day 1, 2 and 3 after treatment (TD+1, 2 & 3). Between day 21 and day 28 post challenge, volunteers 
will be seen only if they have symptoms. 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 8.3.14. 
qPCR assessment of parasite densities will be performed directly in volunteer samples. As 
soon as a qPCR 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 contact the volunteer who will return to the clinic to receive A/P 
treatment. 
For  unexpected  laboratory  abnormalities  during  the  study,  the  laboratory  test  will  be 
repeated. Any elevation of troponin-T outside normal limits will be discussed with the clinical 
supervisor. If the elevation is judged to be significant, a cardiologist is consulted and a full 
cardiac work up is done according to standard medical practice. If there is any ambiguity 
regarding the decision to include or exclude a volunteer, the study physician or 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 


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invited to the next visit. 
All subjects who are exposed to P. falciparum will be treated with A/P either upon positive 
qPCR for malaria or on day 28 after the malaria challenge infection. Additionally, treatment 
can be initiated in any of the following situations: 
1. By decision of study doctor or the safety monitor 
2. On request of the volunteer 
3. In consultation with the cardiologist 
4. When thrombocytes <120 x 109/L 
5. When ALT/AST levels >5x ULN 
 
8.2.4 
Treatment with atovaquone/proguanil after challenge 
After the challenge, all volunteers from cohort A and B will be treated with A/P based on the 
predetermined criteria mentioned above. 
The treatment will consist of the drug A/P. Dosing will be as follows: once daily 4 tablets of 250 
atovaquone/100mg proguanil for three consecutive days according to 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-effects. During treatment, 
complaints of malaria infection will be treated symptomatically. In addition to specific treatment 
with A/P, symptomatic treatment will be administered at the discretion of the study physician. 
After the challenge, during and one day after A/P treatment, qPCR is performed directly in collected 
blood samples. If qPCR remains positive on day three after A/P treatment (usually the result of 
parasite debris remaining in the bloodstream) a thick blood smear may be performed to confirm 
the absence of intact malaria parasites and the volunteer will receive additional follow-up to confirm 
decrease in parasitemia 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. 
8.2.5 
Re-challenge infection 
One year after the last CPS immunization, immunized volunteers of cohort A may undergo a 
second challenge infection to assess the duration of homologous protection. In each of the 
immunized groups of cohort A (1 and 2), a re-challenge will only be performed if at least 50% of 
the volunteers of that group were protected against the primary challenge. To assess the 
duration of homologous protection, volunteers in group 1 and 2 that were protected against 
primary challenge will receive a second challenge with NF135.C10. Previous studies using 
radiation-attenuated sporozoites have not shown a boosting effect of a homologous challenge 
infection [15, 16]. Three new malaria naïve infection controls will be recruited for group 8 (n=3), 
and will be challenged with NF135.C10. There will not be a second challenge infection for cohort B 
(groups 3 and 4). 


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8.2.6 
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 be measured. Body mass index (BMI) will be calculated using the following formula: BMI 
= Body weight (kg) / [Height (m)]2 and converted to an integer. 
8.2.7 
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 baseline, the investigator may obtain two additional readings, so 
that a total of up to three consecutive assessments are made, with the subject seated quietly 
for approximately five minutes preceding each repeat assessment. At least the last reading must 
be within the normal range in order for the subject to qualify. 
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. 
 
8.2.8 
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 in the supine 
position. Calibration should be performed per the local site/requirements. Each ECG tracing should 
be labeled 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. 
 
8.2.9 
Blood sampling and safety laboratory evaluations 
During the study, blood samples will be drawn for screening, safety and research purposes. The 


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blood sampling schedule in the flowchart (section 8.3.14) indicates the  timing  and volume of blood 
drawn. 
Regular hematology safety laboratory evaluations will include: hemoglobin, hematocrit, red blood 
cell count, white blood cell count with differential (e.g. neutrophils, basophils, eosinophils, 
monocytes, lymphocytes) and platelet count. 
Biochemistry safety laboratory evaluations includes, alkaline phosphatase, total bilirubin, creatinine, 
γGT, LDH, potassium, AST, ALT, sodium, highly sensitive troponin T and urea. Glucose, 
triglycerides and cholesterol will be measured only at screening. 
At the screening visit plasma samples will be tested for HIV, hepatitis B surface antigen and 
hepatitis C. Additionally plasma will be tested for reactivity to malaria parasites by ELISA. 
A midstream urine sample (approx. 30 ml) will be obtained at screening and the day before the 
study start. In this sample the presence of cannabis, amphetamines and cocaine will be assessed. 
Additionally, for female participants a commercially available hCG urine test will be used to test 
for pregnancy. HCG urine tests will also be obtained prior to each immunization and prior to CHMI 
1 and 2. 
If a laboratory assessment is outside the reference range for the laboratory, a decision 
regarding whether the result is of clinical significance shall be made by the 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. 
 
8.2.10 
Analysis of parasite densities after challenge infection 
qPCR for assessment of parasite densities will be performed directly in volunteer samples, as 
discussed previously (see section 8.3.3). 
qPCR is performed according to a standard procedure described previously [26, 32]. In short, qPCR 
will be performed on 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 during immunization, and at any time it is deemed necessary by the 
clinical investigator (see section 8.3.3), 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. 
 
8.2.11 
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 


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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. 
 
8.2.12 
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 day 35 after the 
malaria challenge infection. The subject diary will be reviewed at each study visit and used as 
a basis for discussion of possible adverse events or medication use. If the occurrence of an 
adverse event or use of medication is confirmed by the study physician, it is recorded in the subject’s 
study file (see also: section 9.4.1). 
Subjects will also be asked to measure their temperature orally every morning and record this 
temperature in their study diaries from:day 6 until day 10 after each immunization (cohort A), 
from day 6 until day 23 after each immunization (cohort B), and from the day of the malaria 
challenge infection until the third day of A/P treatment, (challenge cohort A and cohort B).  
At the end of the study, the diary will be collected and kept as source data with the subject’s study 
file. 
 
8.2.13 
Exploratory immunological assessments 
Blood will be drawn for exploratory immunological assessments at specific time points (see 
section 8.3.14). From these samples, assays can be performed in whole blood and/or peripheral 
blood mononuclear cell (PBMC) populations and plasma can be isolated. Samples can be analysed 
for phenotype or functionality directly, or PBMCs and plasma can be frozen for possible later 
analyses by the Radboudumc or its collaborating partners as described in the exploratory 
objectives (section 2). If necessary to assess (antigen specific) T cell responses, the HLA-type of 
volunteers may be determined from PBMC samples. 
Phenotype, cytokine profile and function of immune cells can be measured by intracellular flow 
cytometry or cytokine specific ELISA or multiplex assays. T cell, B cell antibody antigen specificity 
can be determined by protein or peptide library screening or by B- or T cell receptor 
sequencing. RNA transcription levels can be analyzed by specific gene RNA-PCR or total mRNA 
sequencing. Epigenetic profiles can be investigated using Chromatin Immunoprecipitation (ChIP) 
followed by sequencing or specific gene PCR. 


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
 
 
8.2.14 
Flow chart study design 
 
COHORT A 
  
Screenin
g visit 
Inclusion/ 
controle 
mefloquin
e 
Mefloquin
e Check 
First immunization 
Second immunization 
Third immunization 
 
Visit Number 
V1 
V2 
V3 
V4 
V5 
V6-10 
V11 
V12 
V13-17 
V18 
V19 
V20-24 
V25 
Reference timepoints 
I-1 
I1 
I1+6 – I1+10 
I2-1 
I2 
I2+6-I2+10 
I3-1 
I3 
I3+6- I3+10 
Number of visits 
1x 
1x 
1x 
1x 
1x 
1x / day 
1x 
1x 
1x / day 
1x 
1x 
1x / day 
1x 
Eligibility criteria + Demographic data1 
X 
 
Physical examination 
X 
X 
X7 
X7 
X7 
X7 
X7 
X7 
 
ECG 
X 
 
Temperature 
X 
X 
X 
X 
X 
X 
X 
X 
Immunization with NF135.C10-infected 
mosquitoes 
X 
X 
X 
Mefloquine prophylaxis 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
A/L treatment 
X14 
X14 
X14 
Haematology tests2 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Biochemistry tests
4 
X (5 ml) 
X (3 ml) 
HsTropT + LDH only 
X 
X 
X 
X 
X 
Serology
5 
X (5 ml) 
X (3 ml) 
Parasitology
6 (2.0ml) 
X 
X 
X 
X 
X 
X 
Exploratory immunology 
Citrate plasma
10 
 
I1-1 
I1+6; I1+7; 
I1+10 
X 
I2+6; I2+10 
X 
I3+7 
X 
PBMC for cellular studies 
 
I1-1: 
80mL
I1+6: 24 ml; 
I1+7: 24mL; 
I2-1: 
40mL
I2+6: 24 ml; 
I2+10: 24mL 
I3-1: 
32ml
I3+7: 24mL 
I3+28: 
40ml 
PBMC for plasmablasts 
 
I1-1 
I1+6 I1+10 
I2-1 
I2+6; I2+10 
I3-1 
 
Whole-blood transcriptomics (2.5 ml) 
 
X 
D28, D32 
X 
D56, D60 
X 
 
Pregnancy and Urine Toxicology 
X 
X 
X 
X 
X 


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
Supply diary 
 
X 
 
Reviewing diaries 
 
X 
X 
X 
X 
X 
X 
X 
A/P or A/L blood levels
12 
 
X12 
X12 
 
Mefloquine blood levels
13 
 
X13 
X13 
X13 
 
 
Blood Draw Estimate 
12 ml 
8 ml 
88.5 ml 
112.5mL 
54mL 
83mL 
46 ml 
94mL 
 
 
Cumulative Blood Drawn 
12 ml 
20 ml 
108.5m
L 
221mL 
275m
L 
358 ml 
404ml 
498 ml 
498ml 
 
1 Visit may be performed 1-3 days earlier 
2 Hemoglobin, hematocrit, platelets, white blood cell count + differentiation 
4 Creatinin, urea, sodium, potassium, bilirubin, AF, γGT, ASAT, ALAT, additional at screening: cholesterol + glucose 
5 Screening: HIV, HBV, HCV, P. falciparum (5.0ml). Inclusion: HIV, HBV, HCV (3.0ml) 
6 Immunization: retrospective qPCR day 6-10; Cohort A: thick smear day 7-9  
7 On indication. 
8 After one positive qPCR 
10 Taken from the same tubes as PBMCs 
11 Only if treated after positive qPCR 
12 1 EDTA tube (2ml) and 1 serum tube (3,5mL) 
13 Mefloquine samples may be collected from volunteers requiring rescue treatment after immunization (cohort A): 1 EDTA tube (2ml) and 1 serum tube (3,5mL) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
COHORT B  
  
Screening 
visit 
Inclusion 
First immunization 
Second immunization 
 
Visit Number 
V1 
V2 
V4 
V5 
V6-10 
V11 
V12 
V13-17 
Reference timepoints 
 
 
I-1 
I1 
I1+6 – I1+10 
I2-1 
I2 
I2+ (6-10); I2+13, 
I2+16, I2+20; I2+2313 
Number of visits 
1x 
1x 
1x 
1x 
1x / day 
1x 
1x 
1x / day 
Eligibility criteria + Demographic 
data1 
X 
 
Physical examination 
X 
X 
X7 
X7 
X7 
X7 
ECG 
X 
 
Temperature 
X 
X 
X 
X 
X 
X 
Immunization with NF135.C10-
infected mosquitoes
 
 
X 
X 
Presumptive treatment (A/L or 
A/P) 
X14 
X14 
Haematology tests3 
X 
X 
X 
X 
X 
X 
Biochemistry tests4 
X (5 ml) 
X (3 ml) 
HsTropT + LDH only 
X 
X 
X 
X 
Serology5 
X (5 ml) 
X (3 ml) 
Parasitology6 (2.0ml) 
X 
X 
X 
X 
Exploratory immunology 
Citrate plasma10 
I1-1 
I1+6; I1+7; I1+10 
X 
I2+6; I2+10 
PBMC for cellular studies 
I1-1: 80mL 
I1+6: 24 ml; I1+7: 24mL; 
I1+10: 24 ml
I2-1: 40mL 
I2+6: 24 ml; I2+10: 
24mL 
PBMC for plasmablasts 
I1-1 
I1+6 I1+10 
I2-1 
I2+6; I2+10 
Whole-blood transcriptomics 
(2.5 ml) 
X 
D28, D32 
X 
D56, D60 
Pregnancy and 
X 
X 
X 
X 
Supply diary 
 
X 
Reviewing diaries 
 
 
X 
X 
X 
X 
A/P or A/L blood levels12 
 
 
X12 
Blood Draw Estimate 
12 ml 
8 ml 
88.5 ml 
74,5mL 
48,5mL 
91mL 


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
Cumulative Blood Drawn 
12 ml 
20 ml 
108.5mL 
215,5mL 
264mL 
355 ml 
 
1 Visit may be performed 1-3 days earlier 
2 Hemoglobin, hematocrit, platelets, white blood cell count + differentiation 
4 Creatinin, urea, sodium, potassium, bilirubin, AF, γGT, ASAT, ALAT, additional at screening: cholesterol + glucose 
5 Screening: HIV, HBV, HCV, P. falciparum (5.0ml). Inclusion: HIV, HBV, HCV (3.0ml) 
6 Immunization: prospective qPCR day 6-10, day 13, day 16, day 20 and day 23 (+/- 1 day) 
7 On indication. 
8 After one positive qPCR 
9 Group 5-8 only 
10 Taken from the same tubes as PBMCs 
11 Only if treated after positive qPCR 
12 1 EDTA tube (2ml) and 1 serum tube (3,5mL) 
13 on day 13, 16, 20 and 23 after second immunization follow-up for prospective qPCR and adverse events 
14 Subjects of Cohort B will be treated presumptively on day 7 after the first immunization with A/L or on day 5 after the second immunization with A/P.  
 
 
 


NL63594.091.17 | CPS135 
Safety and efficacy of NF135 CPS immunization
 
 
 
CHMI 
End of study visit 
 
Number of visits 
fixed
Number of visits depending 
on parasitemia
Number of visits fixed 
Visit Number 
V261
V27
V28-43
V44
V45
V46
V47
V481 
Trial timeline (D=day) 
C-1 
C 
 
C+6 – C+21 
 
TD8 
 
TD8+1 
 
TD8+2 
 
TD8+3 
End of Study 
Number of visits 
1x
1x
1x / day
1x
1x
1x
1x
1x 
 
 
Eligibility criteria + informed consent 
 
Demographic data, medical history 
 
Physical examination and vital signs 
X
X7 
X8 
X7
X7
X7
X7 
ECG 
 
Temperature 
X
X
X
X
X
X
X 
Immunization with NF135.C10-infected mosquitoes 
 
Challenge with 5 infected mosquitoes 
X
 
Mefloquine prophylaxis2
 
A/P treatment 
X
X
X
 
Haematology tests3 (2.0ml)
X
 
X
X
X
X
X
X 
Biochemistry tests4
3 ml
C+6: 3 ml
3 ml
3 ml
3 ml 
HsTropT + LDH only (2.0ml)
X
X
X
X
 
Serology5 
X9 
 
Parasitology6 (2.0ml)
X
X
X
X6 
X 
Exploratory immunology
 
Citrate plasma9
X
X
X
X 
PBMC for cellular studies 
C-1: 80 
 
 
 
TD:80 ml11 
 
TD+3:80 ml11 
80 ml11 
PBMC for plasmablasts
 
Whole-blood transcriptomics (2.5 ml) 
X
X
X
X 
Pregnancy and toxicology urine test 
X
 
Supply diary 
X9 
 
 
 
 
 
 
 
Reviewing diaries 
X
X
X
X
X
X
 
Mefloquine blood levels12 
X 
 
 
 
 
 
 
 
Blood Draw Estimate 
95 ml 
 
C+6: 7 ml 
rest: 6 ml
7 ml 
88.5 ml 
7 ml 
88.5 ml 
88.5 ml 
Cumulative Blood Drawn 
95 ml 
 
192 ml 
199 ml 
287,5 ml 
294,5 ml 
383 ml 
472.5 ml 
1 Visit may be performed 1-3 days earlier 
2 Groups 1-4 only 
3 Hemoglobin, hematocrit, platelets, white blood cell count + differentiation 
4 Creatinin, urea, sodium, potassium, bilirubin, AF, γGT, ASAT, ALAT, additional at screening: cholesterol + glucose 
5 Screening: HIV, HBV, HCV, P. falciparum (5.0ml). Inclusion: HIV, HBV, HCV (3.0ml) 
6 Immunization: retrospective qPCR day 6-10; Cohort A: thick smear day 7-9; Cohort B: thick smear day 10 (end of treatment); Challenge: prospective qPCR daily
  


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7 On indication. 
8 After one positive qPCR 
9 Group 5-8 only 
10 Taken from the same tubes as PBMCs 
11 Only if treated after positive qPCR 
12 Mefloquine will be collected prior to the challenge in all immunized volunteers: 1 EDTA tube (2ml) and 1 serum tube (3,5mL) 
 
 


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CHMI2
 
 
 
 
 
 
Number of visits fixed 
Number of visits 
depending on 
parasitemia
 
Number of visits fixed 
Visit Number 
V49
1
V50
V51-66
V67
V68 
V69
V70
V71
1
Trial timeline (D=day) 
C-1 or 427 
C or 
C+6 – C+21 or 
TD
8 
or
TD
8+1 
or 
TD
8+2 
or
TD
8+3 
or
 
463 
Number of visits 
1x
1x
1x / day
1x
1x 
1x
1x
1x
Eligibility criteria + informed consent 
Demographic data, medical history 
Physical examination and vital signs 
X 
 
X 
X8 
X7 
X7 
X7 
X7 
ECG 
X 
 
Temperature 
X
X
X
X 
X
X
X
Immunization with NF135.C10-
infected mosquitoes
 
 
 
 
 
 
 
 
Challenge with 5 infected mosquitoes 
X
Mefloquine prophylaxis
2
 
A/P treatment 
X
X 
X
Haematology tests
3 (2.0ml)
X
X
X
X 
X
X
X
Biochemistry tests
4
3 ml
C+6: 3 ml
3 ml
3 ml
3 ml
HsTropT + LDH only (2.0ml)
X
X
X 
X
Serology
5
 
X9
Parasitology
6 (2.0ml)
X
X
X
X6
X
Exploratory immunology
Citrate plasma
9
 
X
X 
X
X
PBMC for cellular studies
80 ml
80 ml
11
80 ml
11
80 ml
PBMC for plasmablasts
Whole-blood transcriptomics (2.5 ml) 
X
X 
X
X
Pregnancy and toxicology urine test 
X
Supply diary 
X9
Reviewing diaries
X
X
X
X 
X
X
Blood Draw Estimate 
89.5 ml 
 
C+6: 7 ml 
rest: 6 ml
7 ml 
88.5 ml 
7 ml 
88.5 ml 
88.5 ml 
Cumulative Blood Drawn 
89.5 ml
186.5 ml
193.5 ml
282 ml
289 ml
377.5 ml
467 ml
1 Visit may be performed 1-3 days earlier 
2 Groups 1-2 only 
3 Hemoglobin, hematocrit, platelets, white blood cell count + differentiation 
4 Creatinin, urea, sodium, potassium, bilirubin, AF, γGT, ASAT, ALAT, additional at screening: cholesterol + glucose 
5 Screening: HIV, HBV, HCV, P. falciparum (5.0ml). Inclusion: HIV, HBV, HCV (3.0ml) 
6 Immunization: retrospective qPCR day 6-10; Cohort A: thick smear day 7-9; Cohort B: thick smear day 10 (end of treatment); Challenge: prospective qPCR daily
  
7 On indication. 
8 After one positive qPCR 
9 Group 5-8 only 
10 Taken from the same tubes as PBMCs 
11 Only if treated after positive qPCR 


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8.3 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. 
Safety criteria for Malarone treatment are listen in section 8.3.3. Volunteers can also 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  (i.e. 
immunosuppressive agents) or against P. falciparum 
 Pregnancy 
 Withdrawal of informed consent by volunteer 
 Completely lost to follow-up 
 If, on balance, the investigator or safety monitor believes that continuation would be 
detrimental to the subject’s well-being 
 Any other protocol deviation that results in a significant risk to the subject’s safety 
 
 
If a subject withdrawal occurs for any reason, the investigator must make every effort to 
determine the primary reason for  a subject’s withdrawal from the study and record  this 
information in the study file. 
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 offer curative 
therapy for a malaria episode, and to follow the subject for cardiac manifestations of disease. 
If the subject has already experienced a malaria episode and received curative therapy, 
continued inclusion in the study will be considered. 
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 (i.e. 
documented phone calls, letters 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, 
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8.4 Replacement of individual subjects after withdrawal 
If a subject withdraws before the first immunization (groups 1-4) or before the CHMI (group 5-8), 
he/she will be replaced with an alternate volunteer who passed screening, if possible. 
 
8.5 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 
complete adequate prophylaxis (in the case of subjects withdrawing during CPS immunization) or 
take a curative regimen of A/P or alternative effective anti-malarial treatment should A/P be 
contra-indicated (in the case of subjects withdrawing after CHMI, or subjects withdrawing during 
immunization, for who continued prophylaxis is contraindicated). 
 
8.6 Premature termination of the study 
The study may be discontinued by the sponsor: 
 On advice of the safety monitor 
 On advice of the Safety Monitoring Committee (SMC) 
 On advice of the clinical investigator 
 On advice of the CCMO 
 
 
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 9. 


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9. SAFETY REPORTING 
 
9.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the study if there 
is sufficient ground that continuation of the study will jeopardise subject health or safety. The 
sponsor will notify the accredited METC without undue delay of a temporary halt including the 
reason for such an action. The study will be suspended pending a further positive decision 
by the accredited METC. The investigator will take care that all subjects are kept informed. 
PATH will also be notified of any decisions to prematurely suspend or terminate the study. 
 
 
9.2 AEs, SAEs and SUSARs 
 
9.2.1 Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject during the study, 
whether or not considered related to trial procedure or the experimental intervention. All adverse 
events reported spontaneously by the subject or observed by the investigator or his or her staff 
will be recorded. 
Abnormal laboratory findings (e.g. clinical chemistry 
or haematology) 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 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. 
 
9.2.2 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that 
 
results in death; 
 
is life threatening (at the time of the event); 
 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
 
results in persistent or significant disability or incapacity; 
 
is a congenital anomaly or birth defect; or 


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 
any other important medical event that did not result in any of the outcomes listed above due 
to medical or surgical intervention but could have been, based upon appropriate judgement 
by the investigator. 
An elective hospital admission will not be considered as a serious adverse event. 
 
 
The investigator will report all SAEs to the sponsor without undue delay after obtaining 
knowledge of the events. All SAEs will be reported through the web portal ToetsingOnline to the 
CCMO, within 7 days for SAEs that result in death or are life threatening followed by a period of 
maximum of 8 days to complete the initial preliminary report. All other SAEs will be reported within 
a period of maximum 15 days after the sponsor has first knowledge of the serious adverse 
events. 
 
 
9.3 Follow-up of adverse events 
 
9.3.1 
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 
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If an AE changes in intensity during the specified reporting period, a new description of the AE 
will be added. 
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. 
 
The following signs and symptoms will be solicited at any visit: 
Fever (by examination), headache, fatigue, malaise, chills, myalgia, dizziness, sweats, nausea, 
vomiting, abdominal pain, diarrhoea, chest pain. 
 
The following signs and symptoms local to the site of mosquito bites will be solicited after 
every immunization and CHMI: 
Tenderness, induration (assessed by palpation), erythema, swelling (assessed by direct or 
lateral visualization), and pain, pruritis (subjective symptoms). 
 
9.3.2 
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 
challenge  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. 
 
 
 


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9.3.3 
Follow-up of adverse events 
 
All AEs will be followed until they have abated, or until a stable situation has been reached. 
Depending on the event, follow up may require additional tests or medical procedures as 
indicated, and/or referral to the subject’s general physician or a medical specialist. 
AEs and SAEs will be reported until end of study within the Netherlands, defined as day 35 after 
the malaria challenge infection (see flowchart section 8.3.14). 
 
 
 
 
9.4 Local Safety Monitor (LSM) and Safety Monitoring Committee (SMC) 
 
9.4.1 
Local safety monitor 
For this study, a local safety monitor will be appointed, who is based in the Radboudumc and will 
be involved in the review of 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 and the investigators. 
 
9.4.2 
Safety Monitoring Committee (SMC) 
An independent Safety Monitoring Committee (SMC) will be appointed, including 3 individuals. 
Their main responsibility will be assessing of any serious adverse events and 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 for review by the SMC at the end of 
the immunization period and after each CHMI. 
The advice(s) of the SMC will only be sent to the sponsor of the study. Should the sponsor decide 
not to fully implement the advice of the SMC, the sponsor will send the advice to the CCMO, 
including a note to substantiate why (part of) the advice of the SMC will not be followed. 
 
9.4.3 
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 after each immunization and on day 21 and 35 after 
each CHMI. 
These reports will be prepared by a clinical investigator and sent to the safety monitor and all clinical 
investigators involved. 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 after the last immunization 
and after each CHMI. Responsibilities of the SMC are described in the SMC Charter. The 


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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 60ng/l will be reported to the safety monitor 
within 24 hours. Any abnormal safety laboratory values that lead to immediate anti- malarial 
treatment will be reported to the safety monitor within 24 hours. 
 
 
 
 
9.4.4 
Safety stopping rules 
The study may be placed on safety hold for the following reasons: 
 
On advice of the safety monitor 
 
On advice of the investigators 
 
On advice of the SMC 
 
On advice of the CCMO 
 
One or more participants experience an SAE that is determined to be related to a study 
intervention 
 
Two or more grade 3 adverse events after immunization, which are unexpected and 
possibly, probably or definitively related to the study intervention. 
 
Any clinical cardiac event that does not meet the criteria of SAE 
 
 
The safety monitor, CCMO, or investigators may decide to put the study on hold based on 
adverse events, pending discussion with the safety monitor, SMC, 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 safety monitor will review all 
available safety data on a pre-defined time point after the challenge period. If the CCMO has 
recommended safety hold, re-initiation of the study will require CCMO concurrence. The CCMO 
will be informed of a safety hold by the sponsor. Following discussion, it may be decided to 
terminate the study. 
PATH will be informed of a safety hold by the sponsor. 


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10. STATISTICAL ANALYSIS 
 
 
10.1 Primary study parameter 
The primary endpoint is the number and intensity of adverse events (solicited and unsolicited 
combined) after NF135.C10 CPS immunization. Adverse events will be analyzed by calculating 
the proportion of volunteers in each group who reported mild, moderate or severe adverse events. 
Relatedness of events will be taken into account as well as the time point of the adverse events 
(e.g. during immunization phase or during challenge phase). The frequency of signs and symptoms 
(categorized by severity, relatedness and study phase) will be compared between groups with 
either the chi-square test or Fishers exact test (two-tailed) depending on definitive sample sizes. 
Adverse events will be categorized by ICD-10 coding. Assumptions of statistical tests will be 
evaluated prior to performing the analysis. 
 
10.2 Secondary study parameter 
The secondary endpoints are time to parasitemia, defined as the time to the first single 
positive qPCR, and sterile protection after each CHMI. Volunteers who remain negative until day 
28 after each CHMI will be considered fully protected against malaria. Sterilely protected subjects 
will not be included in the time to parasitemia analysis. Comparison of mean time to first qPCR 
positivity between all groups will be assessed by Mann-Whitney U test (two- sided). Once 
daily qPCR (threshold 100 parasites/ml) has a 99 % sensitivity and a 100 % specificity [26], 
and in the 1% of cases where 100 Pf/mL is not detected, the qPCR test is always repeated. 
The percentage of volunteers protected will be compared with either the chi-square test or 
Fishers exact test (two-tailed) depending on definitive sample sizes. Assumptions of statistical 
tests will be evaluated prior to performing the analysis. 
 
10.3 Exploratory study parameters 
In immunological analyses we will assess differences by comparing mean values between 
groups or time points using either a two-tailed student’s t-test (if comparing two groups) or a one-
way ANOVA (if comparing more than two groups), or non-parametric equivalents. Paired tests will 
be used if pre-intervention values  are compared with post intervention values, unpaired tests 
will be used if comparisons are made between groups. For discrete variables (e.g. the number of 
positive assays), the chi-squared test or Fisher’s exact test will be used (two-tailed). Assumptions 
of statistical tests will be evaluated prior to performing the analysis. 


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11. ETHICAL CONSIDERATIONS 
11.1 Regulation statement 
This study will be conducted in accordance with the latest Fortaleza revision of the Declaration 
of Helsinki (2013), the Medical Research Involving Human Subjects Act (WMO), the ICH Good 
Clinical Practice, and local regulatory requirements. 
The investigators are responsible for obtaining Ethics Committee(s) approval (IRB) of the 
protocol and any subsequent amendments in compliance with local law before the start of the 
study. 
This study will be reviewed by the Central Committee on Research Involving Human Subjects 
(CCMO) and the Western Institutional Review Board (WIRB). 
 
11.2 Recruitment and consent 
As soon as the study is approved, 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 Radboud University. Furthermore, a Facebook page (link: 
http://www.facebook.com/malariavaccin) showing the advertisement text will be used to inform 
people about the trial. This  brief advertisement will indicate a telephone number to call and an 
e-mail address for contact to request further information. It will furthermore indicate a website 
(www.malariavaccin.nl) which contains  a form. This  short questionnaire will be completed using 
the online form. When seemingly suitable 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-approved informed consent. Informed consent must be obtained 
before conducting any study-specific procedures (i.e. all of the procedures described in the 
protocol, including screening procedures). The process of obtaining informed consent should be 
documented in the subject source documents. During the screening visit, the questionnaire 
answers will be discussed and inclusion and exclusion criteria will be checked. Also, a letter for 
the general practitioner will be signed and sent after screening.  Again,  the  investigator  will  
answer  any  questions  the  volunteer  has.  The 


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possibility of withdrawal from the study, at any time, without penalty and without any declaration 
of the reason will be pointed out to the volunteers. 
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. 
 
11.3 Benefits and risks assessment, group relatedness 
Two major areas of ethical concern are contained within this proposal, namely the use of blood 
from humans and the use of human volunteers for P. falciparum CHMI. All partners in this proposal 
are aware of and follow the relevant national and international rules and regulations as they 
pertain to access to material of human origin and clinical research. International agreements 
such as the Declaration of Helsinki will be observed and respected. 
 
11.3.1 
Ethical aspects concerning the production of P. falciparum infected 
mosquitoes 
The human blood and serum used for parasite culture is obtained from screened healthy blood 
donors from Sanquin. Continuous culture of P. falciparum has been routine for the Central 
Animal Facility of the Radboudumc over the past 3 decades. All culture material is checked for 
bacterial contamination, including Mycoplasma, and for blood-borne pathogens (HIV, HBV, HCV, 
HTLV 1+2). The process has become highly standardized as described in standard operating 
procedures and was positively reviewed by an external auditor in 2014. 
 
11.3.2 
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 conducting human infections with mosquitoes 
fed on P. falciparum gametocyte cultures in 1986 [21]. The occurrence of four cardiac events in 
volunteers participating in phase I/IIa malaria vaccine trials in Nijmegen has raised 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 and 13). In 2016 the occurrence of mild, transient liver and kidney injury during CHMI was 
extensively evaluated. Based on recommendations 


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of the CCMO and external experts, the malaria challenge protocol was modified to include extra 
monitoring (see also section 1.4 and 13). 
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 and complementary 
candidate malaria vaccines are of paramount importance with the potential of large-scale application 
in endemic countries. 
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. 
 
11.4 Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of the WMO. 
The sponsor (also) has an insurance which is in accordance with the legal requirements in the 
Netherlands (Article 7 WMO). This insurance provides cover for damage to research subjects 
through injury or death caused by the study. 
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. 
 
11.5 Incentives 
Volunteers in group 1, 2, 3 and 4 will receive 2300 euros in compensation fee for the 
immunisations and the first CHMI. Volunteers participating in the second CHMI will receive an 
additional 800 euros. Volunteers in groups, 5-8 will receive 800 euros. These amounts are based 
on our predefined criteria, i.e. 300 euro reimbursement for each mosquito exposure  
(inconveniences  associated  with  full  day  visit,  mosquito  bites  and  necessary 


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antimalarial prophylaxis/treatment), and a 20 euro reimbursement for each routine follow-up visit 
(maximally one hour, including venapuncture). Additionally, volunteers are compensated for 
completing trials with a long duration (20 euro per month). Travel expenses will not be additionally 
reimbursed. This amount of money is reasonable and in line with Dutch common practice. If a 
subject withdraws from the study prior to completion, they will receive reimbursement proportional 
to number of visits they attended. In case of unexpected medical complications related to 
participation in the study, there will be access to state-of-the-art medical treatment with full costs 
covered by the insurance of Radboudumc. 


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12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
 
12.1 Handling and storage of data and documents 
 
12.1.1 
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 biological samples, with exception of those taken for safety diagnostics, will be labelled only 
with the volunteer study identification  number. Samples  taken for  safety diagnostic (processed 
by the central clinical laboratory of the Radboudumc) will be labelled with part of the subject 
identification code, study identification name and a fictitious birth date (only using the subjects 
actual birth year). The samples will not be labelled with volunteer names or actual birth dates. 
The subject identification code will be kept by the principal investigator. All data will be stored for 
minimally 15 years. All biological samples will be stored for maximally 15 years. 
 
12.1.2 
Data collection 
Designated trial staff will enter the data required by the protocol into the electronic CRF 
(eCRF). 
 
12.1.3 
Database management and quality control 
All data should be recorded, handled and stored in a way that allows its accurate reporting, 
interpretation and verification. 
A data monitor, contracted from an independent CRO, will review the data entered into the 
eCRFs by investigational staff for completeness and accuracy and instruct the site personnel to 
make any required corrections or additions. Queries are made during each monitor visit. 
Designated investigator site staff is required to respond to the query and confirm or correct the 
data. Medical history/current medical conditions and adverse events will be coded using the ICD-
10 terminology. 
 
12.2 Monitoring and Quality Assurance 
Before study initiation, the protocol and eCRFs together with relevant SOPs will be reviewed by 
the sponsor, the investigators and their staff. During and after completion of the study, the data 
monitor will visit the site to check the completeness of records, the accuracy of entries on the 
eCRFs, the adherence to the protocol and to Good Clinical Practice, the progress of 


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enrolment, and to ensure that mefloquine and Malarone are 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, 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 this 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 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 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 from group 1, 2, 3 or 4 and 2 subjects from group 5, 6, 7 
or 8). 
 
 
12.3 Amendments 
Amendments are changes made to the research after a favourable opinion by the CCMO has been 
given. All amendments will be notified to CCMO. 
 
 
12.4 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. 
 
 
12.5 Temporary halt and (prematurely) end of study report 
The sponsor 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 last patient’s last visit. 
The sponsor will notify the CCMO immediately of a temporary halt of the study, including the 
reason of such an action. 
In case the study is ended prematurely, the sponsor will notify the CCMO within 15 days, 
including the reasons for the premature termination. 


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12.6 Public disclosure and publication policy 
This trial will be registered in clinicaltrials.gov prior to study start. The final report will be 
prepared by the investigators at the Radboud university medical center. It will be signed by the 
project leader or the principle investigator. The investigators will make every effort to publish 
the results in a peer-reviewed journal. 


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13. STRUCTURED RISK ANALYSIS 
 
 
13.1 Potential issues of concern 
In this study volunteers will undergo a Controlled Human Malaria Infections by the bites of 
laboratory reared Anopheles mosquitoes infected with the NF135.C10 or NF54 strain of 
Plasmodium falciparum. 
 
a. Level of knowledge about mechanism of action 
The causative organisms of malaria, Plasmodium parasites, were first identified by Laveran in 
1880 and their complete life-cycle in the mammalian host was elucidated by 1947. Extensive clinical 
experience of malaria infection has since been accumulated by the medical 
community. 
Nevertheless, certain aspects of the pathophysiology of (severe) malaria (e.g. cerebral malaria) 
remain incompletely understood. Such manifestations,  however, do not occur during Controlled 
Human Malaria Infections due to the very early treatment of study subjects and hence the 
clearance of parasitemia at extremely low levels (see also below). 
 
b. Previous exposure of human beings with the test product(s) and/or products with a similar 
biological mechanism 
There is extensive clinical experience with 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. Worldwide the majority of these infections have been performed with the laboratory 
NF54 strain or its daughter clone 3D7. 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 [22], in 2007 [23] and in 2011[33]. 
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo human cell 
material? 
In vitro assays cannot capture the complexity of the multi-stage life-cycle of Plasmodium 
parasites and their complex of interaction with the host. Although (parts of) the Plasmodium life-
cycle can be reproduced in (rodent or primate) animal models, these either involve non- human 
Plasmodium-species or non-natural host-parasite combinations. As a result, extrapolation of 
pathophysiological and immunological aspects of infection to human disease are only possible to 
a limited extent [34, 35]. 


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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 [ 3 6 ] . 
 
e. Analysis of potential effect 
Safety and tolerability of CHMI will be assessed by recording adverse events, reported at any time 
during the trial either at a visit or by writing in the diary. Temperature, will be recorded at every visit 
following the CHMI. Assessment of complete blood count, LDH and troponin-T will be performed 
daily starting on day 6 after the CHMI. Assessment of liver and kidney function will be performed 
at baseline, on day 6 post CHMI, at initiation of treatment, on day 2 post treatment and at the 
end of study visit. 
The gold-standard for the detection of blood stage malaria infections is thick blood smear 
microscopy (detection limit 4 parasites/ul). Previously, we have used qPCR as the main tool to 
detect blood-stage malaria parasites [25, 32], which has an even greater sensitivity. 
When treatment is initiated based on thick smear, parasitemia in subjects undergoing CHMIs 
should never exceed ±0.001% [ 2 4 ] . In contrast, manifestations of severe malaria generally only 
occur above 5% parasitemia and never below 1% parasitemia (WHO Guideline Severe Malaria, 
SWAB Richtlijn). 
In two previous CHMI trials (NL48301.091.14; NL48732.091.14) qPCR was performed daily during 
follow-up and volunteers were treated with anti-malarial drugs after a single positive qPCR. 
During these two studies, 39 volunteers developed patent parasitemia. Of these volunteers 26% 
developed no malaria symptoms, 38% had only grade 1 symptoms (no influence on daily 
activities), 28% had grade 2 symptoms (some influence on daily activities) and only 13% had 
grade 3 symptoms (requiring bedrest). All grade 3 symptoms lasted for less than one day. 
 
f. Pharmacokinetic considerations  
Not applicable. 
 
g. Study population 
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, as described in section 8. 


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h. Interaction with other products 
Concurrent use of drugs potentially interacting with atovaquone-proguanil (e.g. artemether- 
lumefantrine, rifampicin, metoclopramide, oral anti-coagulants and certain anti-retrovirals) are 
contra-indicated. 
 
i. Predictability of effect 
In this trial at least some volunteers will experience blood stage malaria infection. Blood stage 
malaria infection has been seen after CHMI in over 3,500 volunteers. The progression and 
symptoms of this parasitemia has been demonstrated to be reproducible and predictable [ 2 2 -
2 4 ] . However, the occurrence of cardiac serious adverse events in Radboudumc CHMI trials 
has led to an increase in safety measures, discussed in a separate section below. 
 
j. 
Can effects be managed? 
Subjects are followed up intensively at Radboudumc on an outpatient basis for clinical and 
parasitological assessment to ensure treatment is started at the earliest possible time point. The 
NF135.C10 andNF54 P. falciparum strains have been tested for susceptibility to atovaquone-
proguanil in vitro. Should treatment with this drug need to be discontinued prematurely in any 
subject for whatever other reason (e.g. intolerability), various other anti-malarial drugs are available, 
both oral and intravenous, to which this strain is also susceptible. 
 
13.2 Cardiac events following Controlled Human Malaria Infections 
In the twenty-three CHMI studies conducted at Radboudumc prior to CPS135* (involving 376 
healthy volunteers), three cardiac events have occurred after infection with the NF54 strain with 
the confirmed or differential diagnosis  of myocarditis. The occurrence of myocarditis in both 
natural uncomplicated malaria and CHMI has never been described elsewhere. However, the three 
cardiac events share no specific factor other than malaria infection.   
With regards to the malaria infection, these myocarditis cases do share a number characteristics: 
i) they took place 1-5 days after start of antimalarial treatment but different anti-malarial drugs 
were used in all cases ii) there was no parasitemia detectable at the time of the event, and iii) 
next to the P. falciparum infection, other and known possibly triggering factors (e.g. preceding 
vaccinations, concomitant infections, cannabis use) were present during or preceding the event. 
The three cases are discussed in detail below. 
*Additionally, after the first immunization in cohort B of the CPS135 trial, a fourth  SAE occurred, 
also described below. 


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Case 1 
In 2007 a 20 year old female was immunized with the candidate malaria vaccine, LSA3 
(NL14715.000.06), underwent a CHMI and was treated with artemether/lumefantrine (Riamet®) 
after a positive thick smear. She presented with retrosternal chest pain three days after treatment, 
blood slide was negative. Elevated troponin-I (maximally 11.80ug/l) and ECG abnormalities were 
suggestive for a cardiac event with minimal cardiac damage and an MRI showed no abnormalities. 
Risk factors for cardiac disease in this volunteer included an acute myocardial infarction in a 
paternal grandfather at age 43 and a paternal family history of dyslipidemia. The final diagnosis 
was acute coronary syndrome or myocarditis. This case is reported in [ 2 7 ] , attachment K4a. 
Following this event, recommendations of the European Malaria Vaccine Development 
Association and the CCMO followed for improved safety of participating volunteers: 
• 
A/P replaced Riamet®  as treatment during CHMIs at Radboudumc. it. 
• 
Individuals  will  be  excluded  from  participation  if  they  have  1st  or  2nd  degree 
relatives who had cardiac events when less than 50 years of age. 
• 
Volunteers will be required to stay very close to the Radboudumc to ensure maximal 
safety from day 5 after CHMI until treatment has been finished (maximum 12 days). 
• 
Monitoring of highly sensitive troponin T (hsTropT), D-dimer, lactate dehydrogenase 
(LDH) and thrombocytes. 
 
Case 2 
In 2013, a healthy male, taking part in the TIP5 clinical trial (NL39541.091.12) underwent a CHMI 
challenge after immunization with cyropreserved sporozoites under chloroquine prophylaxis (CPS 
protocol). The volunteer became thick-smear positive after challenge infection and the volunteer 
was treated with A/P. Per protocol blood examination revealed an elevated troponin-T (maximally 
1115ng/l) on the second day of Malarone treatment, while the volunteer was asymptomatic. He 
was admitted to cardiology and troponin-T values, ECG abnormalities and a cardiac MRI confirmed 
the diagnosis acute myocarditis. During admission he experienced a 20 minute episode of 
retrosternal chest pain. Further diagnostic tests and patient history revealed a concurrent 
rhinovirus infection and a boosting regime of standard travel vaccines 46 days preceding the 
CHMI, with a booster 20 days before the CHMI. This case has been reported in [28], attachment 
K4b. Due to this SAE, new safety recommendations were integrated into our procedures for 
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• 
Exclusion of 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. 
• 
Increased control of hs troponin T as  a marker of cardiac damage; initiation of 
treatment with Malarone when hs troponin T > 0.1 μg/ml or on recommendation of the 
cardiologist. 
• 
Continuation of daily LDH measurements starting from day 5 after CHMI; Malarone 
treatment will be initiated if LDH values are above 1000 U/L. 
• 
Daily measurements of thrombocytes; volunteers will be treated with Malarone when 
thrombocyte levels are < 120x 10^9/L. 
• 
After CHMI we will rely on real-time qPCR for diagnosis of malaria and initiation of 
treatment, using the following criteria: 
 
o 
Two  consecutive  positive  qPCR  results  in  a  volunteer  with  temperature 
<38.0⁰C 
o 
One positive qPCR result in a volunteer with temperature ≥38.0⁰C 
o 
One positive thick  smear (which will be made if  a  volunteers  attends for 
evening follow-up with temperature ≥38.0⁰C or on decision of the trial clinician) 
Case 3 
In 2014, a healthy, 23 year old, male volunteer underwent a CHMI under chloroquine 
prophylaxis (CPS-immunization). On day 10 after exposure to bites of 15 malaria infected 
mosquitoes, routine per protocol blood examinations revealed an elevated troponin-T (maximally 
168ng/l) though the volunteer was asymptomatic. This volunteer had no obvious risk factors for 
cardiac disease other than cigarette smoking and recent cannabis use. This volunteer was 
negative by thick smear but positive by PCR with maximum parasitemia was 1265Pf/ml while 
under chloroquine prophylaxis. Following this cardiac event, further safety measures have been 
integrated into our CHMI protocols: 
• 
Volunteers will be treated with antimalarial after a single positive qPCR after malaria 
challenge infection 
• 
Use of cannabis will be added as an exclusion criterion 
• 
Excessive physical exercise around immunization and during challenge period will be 
prohibited 
Case 4 
In 2020, a 23 year old healthy female volunteer underwent a first immunization with 15 NF135 
infected mosquitoes. On day 7 after exposure she started with presumptive A/L treatment, per 
protocol. On this same day she had parasitaemia, as detected by qPCR, with a density above the 
theoretical detection limit of thick blood smear microscopy. By day 9, her qPCR was again 


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negative. On day 10 after exposure she developed chest pain, a dynamic ECG and elevated 
cardiac biomarkers occurring 1 day after completion of presumptive artemether-lumefantrine 
treatment. Chest pain decreased spontaneously and troponin levels decreased.  MRI, Coronary 
CT-A, CAG and echocardiography showed no evidence of myocarditis, Tako Tsubo or coronary 
occlusion / dissection. The presumptive diagnosis was an inflammatory response with no 
evidence of myocarditis on MRI.  
 
Considerations regarding presumptive A/L treatment during the first immunization phase 
of cohort B 
Both A/L and A/P are recommended in the Dutch national treatment guidelines for uncomplicated 
P. falciparum malaria. After the first cardiac SAE in the LSA-3 trial in 2007, as described in Case 
1, it was decided in consultation with CCMO to favor treatment with A/P over A/L. Although at the 
time of this serious adverse event, A/L treatment was considered unlikely to be a cause/risk 
factor and there was no direct evidence of A/L cardiotoxicity, an alternative treatment was 
preferred based on: 
1. The hypothesis that due to the rapid killing of parasites, A/L would induce an inflammatory 
response with possible predisposition to myocarditis. Further clinical or other evidence to 
support this assumption remains lacking, however. 
2.  A specific case report of vasospasms after A/L use, where the patient’s urine was also 
tested positive for tetrahydrocannabidol [37].  
Since both drugs are registered for malaria treatment, a preference was expressed at that time 
for A/P, on the basis of the above considerations. After the switch to A/P, two other cardio-
ischemic events occurred in CHMI volunteers, which does not support A/L as a possible etiology. 
All things considered, therefore, from the perspective of the safety  of volunteers in this study, the 
substitution of A/P by A/L as presumptive treatment during the first immunization of cohort B is 
considered justifiable.  
Considerations regarding presumptive A/P treatment during the second immunization 
phase of cohort B 
After the cardiac SAE in cohort B, immunization 1, an alternative presumptive treatment based on 
A/P was chosen, starting on day 5 after immunization2. A/P treatment is preferred over A/L 
because of  i) risk of treatment failure after A/L treatment, and ii) the higher inflammatory 
responses after A/L treatment (potentially due to more rapid-acting mechanism) as is also 
illustrated by the higher number and severity of adverse events following cohort B compared to 
the 15 mosquito-bite dose arm in cohort A, despite similar parasite densities. Treatment will be 
initiated on day 5 to potentially block or reduce release of merozoites from the liver, thus further 
reducing inflammation, whilst still allowing late liver-stage development and the consequent 
development of pre-erythrocytic immunity. Even if merozoite release is not fully blocked, the 
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erythrocytic development is suppressed from the start.  
 
 
13.3 Transient liver function test elevations 
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%, <2.5xULN), moderate (10%, ≥2.5-5.0xULN) or severe (12%, 
≥5.0xULN) increases of liver transaminases (ALT/AST). Among these volunteers the median 
increase of ALT was 71.5 U/l (range 19-870 U/l, Q25 of 48 U/l, Q75 of 116 U/l), and median 
increase of AST was 57 U/l (range 31-723 U/l, Q25 of 45 U/l, Q75 of 97 U/l). The liver function 
test (LFT) elevations remained asymptomatic in all volunteers, and the typical pattern is limited 
to ALT/AST elevations without change in bilirubin. The LFT elevations exceeded the upper limit 
of normal (ULN) at the start up to two days after the start of malaria treatment. LFT monitoring 
before treatment was only assessed in 1 CHMI study (n=18), showing that LFT elevations 
typically arise after treatment. The LFTs peak between 2-14 days post treatment with values 
normalized at study end (35–42 days after challenge infection). 
Volunteers treated earlier with positive qPCR rather than thick smear, showed a lower percentage, 
and severity of LFT abnormalities, 13/58 volunteers (22%) (11/58 (19%) mild, 1/58 (2%) 
moderate, and 1/58 (2%) severe). All CHMI-volunteers showed normalized values at study end. 
A clear explanation for the transient elevated transaminases is not obvious. Although higher 
parasitaemia associates with higher LFT at group level, there is no clear relationship on 
individual levels. It also seems unlikely related to choice of anti-malarials, given the diversity of 
drug regimens used. Similarly, the timing, different, 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 these abnormalities. 
According to an independent safety monitoring committee and an independent hepatologist, these 
transient asymptomatic and severe LFT elevations are most likely a  direct consequence of the 
malaria infection and subsequent treatment, rather than antimalarial drugs only or another liver 
nox. Given the rapid and spontaneous resolution and the absence of clearly elevated bilirubin 
levels, this damage was considered transient, and to permit further studies utilising Plasmodium 
falciparum challenge. 
 
Consequently, the CHMI protocol has been adapted to include additional safety measures. 
• 
Volunteers  will  undergo  regular  safety  monitoring  to  assess  asymptomatic  liver 
function test abnormalities 


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• 
Volunteers must avoid additional triggers that may cause elevations in liver enzymes 
including alcohol from baseline up to 1 week post treatment. 
• 
The maximum dose of paracetamol/acetaminophen is 3000 mg per day 
• 
Curative anti-malarial treatment will be initiation of a at ALT/AST levels >5x ULN 
 
13.4 Transient serum creatinine elevations 
Transient, asymptomatic increases in serum creatinine compared to baseline (though within 
normal limits) have also been found during CHMI in the Radboudumc. A retrospective analysis of 
5 CHMI studies showed a small increase of creatinin, relative to the subject’s own baseline (but 
within the accepted range of normal), in 70/80 (88%) of the subjects. Only 5/80 (5%) showed mild 
elevations above the upper limit of normal 2 days after treatment. No moderate or severe 
elevations have been found. No difference in serum creatinine was found between subjects 
that were treated based on thick smear threshold or qPCR threshold. All subjects showed 
normalized values at the end of the study. An independent nephrology expert did not consider 
the transient decrease in renal function as a particular risk for permanent kidney damage. 
 
Based on these findings the CHMI protocol has been adapted to: 
• 
Exclude subjects with abnormal renal function at baseline. 
• 
Avoid additional triggers that may cause renal dysfunction, such as: 
o Alcohol intake from baseline up to 1 week post-treatment 
o Heavy physical exercise 
o Use of recreational drugs 
• 
Add standard monitoring of renal function at specific time points after CHMI 
 
 
13.5 Synthesis 
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 CHMI by the bites of 
mosquitoes fed on cultures of P. falciparum gametocytes to produce sporozoites [21]. This has 
proved to be a reproducible, predictable and safe method of inducing P. falciparum malaria. 
The results of such studies were summarized in 1997 [22], in 2007 [23] and in 2011 [ 2 4 ] . 
Recently, treatment based on qPCR has allowed for a significant decrease in intensity and 
duration of adverse events [26]. 
Following four cardiac events after blood stage parasitemia, our safety procedures for CHMI 
remain strongly intensified. In the current trial, we will adhere to those stringent procedures that 
are relevant, see section 1.4. 


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